WO2015085411A1 - Modulation of the beta-oxidation pathway in the control of rhamnolipid production - Google Patents
Modulation of the beta-oxidation pathway in the control of rhamnolipid production Download PDFInfo
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Definitions
- the present invention relates to the production of rhamnolipids (RLs) by microorganisms, and more particularly to the modulation thereof for different applications.
- RLs rhamnolipids
- Rhamnolipids are glycolipidic biosurfactants produced by various bacterial species.
- the bacterium Pseudomonas aeruginosa is the best-studied producer of rhamnolipids (RLs), surface-active, multipurpose amphiphilic molecules.
- RLs are produced as mixtures of congeners composed of 1 or 2 rhamnose moieties linked to a dimer of R-3-hydroxyfatty acids with chain lengths of Ci2 ⁇ 4 (Deziel et al.,
- RLs are synthesized from two precursors, R-3-hydroxyfatty acids and L-rhamnose, by the actions of three enzymes: RhIA, RhIB, and RhIC.
- RhIA dimerizes R-3-hydroxyfatty acids forming congeners of R-3-((R-3-hydroxyalkanoyl)oxy)alkanoic acids (HAAs); the most abundant of which in P. aeruginosa is C10-C10 (Deziel et al., 2003; Zhu and Rock, 2008).
- RhIB and RhIC catalyze the sequential addition of the first and second rhamnose to the C10-C10 dimer, forming mono- (Rha-Cio-Cio) and di-RL (Rha-Rha-Cio-Cio), respectively (Ochsner et al., 1994; Rahim et al.,
- Rhamnolipids promote the uptake and biodegradation of poorly soluble substrates, act as immune modulators and virulence factors, have antimicrobial activities, and are involved in surface motility and in bacterial biofilm development.
- rhamnolipids as virulence factors contributing to pathogenic infection by RL-producing microorganisms such as Pseudomonas aeruginosa
- the inhibition of the production of RLs constitutes a therapeutic strategy for attenuating the virulence of RL-producing microorganisms.
- the present invention provides a method for decreasing the production of rhamnolipids (RLs) by a cell, for example a microorganism, said method comprising inhibiting the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I) in said cell.
- RLs rhamnolipids
- EH/I R-specific enoyl-CoA hydratases/isomerases
- the present invention provides a method for attenuating the virulence of a rhamnolipid-producing microorganism, said method comprising inhibiting the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I) in said rhamnolipid- producing microorganism.
- EH/I R-specific enoyl-CoA hydratases/isomerases
- the present invention provides the use of an inhibitor of R-specific enoyl-CoA hydratases/isomerases (ECH/I) for decreasing the production of rhamnolipids (RLs) by a cell, for example a microorganism.
- EH/I R-specific enoyl-CoA hydratases/isomerases
- the present invention provides the use of an inhibitor of R-specific enoyl-CoA hydratases/isomerases (ECH/I) for the preparation of a medicament for decreasing the production of rhamnolipids (RLs) by a cell, for example a microorganism.
- the present invention provides the use of an inhibitor of R-specific enoyl-CoA hydratases/isomerases (ECH/I) for attenuating the virulence of a rhamnolipid-producing microorganism.
- EH/I R-specific enoyl-CoA hydratases/isomerases
- the present invention provides the use of an inhibitor of R-specific enoyl-CoA hydratases/isomerases (ECH/I) for the preparation of a medicament for attenuating the virulence of a rhamnolipid-producing microorganism.
- ECH/I R-specific enoyl-CoA hydratases/isomerases
- the above-mentioned inhibitor is an antibody specifically binding to said one or more R-specific ECH/I. In a further embodiment, the above-mentioned antibody is monoclonal. [0018] In another embodiment, the above-mentioned inhibitor is an inactive or dominant- negative form of said one or more R-specific ECH/I.
- the above-mentioned inhibitor is an RNA interference agent targeting said one or more ECH/I.
- the above-mentioned RNA interference agent is a shRNA or siRNA.
- the one or more R-specific ECH/I comprise an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95% or 100% identity with the amino acid sequence of FIG. 15A (SEQ ID NO:2), 16A (SEQ ID NO:4) and/or 17A (SEQ ID NO:6).
- the present invention provides a method for increasing the production of rhamnolipids (RLs) by a cell, for example a microorganism, said method comprising increasing the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I) in said cell.
- the method comprises introducing into said cell one or more nucleic acids encoding said one or more R-specific ECH/I.
- the method further comprises introducing one or more nucleic acids encoding one or more polypeptides involved in the RL biosynthetic pathway.
- the above-mentioned one or more polypeptides involved in the RL biosynthetic pathway are rhamnosyl-transferases, in yet a further embodiment RhIB and/or RhIC.
- the one or more nucleic acids encode the R-specific ECH/I defined above.
- the above-mentioned one or more nucleic acids comprise a sequence having at least 50%, 60%, 70%, 80%, 90%, 95% or 100% identity with the nucleotide of FIG. 15B (SEQ ID NO:1 ), 16B (SEQ ID NO:3) and/or 17B (SEQ ID NO:5).
- the above-mentioned method is an in vivo method. In an embodiment, the above-mentioned method is an in vitro method.
- the present invention provides a method for determining whether an agent may be useful for (i) inhibiting the production of rhamnolipids (RLs) by a cell, for example a microorganism and/or (ii) attenuating the virulence of a rhamnolipid-producing microorganism, said method comprising determining whether said agent inhibits the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I).
- RLs rhamnolipids
- the method comprises (a) contacting said agent with said one or more R-specific ECH/I and (b) determining whether said agent inhibits the expression and/or activity of said one or more R-specific ECH/I, wherein the inhibition of said expression and/or activity is indicative that said agent may be useful for (i) inhibiting the production of RLs by a cell and/or (ii) attenuating the virulence of a rhamnolipid-producing microorganism.
- the present invention provides a method for determining whether an agent may be useful for increasing the production of rhamnolipids (RLs) by a cell, for example a microorganism, said method comprising determining whether said agent increases the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I).
- RLs rhamnolipids
- the method comprises (a) contacting said agent with said one or more R-specific ECH/I and (b) determining whether said agent increases the expression and/or activity of said one or more R-specific ECH/I, wherein the increase of said expression and/or activity is indicative that said agent may be useful for increasing the production of rhamnolipids (RLs) by a cell.
- the above method is a cell-free method.
- the above method is performed in a cell-based system, i.e. the one or more R-specific ECH/I are expressed in a cell.
- the one or more R-specific ECH/I comprise an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95% or 100% identity with the amino acid sequence of FIGs. 15A (SEQ ID NO:2), 16A (SEQ ID NO:4) and/or 17A (SEQ ID NO:6).
- the microorganism is of the Pseudomonas genus, in a further embodiment Pseudomonas aeruginosa, e.g., P. aeruginosa strain PA14.
- FIGs. 1A and 1 B show that specific inhibitors of ⁇ -oxidation decrease RLs production in
- FIG. 1A Dose-dependent inhibition of RLs and HAA production by 2- bromooctanoic acid.
- FIG. 1 B 2-bromohexanoate similarly inhibits RLs production at equivalent molar concentration (5 mM).
- Strain PA14 was grown in MSM-glycerol for 3 days at 34°C. Data represent the mean of triplicate experiments ⁇ S.D.
- FIGs. 2A and 2B show that RhIY and RhIZ are implicated in RLs biosynthesis controlling the supply of lipid precursors.
- FIG. 2A Deletion mutants, ⁇ RhIYZ and RhlZ have similarly reduced RLs production. Complementation of ARWVZ with RhIZ only partially restores RLs production.
- FIG. 2B RhIYZ ⁇ s the main supplier of 3-hydroxyalkanoates (C10+2). Wild-type PA14 strain and mutants were grown for 3 days in MSM-glycerol at 34°C. Lipids were analyzed by GC/MS. Data represent the mean of triplicate experiments ⁇ S.D.
- FIGs. 3A to 3F show that RhIZ is an R-specific enoyl-CoA hydratase/isomerase (ECH/I) whose activity is modulated by RhIY.
- the purified RhIZ alone did not catalyze any 3-hydroxydecanoate formation from trans-2-decenoyl-CoA, therefore, FIG. 3A: Total cell lysate of WT was added in conjunction with RhIZ and this caused enrichment of the R-3-hydroxydecanoate compared to the S- enantiomer in comparison to the total cell lysate of WT alone as in FIG. 3B, which produced a nearly racemic mixture of 3-hydroxydecanoate.
- FIG. 3A Total cell lysate of WT was added in conjunction with RhIZ and this caused enrichment of the R-3-hydroxydecanoate compared to the S- enantiomer in comparison to the total cell lysate of WT alone as in FIG. 3B, which produced a nearly racemic mixture of 3-hydroxyde
- FIG. 3C, 3D The same was observed with total cell lysate of the mutant RhlZ.
- FIG. 3E, 3F The factor in lysates of WT or RhIZ mutant that is indispensable for RhIZ activity appears to be RhIY as pure RhIZ does not produce any 3-hydroxydecanoate in the presence of total cell lysate of RhlYZ. R/S-3-hydroxydecanoate was analyzed using a chiral HPLC-MS/MS method.
- FIG. 4 shows that RhIZ is an R-specific ECH/I and is inhibited by a metabolite of 2- bromooctanoate (2-Br-Ce).
- RhIZ causes R-specific enrichment of 3-hydroxydecanoate, and this enrichment is blocked when using WT lysate of cells cultivated in the presence of 5 mM 2-Br-Cs.
- R/S-3-hydroxydecanoate was analyzed using chiral HPLC-MS/MS. Data represent the mean of triplicate experiments ⁇ S.D.
- FIG. 5 shows an SDS-PAGE of the pull-down assay showing the stable physical protein- protein interaction between RhIY and RhIZ.
- Purified His6-tagged RWZ was mixed with total cell lysate of WT PA14 strain for 1 hour at 4°C, the mixture was then subjected to Nickel affinity chromatographic purification using FPLC using the same gradient of imidazole as previously done for purification of RhIZ (FIG. 14A).
- Lane (1) is the purified RhlZ-H ⁇ se alone (bait).
- Lane (2) is the control lysate which was not exposed to the RWZ-His6.
- Lane (3) is the purified p rotei n -R/7/Z-H i se interaction; this protein was shown to be RhIY as shown from its size compared to the purified RhlY-H ⁇ se in lane (4) as well as by protein sequencing.
- FIGs. 6A and 6B show that RLs and PHA share the same 3-hydroxyalkanoate pool. Deletion of RhIYZ results in a decrease in PHA accumulation, as estimated by GC-MS hydrolyzed intracellular 3-hydroxyfatty acids (Cs to C12) (FIG. 6A) as well as by percentage cross-sectional area of PHA granules relative to the whole cell cross-sectional area on images taken by transmission electron microscope, based on an average measurements of more than 400 cells of both WT and mutant (FIG. 6B).
- Cs to C12 3-hydroxyfatty acids
- FIG. 6C The PHA-negative mutant ⁇ kphaC1phaDphaC2phaDkphaFkphal) causes a slight increase in RLs, mostly with a 100% enrichment of mono-RLs at the expense of di-RLs.
- FIG. 7 is a proposed model of metabolic supply for lipid precursors of RLs.
- a fatty acid carbon source With a fatty acid carbon source, shortening of the chain takes place by ⁇ -oxidation until the formation of the trans-2- decenoyl-CoA.
- a soluble carbon source like glycerol, de novo synthesis of fatty acids via FASII forms fatty acids that are then diverted to ⁇ -oxidation to be processed into the trans-2-decenoyl-CoA intermediate.
- the trans-2-decenoyl-CoA is hydrated by the R-specific enoyl-CoA hydratase RhIYZ to form R-3-hydroxydecanoyl-CoA, which then acts as the direct lipid precursors used by RhIA for synthesis of HAAs then RLs.
- RhIA enoyl-CoA hydratase
- a direct precursor supply from FAS II could also be contributing in RL biosynthesis.
- PHAs share with RLs the same pool of R-3- hydroxyacyl-CoA.
- FabB and FabF 3-ketoacyl-ACP synthetases
- FabG NADPH-dependent 3-ketoacyl- ACP reductase
- FabA FabZ: 3-hydroxyacyl-ACP dehydratases
- Fabl NADH-dependent enoyl-ACP reductase
- FadD acyl-CoA synthetase
- FadE acyl-CoA dehydrogenase
- FadB trans-enoyl-CoA hydratase and S-3-hydrxoyacyl-CoA dehydrogenase
- FadA 3-ketoacyl-CoA thiolase.
- FIGs. 8A and 8B show the effect of different inhibitors of ⁇ -oxidation on RLs and HAA production.
- FIG. 8A 2-bromooctanoate (2-Br-C8) inhibits RLs and HAAs production.
- FIG. 8B Acrylic and 4-pentenoic acid are not inhibiting RLs or HAAs production.
- Strain PA14 was grown in MSM- glycerol for 3 days at 34°C.
- FIGs. 9A and 9B show an investigation of a role of PhaG in RLs production.
- FIG. 9A Role of PhaG in PHA biosynthesis and possible role in RLs biosynthesis.
- PhaG is a R-3- hydroxydecanoyl-ACP:CoA transacylase that converts the R-3-hydroxydecanoyl-ACP intermediate of FAS II into the CoA counterpart to enter in the polyhydroxyalkanoates biosynthesis pathway.
- 2- bromooctanoate-associated inhibition of RLs was suspected to be mediated by PhaG and not by its inhibition of ⁇ -oxidation.
- FIG. 9A Role of PhaG in PHA biosynthesis and possible role in RLs biosynthesis.
- PhaG is a R-3- hydroxydecanoyl-ACP:CoA transacylase that converts the R-3-hydroxydecanoyl-ACP intermediate of FAS II into the CoA counterpart to enter in the polyhydroxyalkanoates biosynthesis pathway.
- RL production was unaffected in the knock-out mutant, t ⁇ phaG (KO), as well as the transposon mutant t ⁇ phaG (Tn) compared to the WT PA14 strain. This indicates that PhaG could not be implicated in RL produciton or be the target of the 2-bromooctanoate- mediated inhibition of RLs.
- Strains were grown in MSM-glycerol for 3 days at 34°C.
- FIGs 10A and 10B show that isotopic tracing demonstrates that lipid precursor of RLs is directly recovered from ⁇ -oxidation.
- FIG. 10A Prediction of isotopic distribution in lipid chains of RLs upon feeding labeled dodecanoate-12,12,12-d3 as sole carbon source. If ⁇ -oxidation is involved in supplying the lipidic precursors for RLs pathway, RLs would be labeled at each lipid chain and would carry an extra 3 Da per chain.
- RLs will be mainly unlabeled because the fatty acid side chains in this case would be constructed from acetate units released from ⁇ -oxidation of the dodecanoate- 12,12,12-d3 and which are mostly unlabeled.
- FIG. 10B Fragmentation pattern of the molecular ion of labeled mono-RLd6, m/z 509 confirms a direct link between ⁇ -oxidation and RLs.
- Fragment ions confirm the identity of the parent molecule as mono-RL-d6 and shows the localization of 3 deuterium atoms in each of the two fatty acid chains of labeled mono-RLs Rha-C10-d3-C10-d3.
- P. aeruginosa PA14 was cultivated for 10 days at 34°C in MSM with dodecanoate-12,12,12-d3 (30 mM) as the sole carbon source.
- FIG. 11 shows a proposed mechanism of incorporation of deuterium-labeled 3- hydroxydecanoate intermediates of dodecanoate-d23 in RLs.
- This ratio arises from the ratio of mono-labeled malonyl-CoA-d1 to unlabeled malonyl-CoA whose abundance is at 2:1 as a result of the carboxylation of mono-labeled acetyl-CoA-d1 replacing a methyl-carried hydrogen atoms (two) or deuterium atom (one).
- FIGs. 12A to 12D shows isotopic tracing of fragment m/z 317 (first column, A1 to D1) and of fragment m/z 201 (second column, A2 to D2) in different labeled carbon source conditions.
- Dodecanoate-d23 (30 mM) (took 7 days to grow) (FIGs. 12-A1 and 12-A2), dodecanoate-d23 (0.375%) with glucose (10 g/L) (FIGs. 12-B1 and 12-B2), dodecanoate-12,12,12-d3 (30 mM) with glucose-13C6 (15 g/L) (FIGs.
- FIGs. 13A to 13C show the impact of mutation and overexpression of different predicted enoyl-CoA hydratases.
- FIG. 13A phaJ2 and phaJ4. Mutants of the PHA-associated R-specific enoyl- CoA hydratases phaJ2 and phaJ4 did not show a decrease in RLs production compared to the WT strain of PA14.
- FIG. 13B RhIK, RhIY, RhIZ.
- the transposon mutants, PA14_40980 ⁇ rhlK ⁇ ), PA14_54640 (rhlY ⁇ ), and the deletion mutant PA14_54660 (M/Z) show reduced RLs production.
- FIG. 13A phaJ2 and phaJ4. Mutants of the PHA-associated R-specific enoyl- CoA hydratases phaJ2 and phaJ4 did not show a decrease in RLs production compared to the WT strain of PA14.
- FIG. 13B Rh
- FIG. 14A and 14B show the purification of N-terminal 6xHis-tagged recombinant RhIZ. Both proteins were purified by nickel affinity chromatography using FPLC.
- RhIZ peak elutes at 160 mM imidazole. It migrated with an apparent molecular mass of 40.6 KDa as determined by SDS-PAGE.
- FIG. 15A and 15B show the amino acid (SEQ ID NO: 2) and encoding nucleotide sequences (SEQ ID NO: 1), respectively, of P. aeruginosa (strain UCBPP-PA14) RhIK (PA14_40980, NCBI-GI: 1 16049771 , UniProt: Q02KW4).
- FIG. 16A and 16B show the amino acid (SEQ ID NO: 4) and encoding nucleotide sequences (SEQ ID NO: 3), respectively, of P. aeruginosa (strain UCBPP-PA14) RhIY (PA14_54640, NCBI-GI: 1 16048660, UniProt: Q02HP6).
- FIG. 17A and 17B show the amino acid (SEQ ID NO: 6) and encoding nucleotide sequences (SEQ ID NO: 5), of P. aeruginosa (strain UCBPP-PA14) RhIZ (PA14_54660, NCBI-GI: 1 16048659, UniProt: Q02R63).
- FIGs. 18A and 18B show the chemical structure of different identified rhamnolipid congeners and homologues (from Abdel-Mawgoud et al., 2010).
- FIGs. 19A and 19B show the taxonomical classification of different bacteria reported to produce rhamnolipids (from Abdel-Mawgoud et al., 2010).
- FIGs. 20A and 20B show the amino acid (SEQ ID NO: 27) and encoding nucleotide sequences (SEQ ID NO: 26), of P. aeruginosa (strain UCBPP-PA14) HAA synthase or Rhamnosyltransferase chain A (RhIA) (PA14J 9110, NCBI-GI: 116051498, UniProt: Q02QW8).
- P. aeruginosa strain UCBPP-PA14
- RhIA Rhamnosyltransferase chain A
- FIGs. 21 A and 21 B show the amino acid (SEQ ID NO: 29) and encoding nucleotide sequences (SEQ ID NO: 28), of P. aeruginosa (strain UCBPP-PA14) Rhamnosyltransferase 1 or Rhamnosyltransferase chain B (RhIB) (PA14J 9110, NCBI-GI: 116051497, UniProt: Q02QW7).
- P. aeruginosa strain UCBPP-PA14
- RhIB Rhamnosyltransferase 1 or Rhamnosyltransferase chain B
- FIGs. 22A and 22B show the amino acid (SEQ ID NO: 31 ) and encoding nucleotide sequences (SEQ ID NO: 30), of P. aeruginosa (strain UCBPP-PA14) Rhamnosyltransferase 2 (RhIC) (PA14_497600, NCBI-GI: 116049062, UniProt: Q02IV0).
- the present inventors have shown that the ⁇ -oxidation pathway is the predominant source of the precursors of RLs. They have also shown that R-specific enoyl-CoA hydratases/isomerases (ECH/I) are involved in the production of these precursors, and consequently of RLs, and thus that the modulation of the expression and/or activity of R-specific ECH/I has an effect on the production of RLs by RL-producing microorganisms such as Pseudomonas aeruginosa.
- ECH/I R-specific enoyl-CoA hydratases/isomerases
- the present invention provides a method for decreasing the production of rhamnolipids (RLs) and/or polyhydroxyalkanoates (PHAs) by a microorganism (e.g., a rhamnolipid- and/or polyhydroxyalkanoate-producing microorganism), said method comprising inhibiting (decreasing, reducing) the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I) in said microorganism.
- RLs rhamnolipids
- PHAs polyhydroxyalkanoates
- the present invention provides a method for decreasing the production of rhamnolipids (RLs) by a rhamnolipid-producing microorganism, said method comprising inhibiting the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I) in said microorganism.
- RLs rhamnolipids
- EH/I R-specific enoyl-CoA hydratases/isomerases
- the present invention provides a method for decreasing the production of polyhydroxyalkanoates (PHA) by a PHA-producing microorganism, said method comprising inhibiting the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I) in said microorganism.
- PHA polyhydroxyalkanoates
- EH/I R-specific enoyl-CoA hydratases/isomerases
- the above inhibition comprises contacting the microorganism with an inhibitor of said one or more R-specific ECH/I.
- the present invention provides the use of an inhibitor of R-specific enoyl-CoA hydratases/isomerases (ECH/I) for decreasing the production of rhamnolipids by a rhamnolipid-producing microorganism.
- EH/I R-specific enoyl-CoA hydratases/isomerases
- the present invention provides the use of an inhibitor of R-specific enoyl-CoA hydratases/isomerases (ECH/I) for the preparation of a medicament for decreasing the production of rhamnolipids by a rhamnolipid-producing microorganism.
- EH/I R-specific enoyl-CoA hydratases/isomerases
- the present invention provides the use of an inhibitor of R-specific enoyl-CoA hydratases/isomerases (ECH/I) for the preparation of a medicament for decreasing the production of PHA by a microorganism.
- rhamnolipids refers to glycolipidic biosurfactants (or surface- active molecules) produced by various bacterial species, and which generally comprises rhamnose moieties (glycon part) and lipid moieties (aglycon part) linked to each other via an O-glycosidic linkage.
- the glycon part is typically composed of one (mono-RLs) or two (di-RLs) rhamnose moieties linked to each other through an alpha-1 ,2-glycosidic linkage.
- the aglycon part is mainly one or two (in few cases three) ⁇ -hydroxy fatty acid chains (saturated, mono-, or polyunsaturated and of different chain length, typically varying from Cs to C16) linked to each other through an ester bond formed between the ⁇ -hydroxyl group of the distal (relative to the glycosidic bond) chain with the carboxyl group of the proximal chain.
- rhamnolipid-producing microorganism refers to any microorganism, such as bacteria, which has the capacity to synthesize/produce rhamnolipids under suitable conditions.
- Various bacterial species have been shown to produce RLs, as depicted in FIGs. 19A-B.
- the rhamnolipid-producing microorganism is of a phylum, class, order, family or genus depicted in FIGs. 19A-B.
- the rhamnolipid-producing microorganism is a bacterium of the Gammaproteobacteria class.
- the rhamnolipid-producing microorganism is a bacterium of the Pseudomonadales order. In a further embodiment, the rhamnolipid- producing microorganism is a bacterium of the Pseudomonadacae family. In a further embodiment, the rhamnolipid-producing microorganism is a bacterium of the Pseudomonas genus, such as P. alcaligenes, P. aeruginosa, P. chlororaphis, P. clemancea, P. collierea, P. fluoresceins, P. luteola, P. putida, P. stutzeri and P. teessidea. In a further embodiment, the rhamnolipid-producing microorganism is P. aeruginosa.
- polyhydroxyalkanoate refers to biopolyesters composed of hydroxy fatty acids (polyoxoesters). PHAs can consist of short-chain-length hydroxyalkanoic acids (PHASCL) or medium-chain-length monomers (PHAMCL), depending on strain and culture conditions. More than 150 PHA monomers have been identified.
- PHASCL short-chain-length hydroxyalkanoic acids
- PHAMCL medium-chain-length monomers
- PHA-producing microorganism refers to any microorganism, such as bacteria, which has the capacity to synthesize/produce PHAs under suitable conditions.
- PHAs are synthesized by a wide range of different Gram-positive (e.g., of the Bacillus, Clostridium, Corynebacterium, Nocardia, Rhodococcus, Streptomyces and Staphylococcus genusj and Gram- negative ⁇ Alcaligenes, Ralstonia and Pseudomonas genusj bacteria, as well as by some Archaea (notably belonging to the genera Haloferax, Haloarcula, Natrialba, Haloterrigena, Halococcus, Haloquadratum, Halorubrum, Natronobacterium, Natronococcus and Halobacterium) (see Poli et al., Archaea 201 1 : 201 1 : 693253; Singh et
- inhibiting refers for example to a decrease of at least 10%, in embodiments a decrease of at least 20, 30, 40, 50, 60, 70, 80, 90 or 100% (complete inhibition) relative to a native condition.
- decreasing the production of RLs refers to a lower production of RLs by the microorganism relative to a corresponding "native" microorganism, i.e. in which an R-specific enoyl-CoA hydratase/isomerase has not been inhibited.
- R-specific enoyl-CoA hydratases/isomerases (also referred to as crotonases) as used herein refers to any enzyme capable of converting a frans-enoyl-CoA precursor to an R-hydroxyfatty acid (e.g., capable of converting a frans-2-enoyl-CoA to an R-3-hydroxyfatty acid).
- R- specific means that the ECH/I generates a RL/PHA precursor in the R configuration [i.e., the ⁇ -hydroxyl groups of the fatty acid chains are present in the R-configuration).
- R-specific ECH/I are also referred to as ECH type 2 or ECH-2 (whereas S-specific ECH/I are referred to as ECH type 1 or ECH-1 ).
- the R-specific ECH/I comprises the amino acid sequence depicted in FIGs. 15A (SEQ ID NO:2), 16A (SEQ ID NO:4) and 17A (SEQ ID NO:6) (herein referred to as RhIK, RhIY and RhIZ, respectively), or a sequence having at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98 or 99% identity with the amino acid sequence depicted in FIGs.
- Identity refers to sequence similarity between two polypeptide or nucleic acid molecules (or fragments thereof). Identity can be determined by comparing each position in the aligned sequences. A degree of identity between amino acid or nucleic acid sequences is a function of the number of identical or matching amino acids or nucleotides at positions shared by the sequences. As used herein, a given percentage of identity between sequences denotes the degree of sequence identity in optimally aligned sequences.
- Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981 , Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wl, U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol.
- HSPs high scoring sequence pairs
- T some positive-valued threshold score
- Initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs.
- the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
- Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
- the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
- the BLAST program may use as defaults a word length (W) of 1 1 , the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci.
- R-specific ECH/I is a homolog of RhIK, RhIY and/or RhIZ from another Pseudonomas aeruginosa strain.
- the R-specific ECH/I is an ortholog of RhIK, RhIY and/or RhIZ.
- An ortholog is a protein with a sequence that has at least a portion with similarity/identity to a portion of the sequence of another protein, but found in a different species (e.g., in another bacterial species or in another Pseudonomas species).
- An ortholog and the other protein originated by vertical descent from a single protein of a common ancestor.
- the ortholog encodes a protein that has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85, 90, 95, 96, 97, 98 or 99% identity of the total length of the sequence of the protein.
- the above-mentioned method comprises inhibiting the expression and/or activity of both RhIY and RhIZ, or orthologs/homologs thereof.
- RhIK proteins having at least 50% amino acid sequence identity with SEQ ID NO: 2 include RhIK from, for example, one or more strains of Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas plecoglossicida, Pseudomonas monteilii, Pseudomonas taiwanensis, Pseudomonas entomophila, Pseudomonas stutzeri, Pseudomonas chloritidismutans, Pseudomonas fluorescens, Pseudomonas brassicacearum, Pseudomonas moraviensis, Pseudomonas corrugate, and Pseudomonas chlororaphis (see UNIPROT Cluster ID No. UniRef50_Q88fQ7 - Enoyl-CoA hydrat
- RhIY proteins having at least 50% amino acid sequence identity with SEQ ID NO: 4 include RhIY from, for example, one or more strains of Pseudomonas aeruginosa, Azotobacter vinelandii, Burkholderia caribensis, Burkholderia phenoliruptrix, Burkholderia ambifaria, Burkholderia dolosa , Burkholderia pseudomallei, Burkholderia multivorans, Burkholderia cenocepacia, Vibrio caribbenthicus, Vibrio alginolyticus, Vibrio parahaemolyticus, Herbaspirillum lusitanum, Herbaspirillum frisingense, Pseudomonas stutzeri, Pseudomonas putrefaciens, Pseudomonas cepacia, Shewanella waksmanii, Shewanella oneidensis, Shewanella baltica, Bur
- RhiZ proteins having at least 50% amino acid sequence identity with SEQ ID NO: 6 include RhiZ from, for example, one or more strains of Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas taiwanensis, Pseudomonas vranovensis, Pseudomonas mosselii, Pseudomonas denitnficans, Pseudomonas syringae, Pseudomonas chlororaphis, Pseudomonas stutzeri, Marinobacterium litorale, Azoarcus toluclasticus, Afipia felis, Halotalea alkalilenta, and Sphaerotilus natans (see UNIPROT Cluster ID No. UniRef50_V9T1 M4 - Eno
- the R-specific ECH/I inhibitor includes any compound able to negatively affect the activity of one or more R-specific ECH/I by reducing for example its expression [i.e., at the transcriptional and/or translational level), the level of one or more ECH/I mRNA and/or protein, or an activity associated with one or more ECH/I. It includes intracellular as well as extracellular inhibitors. Without being so limited, such inhibitors include RNA interference agents (siRNA, shRNA, miRNA), antisense molecules, ribozymes, proteins (e.g., dominant negative, inactive variants), peptides, small molecules, antibodies, antibody fragments, etc.
- RNA interference agents siRNA, shRNA, miRNA
- antisense molecules e.g., antisense molecules
- ribozymes e.g., dominant negative, inactive variants
- proteins e.g., dominant negative, inactive variants
- peptides small molecules, antibodies, antibody fragments, etc.
- the present inventors provide evidence that RhiZ and RhIY interact, and that this interaction is involved in RhiZ activity. Accordingly, in an embodiment, the ECH/I inhibitor blocks or interfere with the interaction between RhiZ and RhIY, or homologs/orthologs thereof. [0076] In an embodiment, the R-specific ECH/I inhibitor is a neutralizing antibody directed against (or specifically binding to) one or more R-specific ECH/I polypeptides.
- antibody or “immunoglobulin” is used in the broadest sense, and covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, humanized antibodies, CDR-grafted antibodies, chimeric antibodies, multispecific antibodies, and antibody fragments so long as they exhibit the desired biological activity (e.g., neutralizing an activity of the R-specific ECH/I polypeptide).
- Antibody fragments comprise a portion of a full length antibody, generally an antigen binding or variable region thereof.
- antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments.
- Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, VH regions (VH, VH-VH), anticalins, PepBodies, antibody-T-cell epitope fusions (Troybodies) or Peptibodies.
- the antibody is a monoclonal antibody.
- the antibody is a humanized or CDR-grafted antibody.
- Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (s.c), intravenous (i.v.) or intraperitoneal (i.p.) injections of the relevant antigen (e.g., an R-specific ECH/I polypeptide, or a fragment thereof) with or without an adjuvant.
- the relevant antigen e.g., an R-specific ECH/I polypeptide, or a fragment thereof
- KLH keyhole limpet hemocyanin
- serum albumin serum albumin
- bovine thyroglobulin bovine thyroglobulin
- soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues
- Animals may be immunized against the antigen (an R-specific ECH/I polypeptide or a fragment thereof), immunogenic conjugates, or derivatives by combining the antigen or conjugate (e.g., 100 ⁇ g for rabbits or 5 ⁇ g for mice) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
- the antigen or conjugate e.g., 100 ⁇ g for rabbits or 5 ⁇ g for mice
- 3 volumes of Freund's complete adjuvant e.g., 100 ⁇ g for rabbits or 5 ⁇ g for mice
- the antigen or conjugate e.g., 100 ⁇ g for rabbits or 5 ⁇ g for mice
- the antigen or conjugate e.g., with 1/5 to 1/10 of the original amount used to immunize
- the serum is assayed for antibody titer. Animals are boosted until the titer plateaus.
- the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent.
- Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.
- Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or may be made by recombinant DNA methods (e.g., U.S. Patent No. 6,204,023). Monoclonal antibodies may also be made using the techniques described in U.S. Patent Nos. 6,025,155 and 6,077,677 as well as U.S. Patent Application Publication Nos. 2002/0160970 and 2003/0083293.
- a mouse or other appropriate host animal such as a rat, hamster or monkey
- lymphocytes may be immunized in vitro.
- Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.
- the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
- the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
- HGPRT hypoxanthine guanine phosphoribosyl transferase
- a human chimeric antibody can be produced in the following manner. cDNA encoding heavy chain variable region (VH) and light chain variable region (VL) obtained from a hybridoma derived from non-human animal cells producing monoclonal antibodies, the cDNA is inserted to each of expression vectors for animal cells having DNA encoding a heavy chain constant region (CH) and light chain constant region (CL) of a human antibody so as to construct a human chimeric antibody expression vector, and this vector is introduced to animal cells to express the human chimeric antibody.
- VH heavy chain variable region
- VL light chain variable region obtained from a hybridoma derived from non-human animal cells producing monoclonal antibodies
- a humanized antibody refers to an antibody which is obtained by grafting the amino acid sequence of the complementary determining region (CDR) of VH and VL of a non-human animal antibody to CDR corresponding to VH and VL of a human antibody.
- the region other than CDR of VH and VL is called a framework region (hereinbelow, described as "FR").
- FR framework region
- cDNA encoding an amino acid sequence of VH which consists of an amino acid sequence of CDR of VH of a non-human antibody and an amino acid sequence of FR of VH of any human antibody and cDNA encoding an amino acid sequence of VL which consists of an amino acid sequence of CDR of VL of a non-human animal antibody and an amino acid sequence of FR of VL of any human antibody are constructed, these cDNAs are inserted respectively into expression vectors for animal cells having DNA encoding CH and CL of a human antibody so as to construct a humanized antibody expression vector, and this vector is inserted into animal cells to express the humanized antibody.
- the R-specific ECH/I inhibitor is an inactive R-specific ECH/I polypeptide (e.g., dominant negative), or a nucleic acid encoding an inactive R-specific ECH/I polypeptide. Such an inactive polypeptide may compete with endogenous R-specific ECH/I and inhibits its activity.
- the R-specific ECH/I inhibitor is an RNA interference agent targeting an mRNA encoding one or more R-specific ECH/I.
- RNA interference agent refers to molecules that specifically binds to a target mRNA and induces its degradation (usually through the RNA-induced silencing complex (RISC) or interferes with its translation, and includes for example microRNA (miRNA) molecules, antisense molecules, small interfering RNA (siRNA) molecules and small/short hairpin RNA (shRNA).
- Chemically modified nucleosides such as 2'-substituted arabinonucleosides (e.g., 2'F-ANA) and 2'-substituted RNA (e.g., 2'F-RNA), may be used for incorporation into RNA interference agents to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for a target RNA.
- 2'-substituted arabinonucleosides e.g., 2'F-ANA
- 2'-substituted RNA e.g., 2'F-RNA
- the RNA interference agent may be expressed from recombinant viral vectors, such as vectors derived from adenoviruses, adeno-associated viruses, lentiviruses, retroviruses, herpesviruses, and the like.
- viral vectors such as vectors derived from adenoviruses, adeno-associated viruses, lentiviruses, retroviruses, herpesviruses, and the like.
- Such vectors typically comprise a sequence encoding an RNA interference agent of interest and a suitable promoter operatively linked to the RNA interference agent for expressing the RNA interference agent.
- the vector may also comprise other sequences, such as regulatory sequences, to allow, for example, expression in a specific cell/tissue/organ, or in a particular intracellular environment/compartment. Methods for generating, selecting and using viral vectors are well known in the art.
- RNA interference agent directed against R-specific ECH/I mRNAs, which in turn may be used to decrease the expression of R-specific ECH/I polypeptides.
- Reagents and kits for performing RNA interference are available commercially from for example Ambion® Inc. (Austin, TX, USA), New England Biolabs® Inc. (Beverly, MA, USA), Sigma- Aldrich® and Invitrogen® (Carlsbad, CA, USA).
- RhIZ is at least partially inhibited by a 2- bromooctanoate-derived metabolite.
- the R-specific ECH/I inhibitor is an analog of 2-bromooctanoate, for example an enoate (e.g., a Ce to Ci6 enoate) substituted with one or more halogens.
- RLs can act as immunumodulators and virulence factors (reviewed in Abdel-Mawgoud et al., 2010), the inhibition of R-specific ECH/I, and consequently of RLs production, may be useful for the treatment of infection with RL-producing microorganisms.
- RLs produced by P. aeruginosa are believed to be implicated in biofilm initiation (probably via modulation of cell surface polarity and hence initial cell attachment), in biofilm structure maturation, in biofilm dispersion and as a biofilm shield for immune-protection.
- Biofilm surface-associated RL layer is suggested to be formed in response to contact with immune cells.
- RL are also reported to slow tracheal ciliary function, and to disrupt the tight junctions of lung epithelial cells as a prelude to invasion. Furthermore, RLs have been shown to induce rapid necrosis of polymorphonuclear (PMN) leukocytes, to be cytolytic to macrophages or at least to inhibit their normal phagocytic response and to act as heat- stable hemolysins.
- PMN polymorphonuclear
- the present invention provides a method for attenuating the virulence of a rhamnolipid-producing microorganism, said method comprising inhibiting (completely or partially) the expression and/or activity of one or more enoyl-CoA hydratases/isomerases (ECH/I) in said rhamnolipid-producing microorganism.
- the present invention also provides the use of an inhibitor of enoyl-CoA hydratases/isomerases (ECH/I) for attenuating the virulence of a rhamnolipid-producing microorganism.
- the amount of the R-specific ECH/I inhibitor which is effective for decreasing the production of RLs and/or PHA, and/or for attenuating the virulence of a rhamnolipid- producing microorganism will depend on several factors including the nature and severity of the disease, the chosen prophylactic/therapeutic regimen, the target site of action, the patient's weight, special diets being followed by the patient, concurrent medications being used, the administration route and other factors that will be recognized by those skilled in the art.
- the dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease, the liver function and different parameters from the patient. Typically, 0.001 to 1000 mg/kg of body weight/day will be administered to the subject.
- a daily dose range of about 0.01 mg/kg to about 500 mg/kg, in a further embodiment of about 0.1 mg/kg to about 200 mg/kg, in a further embodiment of about 1 mg/kg to about 100 mg/kg, in a further embodiment of about 10 mg/kg to about 50 mg/kg, may be used.
- the dose administered to a patient, in the context of the present invention should be sufficient to effect/induce a beneficial prophylactic and/or therapeutic response in the patient over time (decrease in bacterial load, reduction of tissue damage, reduction of one or more symptoms associated with the infection, increased survival time, etc.).
- the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration.
- Effective doses may be extrapolated from dose response curves derived from in vitro or animal model test systems. For example, in order to obtain an effective mg/kg dose for humans based on data generated from rat studies, the effective mg/kg dosage in rat may be divided by six.
- the above-mentioned treatment comprises the use/administration of more than one (i.e. a combination of) active/therapeutic agent, including the above-mentioned R-specific ECH/I inhibitor.
- the combination of prophylactic/therapeutic agents and/or compositions of the present invention may be administered or co-administered (e.g., consecutively, simultaneously, at different times) in any conventional dosage form.
- Co-administration in the context of the present invention refers to the administration of more than one therapeutic in the course of a coordinated treatment to achieve an improved clinical outcome.
- Such co-administration may also be coextensive, that is, occurring during overlapping periods of time.
- a first agent may be administered to a patient before, concomitantly, before and after, or after a second active agent is administered.
- the agents may in an embodiment be combined/formulated in a single composition and thus administered at the same time.
- the one or more active agent(s) is used/administered in combination with one or more agent(s) or treatment currently used to prevent or treat the disorder in question (e.g., agents or treatments currently used in the treatment of infections by rhamnolipid-producing microorganisms, such as P. aeruginosa.
- the above-mentioned R-specific ECH/I inhibitor may be provided in a composition (e.g., a pharmaceutical composition) comprising a carrier, diluent and/or excipient (e.g., a pharmaceutically acceptable carrier, diluent and/or excipient).
- a pharmaceutically acceptable carrier e.g., a pharmaceutically acceptable carrier, diluent and/or excipient.
- pharmaceutically acceptable carrier or “excipient” includes any and all solvents, buffers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
- the carrier can be suitable, for example, for oral, intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal or pulmonary (e.g., aerosol) administration.
- Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
- the use of such media and agents for pharmaceutically active substances is well known in the art (Rowe et al., Handbook of pharmaceutical excipients, 2003, 4 th edition, Pharmaceutical Press, London UK). Except insofar as any conventional media or agent is incompatible with the active agent, use thereof in the pharmaceutical compositions of the invention is contemplated.
- Biosurfactants are in demand for a wide range of industrial applications as they increase solubility, foaming capacity and lower surface tensions. Biosurfactants are attracting much attention because they represent ecological alternatives to their synthetic counterparts: they exhibit lower toxicity, potentially high activities, and stability at extremes of temperature, pH, and salinity. They have a wide variety of structures, and they can be produced from renewable feedstocks by a wide variety of microorganisms. Most importantly, they are biodegradable, making them environmentally friendly, "green” chemicals. In particular, RLs have been used in chemical and pharmaceutical industry or as emulsifier in cosmetics and foods.
- PHAs are linear polyesters that are biodegradeable and are used in the production of bioplastics.
- the present invention provides a method for increasing the production of rhamnolipids by a host cell, said method comprising increasing the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I) in said host cell.
- EH/I R-specific enoyl-CoA hydratases/isomerases
- the present invention provides a method for increasing the production of PHA by a host cell, said method comprising increasing the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I) in said host cell.
- EH/I R-specific enoyl-CoA hydratases/isomerases
- Increasing the expression and/or activity of one or more R-specific ECH/I includes inducing the expression of one or more R-specific ECH/I in host cell that do not express one or more endogenous R-specific ECH/I (or express R-specific ECH/I inappropriate for rhamnolipid production), or increasing the expression of one or more R-specific ECH/I in host cell that endogenously expressed one or more R-specific ECH/I.
- the expression may be induced/increased by either introducing a heterologous R-specific ECH/I nucleic acid into a host cell, or increasing/inducing the expression and/or activity of one or more endogenous R-specific ECH/I.
- a host cell includes any suitable host cell that is capable of producing RLs and/or PHA (either endogenously or heterologously, i.e. following introduction of one or more genes into the host cell).
- the present invention envisages as a host cell, preferably a non-pathogenic host cell (e.g., non-pathogenic for humans) including a unicellular host cell such as a fungal host cell, for example, a yeast.
- the host cell may be a bacterial host cell including non-pathogenic bacterial host cells such as bacterial host cells capable of producing rhamnolipids.
- a host cell according to the invention includes an R-specific ECH/I gene (e.g., RhIK, RhIY and/or RhIZ or an ortholog/homolog thereof).
- the R-specific ECH/I gene may be under the control of a heterologous promoter.
- the R-specific ECH/I gene is an endogenous gene of the bacterial host cell.
- the R-specific ECH/I gene is a heterologous gene.
- heterologous refers to the relationship between two or more nucleic acid or protein sequences that are derived from different sources.
- a promoter is heterologous with respect to a transcribable polynucleotide sequence if such a combination is not normally found in nature.
- a particular sequence may be "heterologous" with respect to a host cell in that it encodes a protein or is included in a protein, for example a recombinant protein, that is not normally expressed by the host cell.
- Such a heterologous protein accordingly generally is or has been inserted into the respective host cell, tissue, or species. Accordingly, a heterologous promoter is not normally coupled in vivo transcriptionally to the coding sequence of the R-specific ECH/I gene.
- the above-mentioned method comprises increasing the expression and/or activity of at least two R-specific ECH/I, preferably RhIY and RhIZ (or orthologs/homologs) thereof, in the cell.
- R-specific ECH/I preferably RhIY and RhIZ (or orthologs/homologs) thereof
- RhIY and RhIZ or orthologs/homologs thereof
- a wide variety of transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host, as long as RLs and/or PHAs are produced.
- the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, cytomegalovirus or simian virus, where the regulatory signals are associated with a particular gene sequence which has a high level of expression.
- promoters from mammalian expression products such as actin, collagen and myosin may be employed.
- Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the gene sequences can be modulated.
- Of interest may in some embodiments also be regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabolite) regulation.
- regulatory sequence includes controllable transcriptional promoters, operators, enhancers, silencers, transcriptional terminators, 5' and 3' untranslated regions which interact with host cellular proteins to carry out transcription and translation and other elements that may control gene expression including initiation and termination codons.
- the regulatory sequences can be native (homologous), or can be foreign (heterologous) to the cell and/or the nucleotide sequence that is used.
- the precise nature of the regulatory sequences needed for gene sequence expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation.
- Such regions will normally include those 5'-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence or CAAT sequence.
- These regulatory sequences are generally individually selected for a certain embodiment, for example for a certain cell to be used. The skilled artisan will be aware that proper expression in a prokaryotic cell also requires the presence of a ribosome-binding site upstream of the gene sequence-encoding sequence.
- a vector may be employed which is capable of integrating the desired R-specific ECH/I gene sequences into the host cell chromosome.
- Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
- the marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like.
- the selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transformation or co-transfection. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.
- the introduced nucleic acid molecule can be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species. [00108] An expression vector, which may include one or more regulatory sequences and be capable of directing the expression of nucleic acids to which it is operably linked.
- An operable linkage is a linkage in which a coding nucleotide sequence of interest is linked to one or more regulatory sequence(s) such that expression of the nucleotide sequence sought to be expressed can be allowed.
- a regulatory sequence operably linked to a coding sequence is capable of effecting the expression of the coding sequence, for instance in an in vitro transcription/ translation system or in a cell when the vector is introduced into the cell.
- a respective regulatory sequence need not be contiguous with the coding sequence, as long as it functions to direct the expression thereof.
- intervening untranslated yet transcribed sequences may be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.
- the DNA construct(s) may be introduced into the host cell by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate-precipitation, direct microinjection, and the like.
- suitable means i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate-precipitation, direct microinjection, and the like.
- recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells.
- the host cell according to the invention is able to produce PHA only to a much lower extent than a corresponding native, wild type host cell.
- the host cell may for example include a mutation in the gene of an enzyme that catalyzes the formation of PHA from the precursors, or a mutation in a regulatory sequence to which the respective gene is operably linked.
- the respective mutation may in some embodiments give rise to the formation of a polyhydroxyalkanoic acid synthase that has a reduced activity when compared to a corresponding wild type polyhydroxyalkanoic acid synthase.
- a capability of the host cell according to the invention to produce PHA is reduced in comparison to a wild type cell, including entirely absent.
- the host cell according to the invention is incapable of synthesizing PHA.
- the PHA biosynthetic pathway of the host cell may for example be curtailed, including inactive.
- the host cell may for example have one or more genes in the PHA synthesis pathway, such as polyhydroxyalkanoic acid synthase gene(s), that is/are disrupted.
- the cell may for instance have a knock-out mutation in an endogenous polyhydroxyalkanoic acid synthase gene.
- the host cell according to the invention is able to produce RLs to a lower extent than a corresponding native, wild-type host cell.
- the host cell may for example include a mutation in the gene of an enzyme that catalyzes the formation of RLs from the precursors (e.g., rhamnosyl-transferases such as RhIB and/or RhIC), or a mutation in a regulatory sequence to which the respective gene is operably linked.
- the respective mutation may in some embodiments give rise to the formation of rhamnosyl-transferases that have a reduced activity when compared to corresponding wild type rhamnosyl-transferases.
- a capability of the host cell according to the invention to produce RLs is reduced in comparison to a wild type cell, including entirely absent.
- the host cell according to the invention is incapable of synthesizing RLs.
- the RL biosynthetic pathway of the bacterial host cell may for example be curtailed, including inactive.
- the host cell may for example have one or more genes in the RL synthesis pathway, such as rhamnosyl- transferase gene(s), that is/are disrupted.
- the cell may for instance have a knock-out mutation in an endogenous rhamnosyl-transferase gene.
- the method further comprises increasing the expression of one or more genes involved in RL and/or PHA biosynthesis.
- the host cell may be modified or treated to produce higher levels of enzymes, e.g., RhIA, RhIB, and/or RhIC (e.g., SEQ ID NOs: 27, 29 and/or 31), or orthologs/homologs thereof (comprising a sequence having at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98 or 99% identity with the amino acid sequence depicted in SEQ ID NOs: 27, 29 and/or 31 , and exhibiting the same enzymatic activity).
- RhIA RhIB
- RhIC e.g., SEQ ID NOs: 27, 29 and/or 31
- orthologs/homologs thereof comprising a sequence having at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98 or 99% identity with the amino acid sequence depicted in SEQ ID NOs: 27, 29 and
- the host cell may be modified or treated to increase higher levels of PHA synthases.
- the method further comprises increasing the expression and/or activity of one or more additional proteins/enzymes involves in rhamnolipid production.
- the method further comprises increasing the level of one or more substrates/precursors involved in rhamnolipid production.
- a host cell such as a bacterial host cell, as defined above may be used in a method of producing one or more RLs or PHAs.
- the host cell is cultured under conditions that allow RL and/or PHA production.
- a variety of carbon source may be used such as a monosaccharide, e.g. glucose, a disaccharide, e.g. sucrose, an alcohol, e.g. glycerol, an alkane, e.g., n-hexadecane, a fatty acid such as caprylic acid (also termed octanoate), vegetable oils (fresh or waste) or mixtures thereof.
- the bacterial host cell will typically be exposed to a fermentation process.
- the bacterial host cell may for instance be in the logarithmic growth phase or in the stationary phase.
- the RL is recovered.
- the RL is secreted by the bacterial host cell, so that recovering the fermentation/culture medium includes recovering the RLs.
- the method may include enriching, isolating and/or purifying the RLs.
- the term "enriched" means that the RLs constitute a significantly higher fraction of the total glycolipids, lipids and saccharides present in the solution of interest than in the solution from which it was taken.
- Enrichment may for instance include membrane filtration, for example for clarification, buffer exchange or concentration purposes. It may also include filtration or dialysis, which may for instance be directed at the removal of molecules below a certain molecular weight, or a precipitation by lowering the pH, or extraction using organic solvents.
- Chromatography may for example be carried out in the form of a liquid chromatography such as capillary electrochromatography, HPLC (high performance liquid chromatography) or UPLC (ultrahigh pressure liquid chromatography) or as a gas chromatography.
- the chromatography technique may be a process of column chromatography, of batch chromatography, of centrifugal chromatography or a method of expanded bed chromatography, as well as electrochromatographic, electrokinetic chromatography.
- adsorption chromatography hydrophobic interaction chromatography or hydrophobic charge induction chromatography
- size exclusion chromatography also termed gel-filtration
- ion exchange chromatography affinity chromatography
- affinity chromatography affinity chromatography
- the criteria that govern the selection of a specific recovery method include: (1 ) the cost associated with the extraction method, which adds to the price of the final product, (2) the proposed purpose of the final product, which influences the level of purity required, and (3) the adaptability of the method to a particular industrial fermentation process.
- One of the simplest methods of recovery is by acid precipitation (Deziel et al. 1999b; Van Dyke et al. 1993; Zhang and Miller 1992) or aluminum sulfate precipitation (Schenk et al. 1995). Acid precipitation depends on acidification of RL to low pH (e.g., around 2), which neutralizes the negative charges on RLs, making them less soluble in the aqueous phase. Aluminum sulfate precipitates RLs by salting out.
- the precipitated RLs can then be recovered by centrifugation.
- Another more commonly used method is recovery by solvent extraction (Lepine et al. 2002; Mata-Sandoval et al. 1999; Schenk et al. 1995).
- molecules are precipitated by acidification and then extracted with organic solvents such as ether or ethyl acetate. Acidification is not a critical step in this method, but it enhances the net yield (Heyd et al. 2008).
- Adsorption methods are based on the use of hydrophobic adsorbent such as Amberlite® XADO-2 or -16, polystyrene resin that retain hydrophobic (or amphiphilic) substances through hydrophobic interactions.
- Adsorbed RLs are then released by elution, e.g., with methanol.
- Ion exchange chromatography exploits the fact that RLs behave as anions at high pHs, which allows their retention on columns of weak anion exchange resins such as (diethylamino)ethyl-Sepharose®.
- RLs are released from these resins by adding at least 0.6 M NaCI to the equilibration buffer. Yet, this method has been improved by Abadi et al. (2009), who applied phospholipid-coated colloidal magnetic nanoparticles ion exchange media for the recovery and purification of RLs from culture mixtures.
- the method comprises solvent extraction.
- An isolation may include the combination of different methods.
- the present inventors have identified R-specific enoyl-CoA hydratases/isomerases (ECH/I) as being involved in the biosynthesis of RLs and/or PHAs.
- ECH/I R-specific enoyl-CoA hydratases/isomerases
- the present invention also provides a method (screening assay), in an embodiment an in vitro method, for determining whether an agent may be useful for (i) inhibiting the production of RLs and/or PHAs by a host cell, such as a rhamnolipid- and/or PHA- producing microorganism and/or (ii) attenuating the virulence of a rhamnolipid-producing microorganism, said method comprising determining whether said agent inhibits the expression and/or activity of one or more R-specific ECH/I.
- the method comprises (a) contacting said agent with said one or more R-specific ECH/I and (b) determining whether said agent inhibits the expression and/or activity of said one or more R-specific ECH/I, wherein the inhibition of said expression and/or activity is indicative that said agent may be useful for (i) inhibiting the production of RLs and/or PHAs by the host cell and/or (ii) attenuating the virulence of a rhamnolipid-producing microorganism.
- the present invention also provides a method (screening assay), in an embodiment an in vitro method, for determining whether an agent may be useful for increasing the production of RLs and/or PHAs by a host cell, such as a rhamnolipid- and/or PHA-producing microorganism, said method comprising determining whether said agent increases the expression and/or activity of one or more R- specific ECH/I.
- a host cell such as a rhamnolipid- and/or PHA-producing microorganism
- the method comprises (a) contacting said agent with said one or more R-specific ECH/I and (b) determining whether said agent increases the expression and/or activity of said one or more R-specific ECH/I, wherein the increase of said expression and/or activity is indicative that said agent may be useful for increasing the production of RLs and/or PHAs by the host cell.
- the determination of the expression and/or activity of R-specific ECH/I in the presence (and/or absence) of the test compound may be performed using any known methods to detect nucleic acids or proteins.
- the expression or activity measured in the presence of the test compound is compared to a control or reference level (e.g., the level obtained in the absence of the test compound) to assess whether the test compound modulates (increases or decreases) the expression or activity of R-specific ECH/I .
- a control or reference level e.g., the level obtained in the absence of the test compound
- nucleic acid amplification methods can be used to detect the level of expression of an R-specific ECH/I.
- the oligonucleotide primers and probes may be used in amplification and detection methods that use nucleic acid substrates isolated by any of a variety of well- known and established methodologies (e.g., Sambrook et al., Molecular Cloning, A laboratory Manual, pp.
- Methods for amplifying nucleic acids include, but are not limited to, for example the polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) (see e.g., U.S. Pat. Nos.
- PCR polymerase chain reaction
- RT-PCR reverse transcription PCR
- ligase chain reaction LCR
- SDA strand displacement amplification
- tSDA Thermophilic SDA (tSDA) (see e.g., European Pat. No. 0 684 315) and methods described in U.S. Pat. No. 5,130,238; Lizardi et al., BioTechnol.
- TMA Transcription Mediated Amplification
- the nucleic acid or amplification product may be detected or quantified by hybridizing a labeled probe to a portion of the R-specific ECH/I nucleic acid or amplified product.
- the labeled probe contains a detectable group that may be, for example, a fluorescent moiety, chemiluminescent moiety, radioisotope, biotin, avidin, enzyme, enzyme substrate, or other reactive group.
- Other well-known detection techniques include, for example, gel filtration, gel electrophoresis and visualization of the amplicons, and High Performance Liquid Chromatography (HPLC).
- HPLC High Performance Liquid Chromatography
- the level of amplified product is detected as the product accumulates.
- the expression and/or activity of an R-specific ECH/I is measured at the protein level.
- Methods to measure the amount/level of proteins are well known in the art. Protein levels may be detected directly using a ligand binding specifically to the protein, such as an antibody or a fragment thereof.
- a binding molecule or reagent e.g., antibody
- is labeled/conjugated e.g., radio-labeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled to facilitate detection and quantification of the complex (direct detection).
- protein levels may be detected indirectly, using a binding molecule or reagent, followed by the detection of the [protein/ binding molecule or reagent] complex using a second ligand (or second binding molecule) specifically recognizing the binding molecule or reagent (indirect detection).
- a second ligand may be radio- labeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled to facilitate detection and quantification of the complex.
- Enzymes used for labeling antibodies for immunoassays are known in the art, and the most widely used are horseradish peroxidase (HRP) and alkaline phosphatase (AP).
- binding molecules or reagents include antibodies (monoclonal or polyclonal), natural or synthetic R-specific ECH/I ligands, glycoproteins, monosaccharides (e.g., galactose, galactosamine, lactose) aptamers and the like.
- Examples of methods to measure the amount/level of protein in a sample include, but are not limited to: Western blot, immunoblot, enzyme-linked immunosorbent assay (ELISA), "sandwich” immunoassays, radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance (SPR), chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical (IHC) analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microarray, antibody array, microscopy (e.g., electron microscopy), flow cytometry, proteomic-based assays, and assays based on a property of the protein including but not limited to ligand binding or interaction with other protein partners, enzymatic activity, fluorescence, etc.
- ELISA enzyme-linked immunosorbent assay
- RIA radioimmunoassay
- SPR surface plasmon
- the level or activity of R-specific ECH/I may be determined by measuring the level of conversion of a substrate of an R-specific ECH/I (e.g., a suitable enoyl-CoA substrate such as trans-2- decenoyl-CoA) into a product (an R-hydroxyfatty acid such as 3-hydroxydecanoate) (see below).
- a substrate of an R-specific ECH/I e.g., a suitable enoyl-CoA substrate such as trans-2- decenoyl-CoA
- a product an R-hydroxyfatty acid such as 3-hydroxydecanoate
- the effect of the test agent on the activity/expression of R- specific ECH/I may be assessed using a reporter gene/protein system.
- a reporter gene may be put under the transcriptional control of a transcriptional control element (e.g., promoter) normally associated with an R-specific ECH/I, and the expression of the reporter protein may be measured to determine if the test agent could modulate the transcription of the R-specific ECH/I. If in the presence of the test agent the level of the reporter protein is increased, it is an indication that the test agent may be useful to increase the expression of the R-specific ECH/I (as the reporter protein is under the transcriptional control of a transcriptional control element normally associated with the the R-specific ECH/I.
- a transcriptional control element e.g., promoter
- reporter gene refers to gene encoding a protein (a reporter protein) that can be detected (e.g., by fluorescence, spectroscopy, luminometry, etc.) easily and that is not present normally (endogenously) in the system used.
- reporter proteins include enzymes such as ⁇ - galactosidase (encoded by the bacterial gene /acZ), luciferase, chloramphenicol acetyltransferase (CAT; from bacteria), GUS ( ⁇ -glucuronidase), bioluminescent proteins and fluorescent proteins. The method to determine the readout signal from the reporter protein depends from the nature of the reporter protein.
- the readout signal corresponds to the intensity of the fluorescent signal.
- the readout signal may be measured using spectroscopy-, fluorometry-, photometry- , and/or luminometry-based methods and detection systems, for example. Such methods and detection systems are well known in the art.
- the determination of the effect of the test compound on the expression and/or activity of R-specific ECH/I may be performed indirectly, for example by assessing the production of RLs and/or PHAs by a cell (e.g., using the methods described in the Examples below).
- the production of RLs and/or PHAs in the presence of the test compound is compared to a control or reference level (e.g., the production measured in the absence of the test compound) to assess whether the test compound modulates (increases or decreases) the expression or activity of R- specific ECH/I .
- a control or reference level e.g., the production measured in the absence of the test compound
- Higher levels/production of RLs and/or PHAs in the presence of the test compound being indicative that the test compound increases the expression or activity of R-specific ECH/I and vice-versa.
- the method/assay may be a cell-free method/assay or a cell-based method/assay.
- any cell capable of expressing the R-specific ECH/I nucleic acid and protein (or the reporter gene construct described above) may be used in the method/system of the invention.
- the above-mentioned cell is a bacterial cell .
- the cell may be naturally expressing the R- specific ECH/I protein, or may be prepared by introducing a nucleic acid encoding the R-specific ECH/I or a reporter gene construct (by any transfection, transduction or transformation method), and providing conditions suitable for the expression of the protein .
- Kits for introducing a nucleic acid into a cell are commercially available.
- the assay may be performed using a native, full-length R-specific ECH/I, or an active variant or fragment thereof.
- Test compounds that may be screened by the method/system of the invention may be obtained from any number of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means.
- Screening assay systems may comprise a variety of means to enable and optimize useful assay conditions.
- Such means may include but are not limited to: suitable buffer solutions, for example, for the control of pH and ionic strength and to provide any necessary components for optimal activity and stability (e.g. , protease inhibitors), temperature control means for optimal activity and/or stability of R-specific ECH/I, and detection means to enable the detection of its activity.
- detection means may be used, including but not limited to one or a combination of the following: radiolabelling, antibody-based detection, fluorescence, chemiluminescence, spectroscopic methods (e.g., generation of a product with altered spectroscopic properties), various reporter enzymes or proteins (e.g., horseradish peroxidase, green fluorescent protein), specific binding reagents (e.g., biotin/(strept)avidin), and others.
- radiolabelling e.g., antibody-based detection, fluorescence, chemiluminescence, spectroscopic methods (e.g., generation of a product with altered spectroscopic properties), various reporter enzymes or proteins (e.g., horseradish peroxidase, green fluorescent protein), specific binding reagents (e.g., biotin/(strept)avidin), and others.
- Bacterial strains and growth conditions [00136] Bacterial strains and plasmids are described in Table 1 .
- MSM mineral salts medium
- Composition of TES is: FeS0 4 H 2 0 (2 g/L), MnS0 4 'H 2 0 (1 .5 g/L), ( ⁇ 4 )6 ⁇ 7 0 24 ⁇ 4 ⁇ 2 0 (0.6 g/L).
- Glycerol (20 g/L) was used as the carbon source unless otherwise stated. Tryptone was omitted when fatty acid carbon sources were tested. Inoculated MSM medium was incubated at 34°C under rotation.
- P. aeruginosa was grown on LB agar (NaCI 5 g/L) at 37°C for mating purposes and for selection of transformants after mating or electroporation. It was also grown in LB-no salt (LBNS, with no NaCI) broth at 30°C for resolution of merodiploids in mutant constructions. Antibiotics were added to maintain or select for plasmids as follows: for £ coli, carbenicillin (Cb) at 100 g/mL, gentamicin (Gm) at 30 ⁇ g/mL, tetracycline (Tc) at 15 ⁇ g/mL, kanamycin (Km) at 50 g/ml; for P.
- Cb carbenicillin
- Gm gentamicin
- Tc tetracycline
- Km kanamycin
- aeruginosa carbenicillin (Cb) at 300 ⁇ g/mL, Gm at 50 ⁇ g/mL, and Tc at 125 ⁇ g/mL for solid media and at 75 ⁇ g/mL for liquid media.
- Triclosan at 10 ⁇ g/mL, was used to counter select E. coli.
- Conjugation spots were spread onto LB agar containing Gm (50 g/ml) and triclosan (10 g/ml) to select for transconjugants of PA14 that had undergone the first homologous allelic replacement.
- Merodiploids were resolved by growing three transconjugant colonies in LBNS broth overnight, dilutions (usually 1 CH, 10 "2 ) of which were plated onto LBNS agar containing Gm (50 g/ml) and sucrose 10% (w/v).
- positive clones, that underwent the second recombination were those growing on LB agar Gm (50 g/ml but not on Cb (300 g/mL).
- the Gm resistance cassette was subsequently excised from produced mutants APA14_54660::Gm-FRT (ArWZ::Gm-FRT), by Flp recombinase. This was performed by conjugation of ArWZ: :Gm-FRT mutant with £ coli SM10(pFLP2).
- PA14 mutant clones that had lost the Gm cassette were recovered by plating dilutions, up to 10 "4 , of suspended conjugation spots onto LB agar containing Cb (300 ⁇ g/mL) and Tr (10 g/ml) to eliminate the £ coli.
- Growing clones were replica plated onto LBNS agar containing sucrose 10% (w/v) to eliminate the pFLP2 plasmid.
- the double knockout mutant hrhlYZ ( ⁇ 14_54640- ⁇ 14_54660) was constructed using the PA14-54640-UpF-GWL and PA14-54640-UpR-Gm primers for amplification of the upstream fragment in combination with PA14-54660-DnF-Gm and PA14-54660- DnR-GWR for the amplification of the downstream fragment.
- the knockout mutant AphaG ( ⁇ 14_54830, ortholog of phaG of P.
- putida KT2440 (Rehm et al., 1998)) was constructed using phaG-UpF-GWL and phaG-UpR-Gm primers for the amplification of the upstream fragment in combination with phaG-DnF-Gm and phaG-DnR-GWR primers for the amplification of the downstream fragment.
- the multiple knockout mutant (ApftaCf, AphaD, kphaC2, kphaD, kphaF, t phaf) was constructed using the PHA-UpF-GWL and PHA-UpR-Gm primers to amplify the upstream fragment and PHA-DnF-Gm and PHA-DnR-GWL primers to amplify the downstream fragment.
- the plasmid pUCP26-rWZ was constructed by PCR amplification of PA14_54660 from wild type PA14 using RhlZ-F- EcoRI and RWZ-R-Hindlll primers including the region from -57 bp (with the native RBS) to 84 bp downstream to the stop codon of RhIZ.
- the EcoRI-Hindlll digested fragment was cloned into the EcoRI- Hindlll-digested pUCP26 vector.
- the expression vector pET28a(+)-RWZ was constructed by PCR amplification of PA14_54660 from the wild-type PA14 using RWZ-F-Ndel and RWZ-R-Hindlll primers. The double digested fragment was cloned into the Ndel-Hindlll digested pET28a(+) to yield the inducible RhIZ expression 56 vector pET28a(+)-RWZ with the His-tag at the N-terminal of RhIZ. The plasmid was transformed in thermocompetent E.coli BL21 (DE3).
- Plasmid DNA and PCR products were sequenced by the DNA Sequencing and Gene Analysis Center at the Genome Quebec, Montreal, CA.
- the plasmids and PCR primers used are listed in Tables 1 and 2, respectively.
- PA14_54660-UpF-GWL TACAAAAAAGCAGGCTctggtcgaacaggtggtg 7
- PA14_54660-UpR-Gm TCAGAGCGCTTTTGAAGCTAATTCGtcttcgaaaagcacgttca 8
- PA14_54660-DnF-Gm AGGAACTTCAAGATCCCCAATTCGctgatcgatcgcgacaac 9
- PA14_54640-UpF-GWL TACAAAAAAGCAGGCTg acatgctcaccg aactca 1 1
- PA14_54640-UpR-Gm TCAGAGCGCTTTTGAAGCTAATTCGcatgaagtccctcactggat 12 phaG-UpF-GWL TACAAAAAAGCAGGCTctttcctgccggccaat 13 phaG-UpR-Gm TCAGAGCGCTTTTGAAGCTAATTCGgatgatggtctgctgcgccc 14 phaG-DnF-Gm AGGAACTTCAAGATCCCCAATTCGcggcgcggaatttcacac 15 phaG-DnR-GWR T AC AAG AAAG CTGGGTccattatacg acg cccg etc 16
- nucleotides are the gene specific sequences for generation of knockout mutants. Underlined nucleotides refer to corresponding restriction sites preceded by a number of base pairs for efficient cleavage as recommended by supplier (New England BioLabs®, Inc).
- Each cell lysates were clarified by centrifugation at 10,000 x g at 4°C for 30-45 min and then applied onto a preconditioned Ni Sepharose® High Performance column (HisTrapHP®-5ml, GE) using Fast Protein Liquid Chromatography® (AKTA® FPLC, GE). Preconditioning of the column was achieved according to instructions of the manufacturer and flow rate was adjusted at 5 ml/min. The column was washed with five column volumes of binding buffer then a linear gradient was initiated. For RhIZ purification, the gradient was from 0% to 50% elution buffer (20 mM NaP04, pH 7.4, 500 mM NaCI, 500 mM imidazole, 20 mM ⁇ -mercaptoethanol) over twenty column volumes.
- RhIY the gradient was from 0% to 100% elution buffer over thirty column volumes. Fractions of 5 ml were collected throughout the chromatography. RhIZ started to elute out of the column at 130 mM, peaked at 160 mM and tailed at 190 mM imidazole (FIG. 14A), RhIY started to elute at 170 mM, peaked at 240 mM and tailed at 335 mM imidazole (FIG. 14B). Fractions of these peaks and flanking fractions were applied to 12% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
- Appearing bands were excised for identification via protein sequencing at the Proteomic services of INRS-lnstitut Armand Frappier (Canada). Fractions containing pure proteins were pooled together and concentrated with Amicon® Ultra-15 (30 kDa, Millipore®) for RhIZ and Amicon® Ultra-15 (15 kDa) for RhIY, and washed with 900 volumes of buffer (50 mM Tris-CI, pH 7.7, 1 mM EDTA, 10 mM ⁇ -mercaptoethanol, 0.5 mM PMSF). The Amicon®-concentrated protein solutions were assayed using Bradford method (protein concentration was 8 mg/ml), then aliquoted and stored at -80°C.
- Intact protein masses were determined by mass spectrometry (Quattro Premier® XE, Micromass®) equipped with an electrospray ionization interface at the positive mode using direct infusion of purified proteins at 800 mg/L that were diluted and washed in 50 mM ammonium carbonate solution. The obtained spectra were integrated using the MaxEnt 1 option of MassLynxTM software. Intact masses of purified proteins were 31 ,933 and 42,648 Da positively identifying the proteins as His- tagged RhIY and RhIZ, respectively, both lacking their N-terminal fMet amino acid.
- Clarified cell lysate of WT PA14 strain and of the mutants ⁇ / ⁇ and RhlYZ were prepared similarly from cells collected from a 25-ml fresh cultures in TSB broth that were then washed and sonicated in lysis buffer (50 mM Tris-CI, pH 7.7, 1 mM EDTA, 10 mM ⁇ -mercaptoethanol, 0.5 mM PMSF) (Davis et al., 2008) to be used freshly or kept at 4°C until use.
- lysis buffer 50 mM Tris-CI, pH 7.7, 1 mM EDTA, 10 mM ⁇ -mercaptoethanol, 0.5 mM PMSF
- Trans-2-decenoyl-Co was synthesized using the mixed anhydride method (Abdel- Mawgoud et al., 2013). An amount of 2-4 ⁇ g of pure enzyme and/or 20 ⁇ of clarified cell lysates was used for the activity assays. Enzymatic reaction set up and enzyme product assay using chiral HPLC- MS/MS were conducted as described by Abdel-Mawgoud et al. (2013).
- RL were analyzed using a high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) as described previously (Deziel et al., 1999).
- 3-hydroxy fatty acids were analyzed using gas chromatography-mass spectrometry (GC/MS).
- GC/MS gas chromatography-mass spectrometry
- PHA was analyzed by the GC/MS estimation of 3-hydroxy fatty acids pool as well as by estimation of the trans-sectional area of PHA granules as visualized by transmission electron microscope.
- HPLC-MS analysis and identification of rhamnolipids HPLC/MS analysis of RL was performed as reported previously (Deziel et al., 1999) with some modifications. RL containing culture broths were centrifuged at 10,000 g for 10 min to prepare cell free supernatants. An aliquot of 250 ⁇ of such supernatant was kept at -20°C until the day of analysis. In HPLC vials, 25 ⁇ of such supernatant was diluted to 500 ⁇ of acetonitrile:water (70:30) to which 2-heptyl-3-hydroxyquinoline-d4 (HHQ-d4) was added at 5.6 mg/L as internal standard (Dubeau et al., 2009).
- HHQ-d4 2-heptyl-3-hydroxyquinoline-d4
- HPLC separation of RLs was performed with a sample injection volume of 20 ⁇ _ and mobile phase at a flow rate of 0.4 ml/min with a gradient of water (A):acetonitrile (B), both containing 2 mM ammonium acetate and adjusted to pH 8.
- the gradient was set according to the following profile: 0 min, 30% A + 70% B; 1 min, 25% A + 75% B; 10 min, 100% B; 16 min, 100% B; 17 min, 30% A + 70% B; 20 min, 30% A + 70% B.
- the mobile phase flow was split through a Valco® Tee with 10% of the flow introduced into the mass spectrometer.
- the mass spectrometer was in the negative ESI mode with capillary voltage set at 3.0 kV, cone voltage at 20 V, source and desolvation temperatures of 120 and 150°C respectively, cone and desolvation gas (N2) flow of 15 and 100 L/Hr, respectively.
- the scanning mass range was from 130 to 800 Da. Quantification was performed by comparing integration data of the pseudomolecular ion [M-H] ⁇ peak areas of different RLs homologues with those of the internal standard using response factors previously determined through calibration curves made with pure mono-RL and pure di-RL.
- CID Collision-induced dissociation
- GC/MS analysis of 3-hydroxy fatty acids and PHA For the analysis of total 3-hydroxy fatty acids, 3 ml of whole culture broths grown in MSM-glycerol were collected in screw-capped glass tubes to which nonanoic acid (Sigma®, USA) was added as the internal standard at 15 mg/L. These broths were then extracted with 3 ml ethyl acetate and vortexed for 10 seconds. Two ml of the organic phase was collected in small glass vials and evaporated to dryness under a stream of nitrogen gas.
- Residues were derivatized by silylation using BSTFA (/V, 0-bis(trimethylsilyl) trifluoroacetamide); Supelco®, PA, USA) in a total reaction volume of 100 ⁇ , composed of 25 ⁇ BSTFA and 75 ⁇ acetonitrile, each added freshly.
- the reaction was incubated at 70°C for 30 min, then cooled to room temperature and diluted to 1000 ⁇ with acetonitrile prior to injection into the gas chromatograph (see below). This way, the final concentrations of 3-hydroxy fatty acids as well as internal standard are twice higher than their original concentrations in the culture broths.
- CHCI3 layers were collected and evaporated to dryness before derivatization by silylation as above.
- the values of free intracellular 3-hydroxyfatty acids obtained from the first batch were subtracted from those of the second batch to obtain the concentration of PHA.
- Analysis of the trimethylsilyl derivatives of the 3-hydroxyfatty acid was performed by gas chromatography/mass spectrometry (GC/MS) (Trace GC Ultra® - Polaris® Q, Thermo Fisher Scientific®, Waltham, MA) equipped with a ZB- 5MS (ZebronTM1 13) capillary column (30 m X 0.25 mm internal diameter, 0.25 ⁇ film thickness, Phenomenex®) using the electron impact ionization mode.
- GC/MS gas chromatography/mass spectrometry
- the GC program started at an oven temperature of 70°C that was increased at 30°C/min to 110°C and held at 1 10°C for 5 min, then increased at 20°C/min to 165°C and held at 165°C for 5 min, then increased at 30°C/min to 310°C and held for 0.5 min with an overall oven run time of 19.42 min.
- the sample injection volume was 1 ⁇ _.
- the Polaris Q MS was operated in full scan positive ion mode covering the mass range of 70-400 with a total scan time of 0.41 sec. MS scan start time was at 6.6 min and the ion source temperature was at 250°C.
- the injector and detector (ion source) temperatures were 200°C and 250°C, respectively.
- Helium was used as the carrier gas at a constant flow rate of 1.1 ml/min. Quantification was performed by comparing integration data of ⁇ + 122 fragment ions resulting from the electron impact of the trimethylsilyl esters of 3-hydroxy fatty acids with those of the internal standard using response factors previously determined through calibration curves made with pure standards. The experiments were conducted in triplicate.
- PBS phosphate buffer saline
- the material was kept overnight in a 1 :1 volume mixture of Spurr resin and acetone, then immersed twice, each 1 hour, in a bath of Spurr mixtures.
- the block containing fixed cells was cut into small pieces, placed in BEEM capsules, filled with Spurr resin and held at room temperature for 16 hours and then polymerized at 60°C for 20-30 hours.
- Ultrathin sections (70-100 nm thick) were examined by a Hitachi H-7100 electron microscope with an accelerating voltage of 75 kV. Estimation of PHA production was made using the photos obtained from the electron microscope and the area measurement tools of Adobe Photoshop® (version CS3 extended).
- Example 2 Inhibition of ⁇ -oxidation decreases RLs production
- FASH de novo fatty acid synthesis
- produced RLs would be mostly unlabeled as its chains would be de novo synthesized from the more abundant unlabeled acetyl-CoA (five unlabeled versus one labeled) units released after complete ⁇ -oxidation of dodecanoic-12,12,12-d 3 acid (FIG. 10A).
- Example 4 ⁇ -oxidation is directly linked to RLs pathway [00163] Using perdeuterooctadecanoic acid (Ci8-d 3 s) and glucose as co-substrate, Zhang et al. (2012) suggested that the fatty acid substrates of RLs are shortened via ⁇ -oxidation to the Cs chain length after which one C2-elongation step via FASII takes place before diversion of corresponding FAS II Cio-intermediate to RLs biosynthesis (Zhang et al., 2012). However, our isotope tracing experiments using dodecanoic-12,12,12-d 3 acid are suggesting instead a direct link between ⁇ -oxidation and RL biosynthesis.
- Example 5 Enoyl-CoA intermediates of ⁇ -oxidation are diverted to RLs biosynthesis
- ⁇ -oxidation implicates that one of its intermediates, namely, trans-2-enoyl- CoA, S-3-hydroxyacyl-CoA or 3-ketoacyl-CoA, is converted via an enoyl-CoA hydratase, an epimerase, or a 3-ketoacyl-CoA reductase, respectively, into R-3-hydroxyfatty acids, the ultimate lipid precursor of RLs.
- RLs production was examined in mutants defective in enzymes carrying out these conversions.
- Transposon mutants of phaJ2 and phaJ4 had no negative effect on RLs production (FIG. 13A).
- three putative enoyl-CoA hydratases/isomerases (ECH/I) mutants PA14_40980 (Tn), PA14_54640 (Tn), and PA14_54660 (KO) were associated with remarkable 35%, 56% and 54% reductions in RLs productions, respectively (FIG. 13B). Because of their impact on RL synthesis, they were respectively named RhIK, RhIY and RhIZ.
- PA14 locus tag PA01 locus tag Transposon mutant available (Liberati et al.,
- PA3302, PA1 018, PA4788 and PA4015 are reported in Pseudomonas aeruginosa as PHA-related hydratases; PhaJ 1 , PhaJ2, PhaJ3 and PhaJ4, respectively.
- RhIY and RhIZ were selected for further studies, as their corresponding mutants were the most affected in RL production. These two genes are arranged in a single computationally predicted operon with RhIZ downstream to RhIY (Winsor et al., 2011 ). To exclude the possibility that the RL phenotype associated with the RhIY transposon mutant (Tn) is ultimately caused by a polar effect on its downstream gene RhIZ, a double RhIYZ knockout mutant (KO) was constructed and its RLs production was compared to that of the simple RhIZ knockout mutant. In addition, RhIZ was expressed in trans from a multicopy plasmid in both mutants.
- Example 6 The ECH/I RhIY and RhIZ ar main suppliers of 3-hydroxydecanoate/octanoate pools
- RhIZ is an R-specific ECH/I
- RhIZ is an R-specific ECH/I (R-ECH/I) converting a 2-decenoyl- CoA intermediate of ⁇ -oxidation into the corresponding R-3-hydroxydecanoyl-CoA precursor of RL (FIG. 9A). This was verified in vitro using purified RhIZ (FIG. 3) and trans-2-decenoyl-CoA as substrate. The relative proportion of the R- and S-stereoisomers of 3-hydroxydecanoyl-CoA (3-OH-Cio-CoA) produced was estimated using a chiral HPLC-MS/MS method.
- RhIZ is indeed an R-ECH/I, but that a factor present in the WT cell lysate is involved in its activity.
- this factor is a protein since heat treatment of the clarified cell lysate abolished RhIZ activity with no any 3-hydroxydecanoate produced.
- this heat-sensitive factor is likely RhIY.
- purified RhIZ was combined with the clarified cell lysates of t RhlYZ or t RhlZ. Consistent with the hypothesis that the heat-sensitive factor is RhIY, the R-3-OH-Cio enrichment was conserved when purified RhIZ was combined with the lysate of t RhiZ (FIG. 3C, D), whereas, no such enrichment was observed when this purified RhIZ was combined with the lysate of the double mutant t RhlYZ (FIG.
- RhIZ is an R-specific ECH/I that is functional in vivo in the presence of RhIY with which it forms a stable complex.
- RhIYZ polyhydroxyalkanoates
- rhIA is required for the production of a novel biosurfactant promoting swarming motility in Pseudomonas aeruginosa: 3-(3- hydroxyalkanoyloxy)alkanoic acids (HAAs), the precursors of rhamnolipids.
- HAAs 3-(3- hydroxyalkanoyloxy)alkanoic acids
- Burkholderia thailandensis harbors two identical rhl gene clusters responsible for the biosynthesis of rhamnolipids. BMC Microbiol 9, 263.
- RhIA converts ⁇ -hydroxyacyl-acyl carrier protein intermediates in fatty acid synthesis to the -hydroxydecanoyl- -hydroxydecanoate component of rhamnolipids in Pseudomonas aeruginosa. Journal of Bacteriology 190, 3147-3154.
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Abstract
Novel methods and agents for modulating the production of rhamnolipids (RLs) by a microorganism, through the modulation of the expression and/or activity of R-specific enoyl-CoA hydratases/isomerases (ECH/I), are described. Assays to identify agents that may be useful for modulating the production of RLs by a microorganism are also described.
Description
MODULATION OF THE BETA-OXIDATION PATHWAY IN THE CONTROL OF RHAMNOLIPID
PRODUCTION
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of United States provisional application serial No. 61/915,049 filed December 12, 2013, which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the production of rhamnolipids (RLs) by microorganisms, and more particularly to the modulation thereof for different applications.
BACKGROUND OF THE INVENTION [0003] Rhamnolipids are glycolipidic biosurfactants produced by various bacterial species. The bacterium Pseudomonas aeruginosa is the best-studied producer of rhamnolipids (RLs), surface-active, multipurpose amphiphilic molecules. RLs are produced as mixtures of congeners composed of 1 or 2 rhamnose moieties linked to a dimer of R-3-hydroxyfatty acids with chain lengths of Ci2±4 (Deziel et al.,
2000) . [0004] RLs are synthesized from two precursors, R-3-hydroxyfatty acids and L-rhamnose, by the actions of three enzymes: RhIA, RhIB, and RhIC. RhIA dimerizes R-3-hydroxyfatty acids forming congeners of R-3-((R-3-hydroxyalkanoyl)oxy)alkanoic acids (HAAs); the most abundant of which in P. aeruginosa is C10-C10 (Deziel et al., 2003; Zhu and Rock, 2008). The rhamnosyl transferases RhIB and RhIC catalyze the sequential addition of the first and second rhamnose to the C10-C10 dimer, forming mono- (Rha-Cio-Cio) and di-RL (Rha-Rha-Cio-Cio), respectively (Ochsner et al., 1994; Rahim et al.,
2001 ) .
[0005] A wide diversity of rhamnolipid congeners and homologues (about 60) that are produced at different concentrations by various Pseudomonas species and by bacteria belonging to other families, classes, or even phyla (Abdel-Mawgoud et al., 2010). For example, various Burkholderia species have been shown to produce rhamnolipids that have longer alkyl chains than those produced by P. aeruginosa.
[0006] Rhamnolipids promote the uptake and biodegradation of poorly soluble substrates, act as immune modulators and virulence factors, have antimicrobial activities, and are involved in surface motility and in bacterial biofilm development. Given the role of rhamnolipids as virulence factors
contributing to pathogenic infection by RL-producing microorganisms such as Pseudomonas aeruginosa, the inhibition of the production of RLs constitutes a therapeutic strategy for attenuating the virulence of RL-producing microorganisms.
[0007] Given the biotechnological importance of RLs, much effort is dedicated to maximize the production via optimization of culture conditions (Abdel-Mawgoud et al., 2011 ). Nonetheless, higher yields are still needed for an economically feasible industrial production. It is becoming obvious that this can only be achieved via metabolic engineering, which requires an exquisite understanding of the metabolic regulation of RLs (Abdel-Mawgoud et al., 2011 ).
[0008] There is thus a need for a better understanding of the metabolic regulation of RLs, and for novel approaches to modulate the biosynthesis of RLs.
[0009] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
[0010] In an aspect, the present invention provides a method for decreasing the production of rhamnolipids (RLs) by a cell, for example a microorganism, said method comprising inhibiting the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I) in said cell.
[0011] In another aspect, the present invention provides a method for attenuating the virulence of a rhamnolipid-producing microorganism, said method comprising inhibiting the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I) in said rhamnolipid- producing microorganism.
[0012] In another aspect, the present invention provides the use of an inhibitor of R-specific enoyl-CoA hydratases/isomerases (ECH/I) for decreasing the production of rhamnolipids (RLs) by a cell, for example a microorganism. [0013] In another aspect, the present invention provides the use of an inhibitor of R-specific enoyl-CoA hydratases/isomerases (ECH/I) for the preparation of a medicament for decreasing the production of rhamnolipids (RLs) by a cell, for example a microorganism.
[0014] In another aspect, the present invention provides the use of an inhibitor of R-specific enoyl-CoA hydratases/isomerases (ECH/I) for attenuating the virulence of a rhamnolipid-producing
microorganism.
[0015] In another aspect, the present invention provides the use of an inhibitor of R-specific enoyl-CoA hydratases/isomerases (ECH/I) for the preparation of a medicament for attenuating the virulence of a rhamnolipid-producing microorganism. [0016] In an embodiment, the above-mentioned inhibiting comprises contacting said microorganism with an inhibitor of said one or more R-specific ECH/I.
[0017] In an embodiment, the above-mentioned inhibitor is an antibody specifically binding to said one or more R-specific ECH/I. In a further embodiment, the above-mentioned antibody is monoclonal. [0018] In another embodiment, the above-mentioned inhibitor is an inactive or dominant- negative form of said one or more R-specific ECH/I.
[0019] In another embodiment, the above-mentioned inhibitor is an RNA interference agent targeting said one or more ECH/I. In a further embodiment, the above-mentioned RNA interference agent is a shRNA or siRNA. [0020] In an embodiment, the one or more R-specific ECH/I comprise an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95% or 100% identity with the amino acid sequence of FIG. 15A (SEQ ID NO:2), 16A (SEQ ID NO:4) and/or 17A (SEQ ID NO:6).
[0021] In another aspect, the present invention provides a method for increasing the production of rhamnolipids (RLs) by a cell, for example a microorganism, said method comprising increasing the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I) in said cell. In an embodiment, the method comprises introducing into said cell one or more nucleic acids encoding said one or more R-specific ECH/I. In an embodiment, the method further comprises introducing one or more nucleic acids encoding one or more polypeptides involved in the RL biosynthetic pathway. In a further embodiment, the above-mentioned one or more polypeptides involved in the RL biosynthetic pathway are rhamnosyl-transferases, in yet a further embodiment RhIB and/or RhIC. In an embodiment, the one or more nucleic acids encode the R-specific ECH/I defined above. In a further embodiment, the above-mentioned one or more nucleic acids comprise a sequence having at least 50%, 60%, 70%, 80%, 90%, 95% or 100% identity with the nucleotide of FIG. 15B (SEQ ID NO:1 ), 16B (SEQ ID NO:3) and/or 17B (SEQ ID NO:5).
[0022] In an embodiment, the above-mentioned method is an in vivo method. In an embodiment, the above-mentioned method is an in vitro method.
[0023] In another aspect, the present invention provides a method for determining whether an agent may be useful for (i) inhibiting the production of rhamnolipids (RLs) by a cell, for example a microorganism and/or (ii) attenuating the virulence of a rhamnolipid-producing microorganism, said method comprising determining whether said agent inhibits the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I). In an embodiment, the method comprises (a) contacting said agent with said one or more R-specific ECH/I and (b) determining whether said agent inhibits the expression and/or activity of said one or more R-specific ECH/I, wherein the inhibition of said expression and/or activity is indicative that said agent may be useful for (i) inhibiting the production of RLs by a cell and/or (ii) attenuating the virulence of a rhamnolipid-producing microorganism.
[0024] In another aspect, the present invention provides a method for determining whether an agent may be useful for increasing the production of rhamnolipids (RLs) by a cell, for example a microorganism, said method comprising determining whether said agent increases the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I). In an embodiment, the method comprises (a) contacting said agent with said one or more R-specific ECH/I and (b) determining whether said agent increases the expression and/or activity of said one or more R-specific ECH/I, wherein the increase of said expression and/or activity is indicative that said agent may be useful for increasing the production of rhamnolipids (RLs) by a cell. [0025] In an embodiment, the above method is a cell-free method.
[0026] In another embodiment, the above method is performed in a cell-based system, i.e. the one or more R-specific ECH/I are expressed in a cell. In a further embodiment, the one or more R- specific ECH/I comprise an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95% or 100% identity with the amino acid sequence of FIGs. 15A (SEQ ID NO:2), 16A (SEQ ID NO:4) and/or 17A (SEQ ID NO:6).
[0027] In an embodiment, the microorganism is of the Pseudomonas genus, in a further embodiment Pseudomonas aeruginosa, e.g., P. aeruginosa strain PA14.
[0028] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] In the appended drawings:
[0030] FIGs. 1A and 1 B show that specific inhibitors of β-oxidation decrease RLs production in
P. aeruginosa strain PA14. FIG. 1A: Dose-dependent inhibition of RLs and HAA production by 2- bromooctanoic acid. FIG. 1 B: 2-bromohexanoate similarly inhibits RLs production at equivalent molar concentration (5 mM). Strain PA14 was grown in MSM-glycerol for 3 days at 34°C. Data represent the mean of triplicate experiments ± S.D.
[0031] FIGs. 2A and 2B show that RhIY and RhIZ are implicated in RLs biosynthesis controlling the supply of lipid precursors. FIG. 2A: Deletion mutants, Δ RhIYZ and RhlZ have similarly reduced RLs production. Complementation of ARWVZ with RhIZ only partially restores RLs production. FIG. 2B: RhIYZ \s the main supplier of 3-hydroxyalkanoates (C10+2). Wild-type PA14 strain and mutants were grown for 3 days in MSM-glycerol at 34°C. Lipids were analyzed by GC/MS. Data represent the mean of triplicate experiments ± S.D.
[0032] FIGs. 3A to 3F show that RhIZ is an R-specific enoyl-CoA hydratase/isomerase (ECH/I) whose activity is modulated by RhIY. The purified RhIZ alone did not catalyze any 3-hydroxydecanoate formation from trans-2-decenoyl-CoA, therefore, FIG. 3A: Total cell lysate of WT was added in conjunction with RhIZ and this caused enrichment of the R-3-hydroxydecanoate compared to the S- enantiomer in comparison to the total cell lysate of WT alone as in FIG. 3B, which produced a nearly racemic mixture of 3-hydroxydecanoate. FIG. 3C, 3D: The same was observed with total cell lysate of the mutant RhlZ. FIG. 3E, 3F: The factor in lysates of WT or RhIZ mutant that is indispensable for RhIZ activity appears to be RhIY as pure RhIZ does not produce any 3-hydroxydecanoate in the presence of total cell lysate of RhlYZ. R/S-3-hydroxydecanoate was analyzed using a chiral HPLC-MS/MS method.
[0033] FIG. 4 shows that RhIZ is an R-specific ECH/I and is inhibited by a metabolite of 2- bromooctanoate (2-Br-Ce). In in vitro enzymatic assays in the presence of WT cell lysate, RhIZ causes R-specific enrichment of 3-hydroxydecanoate, and this enrichment is blocked when using WT lysate of cells cultivated in the presence of 5 mM 2-Br-Cs. R/S-3-hydroxydecanoate was analyzed using chiral HPLC-MS/MS. Data represent the mean of triplicate experiments ± S.D.
[0034] FIG. 5 shows an SDS-PAGE of the pull-down assay showing the stable physical protein- protein interaction between RhIY and RhIZ. Purified His6-tagged RWZ was mixed with total cell lysate of WT PA14 strain for 1 hour at 4°C, the mixture was then subjected to Nickel affinity chromatographic
purification using FPLC using the same gradient of imidazole as previously done for purification of RhIZ (FIG. 14A). Lane (1) is the purified RhlZ-H\se alone (bait). Lane (2) is the control lysate which was not exposed to the RWZ-His6. Lane (3) is the purified p rotei n -R/7/Z-H i se interaction; this protein was shown to be RhIY as shown from its size compared to the purified RhlY-H\se in lane (4) as well as by protein sequencing.
[0035] FIGs. 6A and 6B show that RLs and PHA share the same 3-hydroxyalkanoate pool. Deletion of RhIYZ results in a decrease in PHA accumulation, as estimated by GC-MS hydrolyzed intracellular 3-hydroxyfatty acids (Cs to C12) (FIG. 6A) as well as by percentage cross-sectional area of PHA granules relative to the whole cell cross-sectional area on images taken by transmission electron microscope, based on an average measurements of more than 400 cells of both WT and mutant (FIG. 6B). FIG. 6C: The PHA-negative mutant {kphaC1phaDphaC2phaDkphaFkphal) causes a slight increase in RLs, mostly with a 100% enrichment of mono-RLs at the expense of di-RLs. FIG. 6D: Thin transmission electron microscope images of PHA granules of P. aeruginosa PA14 strains; RhlYZ, WT, t pha. White spheres are PHA granules, whereas black spheres are polyphosphates. Magnification bar = 100 nm. WT strain and deletion mutants were cultured in MSM-glycerol for 2-2.5 days at 34°C with shaking. Data represent the mean of triplicate experiments ± S.D.
[0036] FIG. 7 is a proposed model of metabolic supply for lipid precursors of RLs. With a fatty acid carbon source, shortening of the chain takes place by β-oxidation until the formation of the trans-2- decenoyl-CoA. With a soluble carbon source like glycerol, de novo synthesis of fatty acids via FASII forms fatty acids that are then diverted to β-oxidation to be processed into the trans-2-decenoyl-CoA intermediate. In either case, the trans-2-decenoyl-CoA is hydrated by the R-specific enoyl-CoA hydratase RhIYZ to form R-3-hydroxydecanoyl-CoA, which then acts as the direct lipid precursors used by RhIA for synthesis of HAAs then RLs. Although never demonstrated in vivo, a direct precursor supply from FAS II could also be contributing in RL biosynthesis. PHAs share with RLs the same pool of R-3- hydroxyacyl-CoA. FabB and FabF: 3-ketoacyl-ACP synthetases, FabG: NADPH-dependent 3-ketoacyl- ACP reductase, FabA, FabZ: 3-hydroxyacyl-ACP dehydratases, Fabl: NADH-dependent enoyl-ACP reductase. FadD: acyl-CoA synthetase, FadE: acyl-CoA dehydrogenase, FadB: trans-enoyl-CoA hydratase and S-3-hydrxoyacyl-CoA dehydrogenase, FadA: 3-ketoacyl-CoA thiolase. RhIA: HAA synthetase, RhIB: rhamnosyltransferase 1 , RhIC: rhamnosyltransferase 2. [0037] FIGs. 8A and 8B show the effect of different inhibitors of β-oxidation on RLs and HAA production. FIG. 8A: 2-bromooctanoate (2-Br-C8) inhibits RLs and HAAs production. FIG. 8B: Acrylic and 4-pentenoic acid are not inhibiting RLs or HAAs production. Strain PA14 was grown in MSM-
glycerol for 3 days at 34°C.
[0038] FIGs. 9A and 9B show an investigation of a role of PhaG in RLs production. FIG. 9A: Role of PhaG in PHA biosynthesis and possible role in RLs biosynthesis. PhaG is a R-3- hydroxydecanoyl-ACP:CoA transacylase that converts the R-3-hydroxydecanoyl-ACP intermediate of FAS II into the CoA counterpart to enter in the polyhydroxyalkanoates biosynthesis pathway. 2- bromooctanoate-associated inhibition of RLs was suspected to be mediated by PhaG and not by its inhibition of β-oxidation. FIG. 9B: PhaG in not implicated in RL production and thus the 2- bromooctanoate-mediated inhibition of RLs. RL production was unaffected in the knock-out mutant, t\phaG (KO), as well as the transposon mutant t\phaG (Tn) compared to the WT PA14 strain. This indicates that PhaG could not be implicated in RL produciton or be the target of the 2-bromooctanoate- mediated inhibition of RLs. Strains were grown in MSM-glycerol for 3 days at 34°C.
[0039] FIGs 10A and 10B show that isotopic tracing demonstrates that lipid precursor of RLs is directly recovered from β-oxidation. FIG. 10A: Prediction of isotopic distribution in lipid chains of RLs upon feeding labeled dodecanoate-12,12,12-d3 as sole carbon source. If β-oxidation is involved in supplying the lipidic precursors for RLs pathway, RLs would be labeled at each lipid chain and would carry an extra 3 Da per chain. If de novo fatty acid synthesis (FAS II) is responsible for supplying the lipidic precursors for RLs pathway, RLs will be mainly unlabeled because the fatty acid side chains in this case would be constructed from acetate units released from β-oxidation of the dodecanoate- 12,12,12-d3 and which are mostly unlabeled. FIG. 10B: Fragmentation pattern of the molecular ion of labeled mono-RLd6, m/z 509 confirms a direct link between β-oxidation and RLs. Fragment ions confirm the identity of the parent molecule as mono-RL-d6 and shows the localization of 3 deuterium atoms in each of the two fatty acid chains of labeled mono-RLs Rha-C10-d3-C10-d3. P. aeruginosa PA14 was cultivated for 10 days at 34°C in MSM with dodecanoate-12,12,12-d3 (30 mM) as the sole carbon source. [0040] FIG. 11 shows a proposed mechanism of incorporation of deuterium-labeled 3- hydroxydecanoate intermediates of dodecanoate-d23 in RLs. (Box A) According to a proposed model, 3-hydroxydecanoate will derive directly from β-oxidation, thus would carry the maximum possible deuterium labeling i.e. +17 Da. (Box B) According the model of Zhang et al. (2012), the octanoate species of β-oxidation will bypass through FASII to get lengthened by one two-carbon unit. Theoretically, resulting decanoate species would carry +16 and +15 at a ratio of 2:1 . This ratio arises from the ratio of mono-labeled malonyl-CoA-d1 to unlabeled malonyl-CoA whose abundance is at 2:1 as a result of the carboxylation of mono-labeled acetyl-CoA-d1 replacing a methyl-carried hydrogen atoms
(two) or deuterium atom (one).
[0041] FIGs. 12A to 12D shows isotopic tracing of fragment m/z 317 (first column, A1 to D1) and of fragment m/z 201 (second column, A2 to D2) in different labeled carbon source conditions. Dodecanoate-d23 (30 mM) (took 7 days to grow) (FIGs. 12-A1 and 12-A2), dodecanoate-d23 (0.375%) with glucose (10 g/L) (FIGs. 12-B1 and 12-B2), dodecanoate-12,12,12-d3 (30 mM) with glucose-13C6 (15 g/L) (FIGs. 12-C1 and 12-C2) and tetradecanoate-14,14,14-d3 (30 mM) with glucose-13C6 (15 g/L) (FIGs. 12-D1 and 12-D2). All presented relative abundances of isotopomers are filtered from the nearby isotopic effects. To identify the nature of labeling in the 3-hydroxydecanoate chains of RLs, RLs were acid hydrolyzed, then freed lipid chains were extracted and derivatized using trimethylsilane and analyzed using GC/MS. Two daughters were traced fragments that would help in the identification and localization of labeling particularly in the first two carbon atoms of the lipid chains.
[0042] FIGs. 13A to 13C show the impact of mutation and overexpression of different predicted enoyl-CoA hydratases. FIG. 13A: phaJ2 and phaJ4. Mutants of the PHA-associated R-specific enoyl- CoA hydratases phaJ2 and phaJ4 did not show a decrease in RLs production compared to the WT strain of PA14. FIG. 13B: RhIK, RhIY, RhIZ. The transposon mutants, PA14_40980 {rhlK~), PA14_54640 (rhlY~), and the deletion mutant PA14_54660 (M/Z) show reduced RLs production. FIG. 13C: Overexpression of RhIZ on pUCP26. Unexpectedly, overexpression of RhIZ in the WT strain PA14 does not increase RLs production; tetracycline was added to maintain plasmid. Cultures were grown for 3 days in MSM-glycerol at 34°C under rotation. [0043] FIG. 14A and 14B show the purification of N-terminal 6xHis-tagged recombinant RhIZ. Both proteins were purified by nickel affinity chromatography using FPLC. (A) RhIZ peak elutes at 160 mM imidazole. It migrated with an apparent molecular mass of 40.6 KDa as determined by SDS-PAGE. (B) RhIY peak elutes at 240 mM imidazole. It migrated with an apparent molecular mass of 29.9 KDa as determined by SDS-PAGE. [0044] FIG. 15A and 15B show the amino acid (SEQ ID NO: 2) and encoding nucleotide sequences (SEQ ID NO: 1), respectively, of P. aeruginosa (strain UCBPP-PA14) RhIK (PA14_40980, NCBI-GI: 1 16049771 , UniProt: Q02KW4).
[0045] FIG. 16A and 16B show the amino acid (SEQ ID NO: 4) and encoding nucleotide sequences (SEQ ID NO: 3), respectively, of P. aeruginosa (strain UCBPP-PA14) RhIY (PA14_54640, NCBI-GI: 1 16048660, UniProt: Q02HP6).
[0046] FIG. 17A and 17B show the amino acid (SEQ ID NO: 6) and encoding nucleotide sequences (SEQ ID NO: 5), of P. aeruginosa (strain UCBPP-PA14) RhIZ (PA14_54660, NCBI-GI: 1 16048659, UniProt: Q02R63).
[0047] FIGs. 18A and 18B show the chemical structure of different identified rhamnolipid congeners and homologues (from Abdel-Mawgoud et al., 2010).
[0048] FIGs. 19A and 19B show the taxonomical classification of different bacteria reported to produce rhamnolipids (from Abdel-Mawgoud et al., 2010).
[0049] FIGs. 20A and 20B show the amino acid (SEQ ID NO: 27) and encoding nucleotide sequences (SEQ ID NO: 26), of P. aeruginosa (strain UCBPP-PA14) HAA synthase or Rhamnosyltransferase chain A (RhIA) (PA14J 9110, NCBI-GI: 116051498, UniProt: Q02QW8).
[0050] FIGs. 21 A and 21 B show the amino acid (SEQ ID NO: 29) and encoding nucleotide sequences (SEQ ID NO: 28), of P. aeruginosa (strain UCBPP-PA14) Rhamnosyltransferase 1 or Rhamnosyltransferase chain B (RhIB) (PA14J 9110, NCBI-GI: 116051497, UniProt: Q02QW7).
[0051] FIGs. 22A and 22B show the amino acid (SEQ ID NO: 31 ) and encoding nucleotide sequences (SEQ ID NO: 30), of P. aeruginosa (strain UCBPP-PA14) Rhamnosyltransferase 2 (RhIC) (PA14_497600, NCBI-GI: 116049062, UniProt: Q02IV0).
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0052] In the studies described herein, the present inventors have shown that the β-oxidation pathway is the predominant source of the precursors of RLs. They have also shown that R-specific enoyl-CoA hydratases/isomerases (ECH/I) are involved in the production of these precursors, and consequently of RLs, and thus that the modulation of the expression and/or activity of R-specific ECH/I has an effect on the production of RLs by RL-producing microorganisms such as Pseudomonas aeruginosa. The studies described herein also demonstrate that the pool of precursors synthesized through the activity of R-specific ECH/I is shared between RLs and polyhydroxyalkanoates (PHA), and thus that the modulation of the expression and/or activity of R-specific ECH/I has also an effect on the production of PHA.
Inhibition of enoyl-CoA hydratases/isomerases (ECH/I)
[0053] Accordingly, in an aspect, the present invention provides a method for decreasing the production of rhamnolipids (RLs) and/or polyhydroxyalkanoates (PHAs) by a microorganism (e.g., a
rhamnolipid- and/or polyhydroxyalkanoate-producing microorganism), said method comprising inhibiting (decreasing, reducing) the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I) in said microorganism.
[0054] In another aspect, the present invention provides a method for decreasing the production of rhamnolipids (RLs) by a rhamnolipid-producing microorganism, said method comprising inhibiting the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I) in said microorganism.
[0055] In another aspect, the present invention provides a method for decreasing the production of polyhydroxyalkanoates (PHA) by a PHA-producing microorganism, said method comprising inhibiting the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I) in said microorganism.
[0056] In an embodiment, the above inhibition comprises contacting the microorganism with an inhibitor of said one or more R-specific ECH/I.
[0057] In another aspect, the present invention provides the use of an inhibitor of R-specific enoyl-CoA hydratases/isomerases (ECH/I) for decreasing the production of rhamnolipids by a rhamnolipid-producing microorganism.
[0058] In another aspect, the present invention provides the use of an inhibitor of R-specific enoyl-CoA hydratases/isomerases (ECH/I) for the preparation of a medicament for decreasing the production of rhamnolipids by a rhamnolipid-producing microorganism. [0059] In another aspect, the present invention provides the use of an inhibitor of R-specific enoyl-CoA hydratases/isomerases (ECH/I) for decreasing the production of PHA by a microorganism.
[0060] In another aspect, the present invention provides the use of an inhibitor of R-specific enoyl-CoA hydratases/isomerases (ECH/I) for the preparation of a medicament for decreasing the production of PHA by a microorganism. [0061] The term "rhamnolipids" as used herein refers to glycolipidic biosurfactants (or surface- active molecules) produced by various bacterial species, and which generally comprises rhamnose moieties (glycon part) and lipid moieties (aglycon part) linked to each other via an O-glycosidic linkage.
[0062] The glycon part is typically composed of one (mono-RLs) or two (di-RLs) rhamnose moieties linked to each other through an alpha-1 ,2-glycosidic linkage. The aglycon part, however, is
mainly one or two (in few cases three) β-hydroxy fatty acid chains (saturated, mono-, or polyunsaturated and of different chain length, typically varying from Cs to C16) linked to each other through an ester bond formed between the β-hydroxyl group of the distal (relative to the glycosidic bond) chain with the carboxyl group of the proximal chain. [0063] To date, about 60 different RLs have been described (reviewed in Abdel-Mawgoud et al., 2010). The structures of these RLs are depicted in FIGs. 18A-B.
[0064] The term "rhamnolipid-producing microorganism" as used herein refers to any microorganism, such as bacteria, which has the capacity to synthesize/produce rhamnolipids under suitable conditions. Various bacterial species have been shown to produce RLs, as depicted in FIGs. 19A-B. In an embodiment, the rhamnolipid-producing microorganism is of a phylum, class, order, family or genus depicted in FIGs. 19A-B. In a further embodiment, the rhamnolipid-producing microorganism is a bacterium of the Gammaproteobacteria class. In a further embodiment, the rhamnolipid-producing microorganism is a bacterium of the Pseudomonadales order. In a further embodiment, the rhamnolipid- producing microorganism is a bacterium of the Pseudomonadacae family. In a further embodiment, the rhamnolipid-producing microorganism is a bacterium of the Pseudomonas genus, such as P. alcaligenes, P. aeruginosa, P. chlororaphis, P. clemancea, P. collierea, P. fluoresceins, P. luteola, P. putida, P. stutzeri and P. teessidea. In a further embodiment, the rhamnolipid-producing microorganism is P. aeruginosa.
[0065] The term polyhydroxyalkanoate (PHA) as used herein refers to biopolyesters composed of hydroxy fatty acids (polyoxoesters). PHAs can consist of short-chain-length hydroxyalkanoic acids (PHASCL) or medium-chain-length monomers (PHAMCL), depending on strain and culture conditions. More than 150 PHA monomers have been identified. The general structure of polyhydroxyalkanoates (PHAs) and examples of their structural derivatives are depicted below (from Shrivastav et al., Biomed Res Int. 2013; 2013: 581684).
Poly( 3- hydroxyalkanoates) [ PH A]
R group
Po I y ( 3- hy d rox yal kanoates ) PHA
CH2-CH, Po ly ( 3- h y d roxyval erate ) PHV
(CH2 )2— CH Poly ( 3 - hy d roxy h exanoat ) PHHex
(CH2)4 - CH Poly(3-hydroxy ctanoate) PHO (C¾)r - CH Poly(3-hydroxydecanoate) PHD
Po ly(3 - hyd roxy- 5 - ph eny 1 val e rate ) PHPV
[0066] The term "PHA-producing microorganism" as used herein refers to any microorganism, such as bacteria, which has the capacity to synthesize/produce PHAs under suitable conditions. PHAs are synthesized by a wide range of different Gram-positive (e.g., of the Bacillus, Clostridium, Corynebacterium, Nocardia, Rhodococcus, Streptomyces and Staphylococcus genusj and Gram- negative {Alcaligenes, Ralstonia and Pseudomonas genusj bacteria, as well as by some Archaea (notably belonging to the genera Haloferax, Haloarcula, Natrialba, Haloterrigena, Halococcus, Haloquadratum, Halorubrum, Natronobacterium, Natronococcus and Halobacterium) (see Poli et al., Archaea 201 1 : 201 1 : 693253; Singh et al., Microbial Cell Factories 2009, 8:38), and are deposited as insoluble cytoplasmic inclusions.
[0067] The term "inhibiting" or ("decreasing", "reducing") refers for example to a decrease of at least 10%, in embodiments a decrease of at least 20, 30, 40, 50, 60, 70, 80, 90 or 100% (complete inhibition) relative to a native condition. For example, "decreasing the production of RLs" refers to a lower production of RLs by the microorganism relative to a corresponding "native" microorganism, i.e. in which an R-specific enoyl-CoA hydratase/isomerase has not been inhibited.
[0068] The term R-specific enoyl-CoA hydratases/isomerases (ECH/I) (also referred to as crotonases) as used herein refers to any enzyme capable of converting a frans-enoyl-CoA precursor to an R-hydroxyfatty acid (e.g., capable of converting a frans-2-enoyl-CoA to an R-3-hydroxyfatty acid). "R- specific" means that the ECH/I generates a RL/PHA precursor in the R configuration [i.e., the β-hydroxyl groups of the fatty acid chains are present in the R-configuration). R-specific ECH/I are also referred to as ECH type 2 or ECH-2 (whereas S-specific ECH/I are referred to as ECH type 1 or ECH-1 ).
[0069] In an embodiment, the R-specific ECH/I comprises the amino acid sequence depicted in FIGs. 15A (SEQ ID NO:2), 16A (SEQ ID NO:4) and 17A (SEQ ID NO:6) (herein referred to as RhIK, RhIY and RhIZ, respectively), or a sequence having at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98 or 99% identity with the amino acid sequence depicted in FIGs. 15A (SEQ ID NO:2), 16A (SEQ ID NO:4) and 17A (SEQ ID NO:6) (and exhibiting hydratase and/or isomerase activity), or with the active domain thereof (i.e. the domain responsible for the hydratase and/or isomerase activity). "Identity" refers to sequence similarity between two polypeptide or nucleic acid molecules (or fragments thereof). Identity can be determined by comparing each position in the aligned sequences. A degree of identity between amino acid or nucleic acid sequences is a function of the number of identical or matching amino acids or nucleotides at positions shared by the sequences. As used herein, a given percentage of identity between sequences denotes the degree of sequence identity in optimally aligned sequences. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981 , Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wl, U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information. The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold. Initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program may use as defaults a word length (W) of 1 1 , the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (or 1 or 0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an
indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In another embodiment, the R-specific ECH/I is a homolog of RhIK, RhIY and/or RhIZ from another Pseudonomas aeruginosa strain. In another embodiment, the R-specific ECH/I is an ortholog of RhIK, RhIY and/or RhIZ. An ortholog is a protein with a sequence that has at least a portion with similarity/identity to a portion of the sequence of another protein, but found in a different species (e.g., in another bacterial species or in another Pseudonomas species). An ortholog and the other protein originated by vertical descent from a single protein of a common ancestor. In an embodiment, the ortholog encodes a protein that has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85, 90, 95, 96, 97, 98 or 99% identity of the total length of the sequence of the protein.
[0070] In an embodiment, the above-mentioned method comprises inhibiting the expression and/or activity of both RhIY and RhIZ, or orthologs/homologs thereof.
[0071] Other RhIK proteins having at least 50% amino acid sequence identity with SEQ ID NO: 2 include RhIK from, for example, one or more strains of Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas plecoglossicida, Pseudomonas monteilii, Pseudomonas taiwanensis, Pseudomonas entomophila, Pseudomonas stutzeri, Pseudomonas chloritidismutans, Pseudomonas fluorescens, Pseudomonas brassicacearum, Pseudomonas moraviensis, Pseudomonas corrugate, and Pseudomonas chlororaphis (see UNIPROT Cluster ID No. UniRef50_Q88fQ7 - Enoyl-CoA hydratase/isomerase family protein for the list and UniProt reference Nos. of RhIK proteins having at least 50% amino acid sequence identity with SEQ ID NO: 2).
[0072] Other RhIY proteins having at least 50% amino acid sequence identity with SEQ ID NO: 4 include RhIY from, for example, one or more strains of Pseudomonas aeruginosa, Azotobacter vinelandii, Burkholderia caribensis, Burkholderia phenoliruptrix, Burkholderia ambifaria, Burkholderia dolosa , Burkholderia pseudomallei, Burkholderia multivorans, Burkholderia cenocepacia, Vibrio caribbenthicus, Vibrio alginolyticus, Vibrio parahaemolyticus, Herbaspirillum lusitanum, Herbaspirillum frisingense, Pseudomonas stutzeri, Pseudomonas putrefaciens, Pseudomonas cepacia, Shewanella waksmanii, Shewanella oneidensis, Shewanella baltica, Burkholderia graminis, Burkholderia xenovorans, Shewanella decolorationis, Marinobacter santoriniensis, Amphritea japonica, marine gamma proteobacterium, Photobacterium profundum, Pseudoalteromonas lipolytica, Pseudoalteromonas luteoviolacea, Pseudoalteromonas rubra, Pseudoalteromonas piscicida, Pseudoalteromonas citrea, Pseudoalteromonas flavipulchra Lysobacter antibioticus, Lysobacter capsici, Colwellia piezophila, Janthinobacterium agaricidamnosum, Endozoicomonas elysicola, Shewanella
denitnficans, Oceanospirillum beijerinckii, Ferrimonas kyonanensis, Pseudomonas fulva, Xanthomonas campestris, Xanthomonas hortorum, Xanthomonas cassava, Simiduia agarivorans, Shewanella woodyi, Shewanella sedimini, and Psychrobacter lutiphocae (see UNIPROT Cluster ID No. UniRef50_C1 DQQ6 - Enoyl-CoA hydratase/isomerase family protein for the list and UniProt reference Nos. of RhIY proteins having at least 50% amino acid sequence identity with SEQ ID NO: 4).
[0073] Other RhiZ proteins having at least 50% amino acid sequence identity with SEQ ID NO: 6 include RhiZ from, for example, one or more strains of Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas taiwanensis, Pseudomonas vranovensis, Pseudomonas mosselii, Pseudomonas denitnficans, Pseudomonas syringae, Pseudomonas chlororaphis, Pseudomonas stutzeri, Marinobacterium litorale, Azoarcus toluclasticus, Afipia felis, Halotalea alkalilenta, and Sphaerotilus natans (see UNIPROT Cluster ID No. UniRef50_V9T1 M4 - Enoyl-CoA hydratase for the list and UniProt reference Nos. of RhiZ proteins having at least 50% amino acid sequence identity with SEQ ID NO: 6).
[0074] The R-specific ECH/I inhibitor includes any compound able to negatively affect the activity of one or more R-specific ECH/I by reducing for example its expression [i.e., at the transcriptional and/or translational level), the level of one or more ECH/I mRNA and/or protein, or an activity associated with one or more ECH/I. It includes intracellular as well as extracellular inhibitors. Without being so limited, such inhibitors include RNA interference agents (siRNA, shRNA, miRNA), antisense molecules, ribozymes, proteins (e.g., dominant negative, inactive variants), peptides, small molecules, antibodies, antibody fragments, etc.
[0075] In the studies described herein, the present inventors provide evidence that RhiZ and RhIY interact, and that this interaction is involved in RhiZ activity. Accordingly, in an embodiment, the ECH/I inhibitor blocks or interfere with the interaction between RhiZ and RhIY, or homologs/orthologs thereof. [0076] In an embodiment, the R-specific ECH/I inhibitor is a neutralizing antibody directed against (or specifically binding to) one or more R-specific ECH/I polypeptides. The term "antibody" or "immunoglobulin" is used in the broadest sense, and covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, humanized antibodies, CDR-grafted antibodies, chimeric antibodies, multispecific antibodies, and antibody fragments so long as they exhibit the desired biological activity (e.g., neutralizing an activity of the R-specific ECH/I polypeptide). Antibody fragments comprise a portion of a full length antibody, generally an antigen binding or variable region thereof.
Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, VH regions (VH, VH-VH), anticalins, PepBodies, antibody-T-cell epitope fusions (Troybodies) or Peptibodies. In an embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a humanized or CDR-grafted antibody.
[0077] In general, techniques for preparing antibodies (including monoclonal antibodies and hybridomas) and for detecting antigens using antibodies are well known in the art (Campbell, 1984, In "Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology", Elsevier Science Publisher, Amsterdam, The Netherlands) and in Harlow et al., 1988 (in: Antibody A Laboratory Manual, CSH Laboratories).
[0078] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (s.c), intravenous (i.v.) or intraperitoneal (i.p.) injections of the relevant antigen (e.g., an R-specific ECH/I polypeptide, or a fragment thereof) with or without an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCI2, or R1N=C=NR, where R and R1 are different alkyl groups.
[0079] Animals may be immunized against the antigen (an R-specific ECH/I polypeptide or a fragment thereof), immunogenic conjugates, or derivatives by combining the antigen or conjugate (e.g., 100 μg for rabbits or 5 μg for mice) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with the antigen or conjugate (e.g., with 1/5 to 1/10 of the original amount used to immunize) in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, for conjugate immunizations, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.
[0080] Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or may be made by recombinant DNA methods (e.g., U.S. Patent No. 6,204,023). Monoclonal antibodies may also be made using the techniques described in U.S. Patent Nos. 6,025,155 and 6,077,677 as well as U.S. Patent Application Publication Nos. 2002/0160970 and 2003/0083293.
[0081] In the hybridoma method, a mouse or other appropriate host animal, such as a rat, hamster or monkey, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the antigen used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
[0082] A human chimeric antibody can be produced in the following manner. cDNA encoding heavy chain variable region (VH) and light chain variable region (VL) obtained from a hybridoma derived from non-human animal cells producing monoclonal antibodies, the cDNA is inserted to each of expression vectors for animal cells having DNA encoding a heavy chain constant region (CH) and light chain constant region (CL) of a human antibody so as to construct a human chimeric antibody expression vector, and this vector is introduced to animal cells to express the human chimeric antibody.
[0083] A humanized antibody refers to an antibody which is obtained by grafting the amino acid sequence of the complementary determining region (CDR) of VH and VL of a non-human animal antibody to CDR corresponding to VH and VL of a human antibody. The region other than CDR of VH and VL is called a framework region (hereinbelow, described as "FR"). A humanized antibody can be produced in the following manner. cDNA encoding an amino acid sequence of VH which consists of an amino acid sequence of CDR of VH of a non-human antibody and an amino acid sequence of FR of VH of any human antibody, and cDNA encoding an amino acid sequence of VL which consists of an amino acid sequence of CDR of VL of a non-human animal antibody and an amino acid sequence of FR of VL of any human antibody are constructed, these cDNAs are inserted respectively into expression vectors for animal cells having DNA encoding CH and CL of a human antibody so as to construct a humanized antibody expression vector, and this vector is inserted into animal cells to express the humanized
antibody.
[0084] Based on the sequences of R-specific ECH/I polypeptides (see, e.g., FIGs. 15A - SEQ ID NO:2), 16A - SEQ ID NO:4 and 17A - SEQ ID NO:6), the skilled person would be able to generate antibodies directed against this polypeptide, which in turn may be used to neutralize its activity. [0085] In an embodiment, the R-specific ECH/I inhibitor is an inactive R-specific ECH/I polypeptide (e.g., dominant negative), or a nucleic acid encoding an inactive R-specific ECH/I polypeptide. Such an inactive polypeptide may compete with endogenous R-specific ECH/I and inhibits its activity.
[0086] In another embodiment, the R-specific ECH/I inhibitor is an RNA interference agent targeting an mRNA encoding one or more R-specific ECH/I. The term "RNA interference agent" as used herein refers to molecules that specifically binds to a target mRNA and induces its degradation (usually through the RNA-induced silencing complex (RISC) or interferes with its translation, and includes for example microRNA (miRNA) molecules, antisense molecules, small interfering RNA (siRNA) molecules and small/short hairpin RNA (shRNA). Chemically modified nucleosides, such as 2'-substituted arabinonucleosides (e.g., 2'F-ANA) and 2'-substituted RNA (e.g., 2'F-RNA), may be used for incorporation into RNA interference agents to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for a target RNA.
[0087] The RNA interference agent may be expressed from recombinant viral vectors, such as vectors derived from adenoviruses, adeno-associated viruses, lentiviruses, retroviruses, herpesviruses, and the like. Such vectors typically comprise a sequence encoding an RNA interference agent of interest and a suitable promoter operatively linked to the RNA interference agent for expressing the RNA interference agent. The vector may also comprise other sequences, such as regulatory sequences, to allow, for example, expression in a specific cell/tissue/organ, or in a particular intracellular environment/compartment. Methods for generating, selecting and using viral vectors are well known in the art.
[0088] Based on the sequences of nucleic acids encoding R-specific ECH/I polypeptides (see, e.g., FIGs. 15B - SEQ ID NO:1 , 16B - SEQ ID NO:3 and 17B - SEQ ID NO:5), the skilled person would be able to generate RNA interference agent directed against R-specific ECH/I mRNAs, which in turn may be used to decrease the expression of R-specific ECH/I polypeptides. [0089] Reagents and kits for performing RNA interference are available commercially from for
example Ambion® Inc. (Austin, TX, USA), New England Biolabs® Inc. (Beverly, MA, USA), Sigma- Aldrich® and Invitrogen® (Carlsbad, CA, USA).
[0090] The present inventors have shown that RhIZ is at least partially inhibited by a 2- bromooctanoate-derived metabolite. Accordingly, in another embodiment, the R-specific ECH/I inhibitor is an analog of 2-bromooctanoate, for example an enoate (e.g., a Ce to Ci6 enoate) substituted with one or more halogens.
Therapeutic applications
[0091] In view of the evidence that RLs can act as immunumodulators and virulence factors (reviewed in Abdel-Mawgoud et al., 2010), the inhibition of R-specific ECH/I, and consequently of RLs production, may be useful for the treatment of infection with RL-producing microorganisms. For example, RLs produced by P. aeruginosa are believed to be implicated in biofilm initiation (probably via modulation of cell surface polarity and hence initial cell attachment), in biofilm structure maturation, in biofilm dispersion and as a biofilm shield for immune-protection. Biofilm surface-associated RL layer is suggested to be formed in response to contact with immune cells. RL are also reported to slow tracheal ciliary function, and to disrupt the tight junctions of lung epithelial cells as a prelude to invasion. Furthermore, RLs have been shown to induce rapid necrosis of polymorphonuclear (PMN) leukocytes, to be cytolytic to macrophages or at least to inhibit their normal phagocytic response and to act as heat- stable hemolysins. Accordingly, in another aspect, the present invention provides a method for attenuating the virulence of a rhamnolipid-producing microorganism, said method comprising inhibiting (completely or partially) the expression and/or activity of one or more enoyl-CoA hydratases/isomerases (ECH/I) in said rhamnolipid-producing microorganism. In another aspect, the present invention also provides the use of an inhibitor of enoyl-CoA hydratases/isomerases (ECH/I) for attenuating the virulence of a rhamnolipid-producing microorganism.
[0092] For in vivo applications, the amount of the R-specific ECH/I inhibitor which is effective for decreasing the production of RLs and/or PHA, and/or for attenuating the virulence of a rhamnolipid- producing microorganism, will depend on several factors including the nature and severity of the disease, the chosen prophylactic/therapeutic regimen, the target site of action, the patient's weight, special diets being followed by the patient, concurrent medications being used, the administration route and other factors that will be recognized by those skilled in the art. The dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease, the liver function and different parameters from the patient. Typically, 0.001 to 1000 mg/kg of body weight/day will be
administered to the subject. In an embodiment, a daily dose range of about 0.01 mg/kg to about 500 mg/kg, in a further embodiment of about 0.1 mg/kg to about 200 mg/kg, in a further embodiment of about 1 mg/kg to about 100 mg/kg, in a further embodiment of about 10 mg/kg to about 50 mg/kg, may be used. The dose administered to a patient, in the context of the present invention should be sufficient to effect/induce a beneficial prophylactic and/or therapeutic response in the patient over time (decrease in bacterial load, reduction of tissue damage, reduction of one or more symptoms associated with the infection, increased survival time, etc.). The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration. Effective doses may be extrapolated from dose response curves derived from in vitro or animal model test systems. For example, in order to obtain an effective mg/kg dose for humans based on data generated from rat studies, the effective mg/kg dosage in rat may be divided by six.
[0093] In an embodiment, the above-mentioned treatment comprises the use/administration of more than one (i.e. a combination of) active/therapeutic agent, including the above-mentioned R-specific ECH/I inhibitor. The combination of prophylactic/therapeutic agents and/or compositions of the present invention may be administered or co-administered (e.g., consecutively, simultaneously, at different times) in any conventional dosage form. Co-administration in the context of the present invention refers to the administration of more than one therapeutic in the course of a coordinated treatment to achieve an improved clinical outcome. Such co-administration may also be coextensive, that is, occurring during overlapping periods of time. For example, a first agent may be administered to a patient before, concomitantly, before and after, or after a second active agent is administered. The agents may in an embodiment be combined/formulated in a single composition and thus administered at the same time. In an embodiment, the one or more active agent(s) is used/administered in combination with one or more agent(s) or treatment currently used to prevent or treat the disorder in question (e.g., agents or treatments currently used in the treatment of infections by rhamnolipid-producing microorganisms, such as P. aeruginosa.
[0094] The above-mentioned R-specific ECH/I inhibitor may be provided in a composition (e.g., a pharmaceutical composition) comprising a carrier, diluent and/or excipient (e.g., a pharmaceutically acceptable carrier, diluent and/or excipient). As used herein "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, buffers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier can be suitable, for example, for oral, intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal or pulmonary (e.g., aerosol) administration.
[0095] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art (Rowe et al., Handbook of pharmaceutical excipients, 2003, 4th edition, Pharmaceutical Press, London UK). Except insofar as any conventional media or agent is incompatible with the active agent, use thereof in the pharmaceutical compositions of the invention is contemplated.
Increasing the production of RLs and/or PHA
[0096] Biosurfactants are in demand for a wide range of industrial applications as they increase solubility, foaming capacity and lower surface tensions. Biosurfactants are attracting much attention because they represent ecological alternatives to their synthetic counterparts: they exhibit lower toxicity, potentially high activities, and stability at extremes of temperature, pH, and salinity. They have a wide variety of structures, and they can be produced from renewable feedstocks by a wide variety of microorganisms. Most importantly, they are biodegradable, making them environmentally friendly, "green" chemicals. In particular, RLs have been used in chemical and pharmaceutical industry or as emulsifier in cosmetics and foods.
[0097] PHAs are linear polyesters that are biodegradeable and are used in the production of bioplastics.
[0098] The demonstration by the present inventors that R-specific ECH/I are involved in the biosynthesis of RLs and PHA provides a novel approach for modulating the production of RLs and PHA through metabolic engineering.
[0099] Accordingly, in another aspect, the present invention provides a method for increasing the production of rhamnolipids by a host cell, said method comprising increasing the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I) in said host cell.
[00100] In another aspect, the present invention provides a method for increasing the production of PHA by a host cell, said method comprising increasing the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I) in said host cell.
[00101] Increasing the expression and/or activity of one or more R-specific ECH/I includes inducing the expression of one or more R-specific ECH/I in host cell that do not express one or more endogenous R-specific ECH/I (or express R-specific ECH/I inappropriate for rhamnolipid production), or increasing the expression of one or more R-specific ECH/I in host cell that endogenously expressed one
or more R-specific ECH/I. The expression may be induced/increased by either introducing a heterologous R-specific ECH/I nucleic acid into a host cell, or increasing/inducing the expression and/or activity of one or more endogenous R-specific ECH/I.
[00102] A host cell includes any suitable host cell that is capable of producing RLs and/or PHA (either endogenously or heterologously, i.e. following introduction of one or more genes into the host cell). Accordingly, the present invention envisages as a host cell, preferably a non-pathogenic host cell (e.g., non-pathogenic for humans) including a unicellular host cell such as a fungal host cell, for example, a yeast. The host cell may be a bacterial host cell including non-pathogenic bacterial host cells such as bacterial host cells capable of producing rhamnolipids. [00103] A host cell according to the invention includes an R-specific ECH/I gene (e.g., RhIK, RhIY and/or RhIZ or an ortholog/homolog thereof). The R-specific ECH/I gene may be under the control of a heterologous promoter. In some embodiments the R-specific ECH/I gene is an endogenous gene of the bacterial host cell. In some embodiments the R-specific ECH/I gene is a heterologous gene. The term "heterologous" refers to the relationship between two or more nucleic acid or protein sequences that are derived from different sources. For example, a promoter is heterologous with respect to a transcribable polynucleotide sequence if such a combination is not normally found in nature. In addition, a particular sequence may be "heterologous" with respect to a host cell in that it encodes a protein or is included in a protein, for example a recombinant protein, that is not normally expressed by the host cell. Such a heterologous protein accordingly generally is or has been inserted into the respective host cell, tissue, or species. Accordingly, a heterologous promoter is not normally coupled in vivo transcriptionally to the coding sequence of the R-specific ECH/I gene.
[00104] In an embodiment, the above-mentioned method comprises increasing the expression and/or activity of at least two R-specific ECH/I, preferably RhIY and RhIZ (or orthologs/homologs) thereof, in the cell. [00105] A wide variety of transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host, as long as RLs and/or PHAs are produced. The transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, cytomegalovirus or simian virus, where the regulatory signals are associated with a particular gene sequence which has a high level of expression. Alternatively, promoters from mammalian expression products, such as actin, collagen and myosin may be employed. Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so
that expression of the gene sequences can be modulated. Of interest may in some embodiments also be regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabolite) regulation. The term "regulatory sequence" includes controllable transcriptional promoters, operators, enhancers, silencers, transcriptional terminators, 5' and 3' untranslated regions which interact with host cellular proteins to carry out transcription and translation and other elements that may control gene expression including initiation and termination codons. The regulatory sequences can be native (homologous), or can be foreign (heterologous) to the cell and/or the nucleotide sequence that is used. The precise nature of the regulatory sequences needed for gene sequence expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation. Such regions will normally include those 5'-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence or CAAT sequence. These regulatory sequences are generally individually selected for a certain embodiment, for example for a certain cell to be used. The skilled artisan will be aware that proper expression in a prokaryotic cell also requires the presence of a ribosome-binding site upstream of the gene sequence-encoding sequence.
[00106] A vector may be employed which is capable of integrating the desired R-specific ECH/I gene sequences into the host cell chromosome. Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transformation or co-transfection. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.
[00107] The introduced nucleic acid molecule can be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
[00108] An expression vector, which may include one or more regulatory sequences and be capable of directing the expression of nucleic acids to which it is operably linked. An operable linkage is a linkage in which a coding nucleotide sequence of interest is linked to one or more regulatory sequence(s) such that expression of the nucleotide sequence sought to be expressed can be allowed. Thus, a regulatory sequence operably linked to a coding sequence is capable of effecting the expression of the coding sequence, for instance in an in vitro transcription/ translation system or in a cell when the vector is introduced into the cell. A respective regulatory sequence need not be contiguous with the coding sequence, as long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences may be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.
[00109] Once the vector or nucleic acid molecule that contains the construct(s) has been prepared for expression, the DNA construct(s) may be introduced into the host cell by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate-precipitation, direct microinjection, and the like. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells.
[00110] In view of the demonstration that the pool of precursors synthesized through the activity of R-specific ECH/I is shared between RLs and PHAs, it might be desirable to reduce the biosynthesis of PHAs to increase the production of RLs, and vice versa.
[00111] Reduction of the biosynthesis of PHAs to increase the production of RLs. Thus, in some embodiments the host cell according to the invention is able to produce PHA only to a much lower extent than a corresponding native, wild type host cell. The host cell may for example include a mutation in the gene of an enzyme that catalyzes the formation of PHA from the precursors, or a mutation in a regulatory sequence to which the respective gene is operably linked. The respective mutation may in some embodiments give rise to the formation of a polyhydroxyalkanoic acid synthase that has a reduced activity when compared to a corresponding wild type polyhydroxyalkanoic acid synthase. In some embodiments a capability of the host cell according to the invention to produce PHA is reduced in comparison to a wild type cell, including entirely absent. In some embodiments the host cell according to the invention is incapable of synthesizing PHA. The PHA biosynthetic pathway of the host cell may for example be curtailed, including inactive. The host cell may for example have one or more genes in the PHA synthesis pathway, such as polyhydroxyalkanoic acid synthase gene(s), that is/are disrupted. The
cell may for instance have a knock-out mutation in an endogenous polyhydroxyalkanoic acid synthase gene.
[00112] Reduction of the biosynthesis of RLs to increase the production of PHAs. In other embodiments, the host cell according to the invention is able to produce RLs to a lower extent than a corresponding native, wild-type host cell. The host cell may for example include a mutation in the gene of an enzyme that catalyzes the formation of RLs from the precursors (e.g., rhamnosyl-transferases such as RhIB and/or RhIC), or a mutation in a regulatory sequence to which the respective gene is operably linked. The respective mutation may in some embodiments give rise to the formation of rhamnosyl-transferases that have a reduced activity when compared to corresponding wild type rhamnosyl-transferases. In some embodiments a capability of the host cell according to the invention to produce RLs is reduced in comparison to a wild type cell, including entirely absent. In some embodiments the host cell according to the invention is incapable of synthesizing RLs. The RL biosynthetic pathway of the bacterial host cell may for example be curtailed, including inactive. The host cell may for example have one or more genes in the RL synthesis pathway, such as rhamnosyl- transferase gene(s), that is/are disrupted. The cell may for instance have a knock-out mutation in an endogenous rhamnosyl-transferase gene.
[00113] In embodiments, the method further comprises increasing the expression of one or more genes involved in RL and/or PHA biosynthesis. For example, to increase the production of RLs, the host cell may be modified or treated to produce higher levels of enzymes, e.g., RhIA, RhIB, and/or RhIC (e.g., SEQ ID NOs: 27, 29 and/or 31), or orthologs/homologs thereof (comprising a sequence having at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98 or 99% identity with the amino acid sequence depicted in SEQ ID NOs: 27, 29 and/or 31 , and exhibiting the same enzymatic activity). To increase the production of PHAs, the host cell may be modified or treated to increase higher levels of PHA synthases. In an embodiment, the method further comprises increasing the expression and/or activity of one or more additional proteins/enzymes involves in rhamnolipid production. In an embodiment, the method further comprises increasing the level of one or more substrates/precursors involved in rhamnolipid production.
[00114] A host cell, such as a bacterial host cell, as defined above may be used in a method of producing one or more RLs or PHAs. The host cell is cultured under conditions that allow RL and/or PHA production. A variety of carbon source may be used such as a monosaccharide, e.g. glucose, a disaccharide, e.g. sucrose, an alcohol, e.g. glycerol, an alkane, e.g., n-hexadecane, a fatty acid such as caprylic acid (also termed octanoate), vegetable oils (fresh or waste) or mixtures thereof. The bacterial host cell will typically be exposed to a fermentation process. The bacterial host cell may for instance be
in the logarithmic growth phase or in the stationary phase.
[00115] In the method the RL is recovered. Typically the RL is secreted by the bacterial host cell, so that recovering the fermentation/culture medium includes recovering the RLs. Further the method may include enriching, isolating and/or purifying the RLs. The term "enriched" means that the RLs constitute a significantly higher fraction of the total glycolipids, lipids and saccharides present in the solution of interest than in the solution from which it was taken. Enrichment may for instance include membrane filtration, for example for clarification, buffer exchange or concentration purposes. It may also include filtration or dialysis, which may for instance be directed at the removal of molecules below a certain molecular weight, or a precipitation by lowering the pH, or extraction using organic solvents. Chromatography may for example be carried out in the form of a liquid chromatography such as capillary electrochromatography, HPLC (high performance liquid chromatography) or UPLC (ultrahigh pressure liquid chromatography) or as a gas chromatography. The chromatography technique may be a process of column chromatography, of batch chromatography, of centrifugal chromatography or a method of expanded bed chromatography, as well as electrochromatographic, electrokinetic chromatography. It may be based on any underlying separation technique, such as adsorption chromatography, hydrophobic interaction chromatography or hydrophobic charge induction chromatography, size exclusion chromatography (also termed gel-filtration), ion exchange chromatography or affinity chromatography and may also be a method of capillary gas chromatography.
[00116] Methods for extracting RLs are well known in the art and are reviewed in Heyd, M. et al. (Development and trends of biosurfactant analysis and purification using rhamnolipids as an example. Anal Bioanal Chem 391 , 1579-1590 (2008)) and Abdel-Mawgoud et al. (2011 ). These methods range from those yielding mixtures of different RL congeners to those yielding specific congeners in pure forms. The criteria that govern the selection of a specific recovery method include: (1 ) the cost associated with the extraction method, which adds to the price of the final product, (2) the proposed purpose of the final product, which influences the level of purity required, and (3) the adaptability of the method to a particular industrial fermentation process. One of the simplest methods of recovery is by acid precipitation (Deziel et al. 1999b; Van Dyke et al. 1993; Zhang and Miller 1992) or aluminum sulfate precipitation (Schenk et al. 1995). Acid precipitation depends on acidification of RL to low pH (e.g., around 2), which neutralizes the negative charges on RLs, making them less soluble in the aqueous phase. Aluminum sulfate precipitates RLs by salting out. The precipitated RLs can then be recovered by centrifugation. Another more commonly used method is recovery by solvent extraction (Lepine et al. 2002; Mata-Sandoval et al. 1999; Schenk et al. 1995). In this method, molecules are precipitated by acidification and then extracted with organic solvents such as ether or ethyl acetate. Acidification is not
a critical step in this method, but it enhances the net yield (Heyd et al. 2008).
[00117] Other methods adapted to downstream processing in continuous fermentative production processes include: adsorption (Dubey et al. 2005), ion exchange chromatography (Abadi et al. 2009; Reiling et al. 1986; Schenk et al. 1995), ultrafiltration (Haussler et al. 1998; Mulligan and Gibbs 1990), and foam fractionation (Gruber 1991 ; Sarachat et al. 2010). Adsorption methods are based on the use of hydrophobic adsorbent such as Amberlite® XADO-2 or -16, polystyrene resin that retain hydrophobic (or amphiphilic) substances through hydrophobic interactions. Adsorbed RLs are then released by elution, e.g., with methanol. Ion exchange chromatography exploits the fact that RLs behave as anions at high pHs, which allows their retention on columns of weak anion exchange resins such as (diethylamino)ethyl-Sepharose®. RLs are released from these resins by adding at least 0.6 M NaCI to the equilibration buffer. Yet, this method has been improved by Abadi et al. (2009), who applied phospholipid-coated colloidal magnetic nanoparticles ion exchange media for the recovery and purification of RLs from culture mixtures. Ultrafiltration with a membrane cutoff of 10 kDa leads to an almost complete retention of RLs even at neutral pH. Foam fractionation depends on the foaming capabilities of RLs; the foam is directed out of the fermentation vessel to a fractionation column where it collapses in a separate receptacle by the action of acids or shear forces, The water in the film surface, known as the lamella, is then allowed to drain by gravitational force, causing a higher concentration of the surfactant in the collapsed foam (Heyd et al. 2008; Sarachat et al. 2010).
[00118] Most of the aforementioned methods result in the recovery of mixtures of different RLs congeners. Alternatively, chromatographic methods are usually the best solutions for separation of specific RL congeners in a pure form. These methods, however, work better after application of one of the extraction methods mentioned above. On the small scale, preparative TLC is a good choice (Monteiro et al. 2007; Sim et al. 1997); however, for large scale downstream processing, preparative column chromatography using silica gel is a better option (Burger et al. 1966; Monteiro et al. 2007). Recrystallization or repurification using TLC can be applied, if necessary (Heyd et al. 2008)
[00119] In an embodiment, the method comprises solvent extraction.
[00120] An isolation may include the combination of different methods.
Screening assays for modulators of RL and/or PHA production
[00121] The present inventors have identified R-specific enoyl-CoA hydratases/isomerases (ECH/I) as being involved in the biosynthesis of RLs and/or PHAs.
[00122] Accordingly, in another aspect, the present invention also provides a method (screening assay), in an embodiment an in vitro method, for determining whether an agent may be useful for (i) inhibiting the production of RLs and/or PHAs by a host cell, such as a rhamnolipid- and/or PHA- producing microorganism and/or (ii) attenuating the virulence of a rhamnolipid-producing microorganism, said method comprising determining whether said agent inhibits the expression and/or activity of one or more R-specific ECH/I. In an embodiment, the method comprises (a) contacting said agent with said one or more R-specific ECH/I and (b) determining whether said agent inhibits the expression and/or activity of said one or more R-specific ECH/I, wherein the inhibition of said expression and/or activity is indicative that said agent may be useful for (i) inhibiting the production of RLs and/or PHAs by the host cell and/or (ii) attenuating the virulence of a rhamnolipid-producing microorganism.
[00123] The present invention also provides a method (screening assay), in an embodiment an in vitro method, for determining whether an agent may be useful for increasing the production of RLs and/or PHAs by a host cell, such as a rhamnolipid- and/or PHA-producing microorganism, said method comprising determining whether said agent increases the expression and/or activity of one or more R- specific ECH/I. In an embodiment, the method comprises (a) contacting said agent with said one or more R-specific ECH/I and (b) determining whether said agent increases the expression and/or activity of said one or more R-specific ECH/I, wherein the increase of said expression and/or activity is indicative that said agent may be useful for increasing the production of RLs and/or PHAs by the host cell. [00124] The determination of the expression and/or activity of R-specific ECH/I in the presence (and/or absence) of the test compound may be performed using any known methods to detect nucleic acids or proteins. In embodiments, the expression or activity measured in the presence of the test compound is compared to a control or reference level (e.g., the level obtained in the absence of the test compound) to assess whether the test compound modulates (increases or decreases) the expression or activity of R-specific ECH/I .
[00125] The levels of nucleic acid corresponding to an R-specific ECH/I can then be evaluated according to the methods disclosed below, e.g., with or without the use of nucleic acid amplification methods. In some embodiments, nucleic acid amplification methods can be used to detect the level of expression of an R-specific ECH/I. For example, the oligonucleotide primers and probes may be used in amplification and detection methods that use nucleic acid substrates isolated by any of a variety of well- known and established methodologies (e.g., Sambrook et al., Molecular Cloning, A laboratory Manual, pp. 7.37-7.57 (2nd ed., 1989); Lin et al., in Diagnostic Molecular Microbiology, Principles and
Applications, pp. 605-16 (Persing et al., eds. (1993); Ausubel et al., Current Protocols in Molecular Biology (2001 and later updates thereto)). Methods for amplifying nucleic acids include, but are not limited to, for example the polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) (see e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188), ligase chain reaction (LCR) (see, e.g., Weiss, Science 254: 1292-93 (1991)), strand displacement amplification (SDA) (see e.g., Walker et al, Proc. Natl. Acad. Sci. USA 89:392-396 (1992); U.S. Pat. Nos. 5,270,184 and 5,455,166), Thermophilic SDA (tSDA) (see e.g., European Pat. No. 0 684 315) and methods described in U.S. Pat. No. 5,130,238; Lizardi et al., BioTechnol. 6:1197-1202 (1988); Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173-77 (1989); Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-78 (1990); U.S. Pat. Nos. 5,480,784; 5,399,491 ; U.S. Publication No. 2006/46265. The methods include the use of Transcription Mediated Amplification (TMA), which employs an RNA polymerase to produce multiple RNA transcripts of a target region (see, e.g., U.S. Pat. Nos. 5,480,784; 5,399,491 and US Publication No. 2006/46265).
[00126] The nucleic acid or amplification product may be detected or quantified by hybridizing a labeled probe to a portion of the R-specific ECH/I nucleic acid or amplified product. The labeled probe contains a detectable group that may be, for example, a fluorescent moiety, chemiluminescent moiety, radioisotope, biotin, avidin, enzyme, enzyme substrate, or other reactive group. Other well-known detection techniques include, for example, gel filtration, gel electrophoresis and visualization of the amplicons, and High Performance Liquid Chromatography (HPLC). In certain embodiments, for example using real-time TMA or real-time PCR, the level of amplified product is detected as the product accumulates.
[00127] In another embodiment, the expression and/or activity of an R-specific ECH/I is measured at the protein level. Methods to measure the amount/level of proteins are well known in the art. Protein levels may be detected directly using a ligand binding specifically to the protein, such as an antibody or a fragment thereof. In embodiments, such a binding molecule or reagent (e.g., antibody) is labeled/conjugated, e.g., radio-labeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled to facilitate detection and quantification of the complex (direct detection). Alternatively, protein levels may be detected indirectly, using a binding molecule or reagent, followed by the detection of the [protein/ binding molecule or reagent] complex using a second ligand (or second binding molecule) specifically recognizing the binding molecule or reagent (indirect detection). Such a second ligand may be radio- labeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled to facilitate detection and quantification of the complex. Enzymes used for labeling antibodies for immunoassays are known in the art, and the most widely used are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Examples of binding molecules or reagents include antibodies (monoclonal or polyclonal), natural or
synthetic R-specific ECH/I ligands, glycoproteins, monosaccharides (e.g., galactose, galactosamine, lactose) aptamers and the like.
[00128] Examples of methods to measure the amount/level of protein in a sample include, but are not limited to: Western blot, immunoblot, enzyme-linked immunosorbent assay (ELISA), "sandwich" immunoassays, radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance (SPR), chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical (IHC) analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microarray, antibody array, microscopy (e.g., electron microscopy), flow cytometry, proteomic-based assays, and assays based on a property of the protein including but not limited to ligand binding or interaction with other protein partners, enzymatic activity, fluorescence, etc. For example, the level or activity of R-specific ECH/I may be determined by measuring the level of conversion of a substrate of an R-specific ECH/I (e.g., a suitable enoyl-CoA substrate such as trans-2- decenoyl-CoA) into a product (an R-hydroxyfatty acid such as 3-hydroxydecanoate) (see below).
[00129] In another embodiment, the effect of the test agent on the activity/expression of R- specific ECH/I may be assessed using a reporter gene/protein system. For example, a reporter gene may be put under the transcriptional control of a transcriptional control element (e.g., promoter) normally associated with an R-specific ECH/I, and the expression of the reporter protein may be measured to determine if the test agent could modulate the transcription of the R-specific ECH/I. If in the presence of the test agent the level of the reporter protein is increased, it is an indication that the test agent may be useful to increase the expression of the R-specific ECH/I (as the reporter protein is under the transcriptional control of a transcriptional control element normally associated with the the R-specific ECH/I. The term "reporter gene" refers to gene encoding a protein (a reporter protein) that can be detected (e.g., by fluorescence, spectroscopy, luminometry, etc.) easily and that is not present normally (endogenously) in the system used. Commonly used reporter proteins include enzymes such as β- galactosidase (encoded by the bacterial gene /acZ), luciferase, chloramphenicol acetyltransferase (CAT; from bacteria), GUS (β-glucuronidase), bioluminescent proteins and fluorescent proteins. The method to determine the readout signal from the reporter protein depends from the nature of the reporter protein. For example, for fluorescent reporter proteins, the readout signal corresponds to the intensity of the fluorescent signal. The readout signal may be measured using spectroscopy-, fluorometry-, photometry- , and/or luminometry-based methods and detection systems, for example. Such methods and detection systems are well known in the art.
[00130] In other embodiments, the determination of the effect of the test compound on the
expression and/or activity of R-specific ECH/I may be performed indirectly, for example by assessing the production of RLs and/or PHAs by a cell (e.g., using the methods described in the Examples below). In embodiments, the production of RLs and/or PHAs in the presence of the test compound is compared to a control or reference level (e.g., the production measured in the absence of the test compound) to assess whether the test compound modulates (increases or decreases) the expression or activity of R- specific ECH/I . Higher levels/production of RLs and/or PHAs in the presence of the test compound being indicative that the test compound increases the expression or activity of R-specific ECH/I and vice-versa.
[00131] The method/assay may be a cell-free method/assay or a cell-based method/assay. For the cell-based assay, any cell capable of expressing the R-specific ECH/I nucleic acid and protein (or the reporter gene construct described above) may be used in the method/system of the invention. In an embodiment, the above-mentioned cell is a bacterial cell . The cell may be naturally expressing the R- specific ECH/I protein, or may be prepared by introducing a nucleic acid encoding the R-specific ECH/I or a reporter gene construct (by any transfection, transduction or transformation method), and providing conditions suitable for the expression of the protein . Methods and systems for introducing a nucleic acid into a cell are well known in the art, and include for example conjugation, chemical-based transfection (using calcium phosphate, liposomes, cationic polymers such as DEAE-dextran or polyethylenimine), electroporation, gene gun, viral transduction. Kits for introducing a nucleic acid into a cell are commercially available. The assay may be performed using a native, full-length R-specific ECH/I, or an active variant or fragment thereof.
[00132] Test compounds (drug candidates) that may be screened by the method/system of the invention may be obtained from any number of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means.
[00133] Screening assay systems may comprise a variety of means to enable and optimize useful assay conditions. Such means may include but are not limited to: suitable buffer solutions, for example, for the control of pH and ionic strength and to provide any necessary components for optimal activity and stability (e.g. , protease inhibitors), temperature control means for optimal activity and/or stability of R-specific ECH/I, and detection means to enable the detection of its activity. A variety of such
detection means may be used, including but not limited to one or a combination of the following: radiolabelling, antibody-based detection, fluorescence, chemiluminescence, spectroscopic methods (e.g., generation of a product with altered spectroscopic properties), various reporter enzymes or proteins (e.g., horseradish peroxidase, green fluorescent protein), specific binding reagents (e.g., biotin/(strept)avidin), and others.
[00134] The present invention is illustrated in further details by the following non-limiting examples.
Example 1 : Materials and Methods
[00135] Bacterial strains and growth conditions. [00136] Bacterial strains and plasmids are described in Table 1 .
Table 1 : Bacterial strains and plasmids used in the experiments described herein
[00137] Seed cultures of P. aeruginosa PA14 in Tryptic Soy Broth (TSB, Difco®) incubated overnight at 37°C under rotation were used to inoculate mineral salts medium (MSM) at a starting Οϋβοο of 0.1 . MSM has the following composition: Na2HP04 (0.9 g/L), KH2P04 (0.7 g/L), CaCI2'2H20 (0.1 g/L), MgS04 H20 (0.4 g/L), NaN03 (2 g/L), tryptone (1 g/L), trace element solution (TES, 2 ml/L), pH adjusted to 7. Composition of TES is: FeS04 H20 (2 g/L), MnS04'H20 (1 .5 g/L), (ΝΗ4)6Μο7024·4Η20 (0.6 g/L). Glycerol (20 g/L) was used as the carbon source unless otherwise stated. Tryptone was omitted when fatty acid carbon sources were tested. Inoculated MSM medium was incubated at 34°C under rotation.
[00138] P. aeruginosa was grown on LB agar (NaCI 5 g/L) at 37°C for mating purposes and for selection of transformants after mating or electroporation. It was also grown in LB-no salt (LBNS, with
no NaCI) broth at 30°C for resolution of merodiploids in mutant constructions. Antibiotics were added to maintain or select for plasmids as follows: for £ coli, carbenicillin (Cb) at 100 g/mL, gentamicin (Gm) at 30 μg/mL, tetracycline (Tc) at 15 μg/mL, kanamycin (Km) at 50 g/ml; for P. aeruginosa, carbenicillin (Cb) at 300 μg/mL, Gm at 50 μg/mL, and Tc at 125 μg/mL for solid media and at 75 μg/mL for liquid media. Triclosan, at 10 μg/mL, was used to counter select E. coli.
[00139] Genes mutation and cloning.
[00140] The P. aeruginosa strain PA14 deletion markerless mutants were constructed by allelic replacement using Gateway technology as described previously (Choi and Schweizer, 2005), with some modifications. [00141] To generate the ArhIZ knockout, the following two pairs of primers, PA14-54660-UpF- GWL and PA14-54660-UpR-Gm with PA14-54660-DnF-Gm and PA14-54660-DnR-GWR, were used to amplify the upstream (5') and the downstream (3') fragments, respectively. After construction of the destination vector pEX18ApGW-APA14_54660::Gm-FRT, it was transformed into E. coli DH5a- subcloning efficiency generating E. coli DH5a-pEX18ApGW- APA14_54660::Gm-FRT. Plasmids from correct DH5a clones, growing on Gm (30 μg/mL) but neither on Km (50 g/ml) nor Cb 100 μg/mL, were then transformed by conjugation into strain PA14 on LB agar using triparental mating with the help of DH5a(pRK2013) or diparental mating using E. coli SM10 Apir carrying the plasmid. Conjugation spots were spread onto LB agar containing Gm (50 g/ml) and triclosan (10 g/ml) to select for transconjugants of PA14 that had undergone the first homologous allelic replacement. Merodiploids were resolved by growing three transconjugant colonies in LBNS broth overnight, dilutions (usually 1 CH, 10"2) of which were plated onto LBNS agar containing Gm (50 g/ml) and sucrose 10% (w/v). Upon replica plating, positive clones, that underwent the second recombination, were those growing on LB agar Gm (50 g/ml but not on Cb (300 g/mL). The Gm resistance cassette was subsequently excised from produced mutants APA14_54660::Gm-FRT (ArWZ::Gm-FRT), by Flp recombinase. This was performed by conjugation of ArWZ: :Gm-FRT mutant with £ coli SM10(pFLP2). PA14 mutant clones that had lost the Gm cassette were recovered by plating dilutions, up to 10"4, of suspended conjugation spots onto LB agar containing Cb (300 μg/mL) and Tr (10 g/ml) to eliminate the £ coli. Growing clones were replica plated onto LBNS agar containing sucrose 10% (w/v) to eliminate the pFLP2 plasmid. Growing clones were further confirmed by replica plating onto LB agar containing Cb (250 μg/mL) and on LB agar containing Gm (50 g/ml) onto which correct clones should not grow and the markerless t rhll (ΔΡΑ14_54660) mutant is generated. The chromosomal deletion was finally confirmed by sequencing after amplification of the mutated region using the two extremities primers, PA14-54660-
UpF-GWL and PA14-54660-DnR-GWR. Similarly, the double knockout mutant hrhlYZ (ΔΡΑ14_54640- ΔΡΑ14_54660) was constructed using the PA14-54640-UpF-GWL and PA14-54640-UpR-Gm primers for amplification of the upstream fragment in combination with PA14-54660-DnF-Gm and PA14-54660- DnR-GWR for the amplification of the downstream fragment. [00142] The knockout mutant AphaG (ΔΡΑ14_54830, ortholog of phaG of P. putida KT2440 (Rehm et al., 1998)) was constructed using phaG-UpF-GWL and phaG-UpR-Gm primers for the amplification of the upstream fragment in combination with phaG-DnF-Gm and phaG-DnR-GWR primers for the amplification of the downstream fragment.
[00143] The multiple knockout mutant (ApftaCf, AphaD, kphaC2, kphaD, kphaF, t phaf) was constructed using the PHA-UpF-GWL and PHA-UpR-Gm primers to amplify the upstream fragment and PHA-DnF-Gm and PHA-DnR-GWL primers to amplify the downstream fragment. The plasmid pUCP26-rWZ was constructed by PCR amplification of PA14_54660 from wild type PA14 using RhlZ-F- EcoRI and RWZ-R-Hindlll primers including the region from -57 bp (with the native RBS) to 84 bp downstream to the stop codon of RhIZ. The EcoRI-Hindlll digested fragment was cloned into the EcoRI- Hindlll-digested pUCP26 vector.
[00144] The expression vector pET28a(+)-RWZ was constructed by PCR amplification of PA14_54660 from the wild-type PA14 using RWZ-F-Ndel and RWZ-R-Hindlll primers. The double digested fragment was cloned into the Ndel-Hindlll digested pET28a(+) to yield the inducible RhIZ expression 56 vector pET28a(+)-RWZ with the His-tag at the N-terminal of RhIZ. The plasmid was transformed in thermocompetent E.coli BL21 (DE3). Similar steps were followed to construct the expression vector pET28a(+)-RWY with an N59 terminal His-tag, by cloning the gene PA14_54640 amplified using RWY-F-Ndel and RWY-R-Hindlll primers.
[00145] Plasmid DNA and PCR products were sequenced by the DNA Sequencing and Gene Analysis Center at the Genome Quebec, Montreal, CA. The plasmids and PCR primers used are listed in Tables 1 and 2, respectively.
Table 2: PCR primers* used in the experiments described herein
Name Sequence (5'-3') SEQ ID NO:
PA14_54660-UpF-GWL TACAAAAAAGCAGGCTctggtcgaacaggtggtg 7
PA14_54660-UpR-Gm TCAGAGCGCTTTTGAAGCTAATTCGtcttcgaaaagcacgttca 8
PA14_54660-DnF-Gm AGGAACTTCAAGATCCCCAATTCGctgatcgatcgcgacaac 9
PA14_54660-DnR-GWR TACAAGAAAGCTGGGTgaagacgttcagcaggtagc 10
PA14_54640-UpF-GWL TACAAAAAAGCAGGCTg acatgctcaccg aactca 1 1
PA14_54640-UpR-Gm TCAGAGCGCTTTTGAAGCTAATTCGcatgaagtccctcactggat 12 phaG-UpF-GWL TACAAAAAAGCAGGCTctttcctgcccggccaat 13 phaG-UpR-Gm TCAGAGCGCTTTTGAAGCTAATTCGgatgatggtctgctgcgcc 14 phaG-DnF-Gm AGGAACTTCAAGATCCCCAATTCGcggcgcggaatttcacac 15 phaG-DnR-GWR T AC AAG AAAG CTGGGTccattatacg acg cccg etc 16
PHA-UpF-GWL TACAAAAAAGCAGGCTtcaaaagaacaataacgagctt 17
PHA-UpR-Gm TCAGAGCGCTTTTGAAGCTAATTCGgtcagcaggttgatgacg 18
PHA-DnF-Gm AGGAACTTCAAGATCCCCAATTCGgtatgcctatccggttcaa 19
PHA-DnR-GWR TACAAGAAAGCTGGGTcgtttccgaatccacttc 20 rhIZ-F-EcoRI CGrAGAATTCATGAACGTGCTTTTCGAA 21 rhIZ-R-Hindlll ACCCAAGCTTGAAACAGCTTAGACGCTATG 22 rhIZ-F-Ndel CACACA CAT ATGAACGTGCTTTTCGAAGAA 23 rhlY-F-Ndel CACACA CAT ATGAACACTGCCGTCGAAC 24 rhIY-R-Hindlll ACCCAAGCTTTCAGCAGTTGCGCCACTT 25
* Small letter nucleotides are the gene specific sequences for generation of knockout mutants. Underlined nucleotides refer to corresponding restriction sites preceded by a number of base pairs for efficient cleavage as recommended by supplier (New England BioLabs®, Inc).
[00146] Protein expression and purification
[00147] The expression of recombinant RhIZ or RhIY proteins with an N-terminal 6xHis-tag encoded by pET28a(+)-RWZ was induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) in a fresh subculture (starting Οϋβοο of 0.05) of E. coli BL21 (DE3) when the Οϋβοο = 0.8. Incubation was then continued at 34°C under rotation of 150 rpm for 2-3 hours. Cells were then collected by centrifugation, resuspended in the binding buffer (20 mM NaP04, pH 7.4, 500 mM NaCI, 25 mM imidazole, 20 mM β-mercaptoethanol) freshly supplemented with Mini Protease Inhibitor Cocktail® Tablets (Roche) and 0.5 mM phenylmethanesulfonylfluoride (PMSF) and lysed by sonication for 9 cycles each of 30 seconds interrupted by 30 seconds of rest on ice. Each cell lysates were clarified by centrifugation at 10,000 x g at 4°C for 30-45 min and then applied onto a preconditioned Ni Sepharose® High Performance column (HisTrapHP®-5ml, GE) using Fast Protein Liquid Chromatography® (AKTA® FPLC, GE). Preconditioning of the column was achieved according to instructions of the manufacturer and flow rate was adjusted at 5 ml/min. The column was washed with five column volumes of binding buffer then a linear gradient was initiated. For RhIZ purification, the gradient was from 0% to 50% elution buffer (20 mM NaP04, pH 7.4, 500 mM NaCI, 500 mM imidazole, 20 mM β-mercaptoethanol) over twenty column volumes. For RhIY, the gradient was from 0% to 100% elution buffer over thirty column volumes. Fractions of 5 ml were collected throughout the chromatography. RhIZ started to elute out of the column at 130 mM, peaked at 160 mM and tailed at 190 mM imidazole (FIG. 14A), RhIY started to elute at 170 mM, peaked at 240 mM and tailed at 335 mM imidazole (FIG. 14B). Fractions of these peaks and flanking fractions were applied to 12% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Appearing bands were excised for identification via protein sequencing at
the Proteomic services of INRS-lnstitut Armand Frappier (Canada). Fractions containing pure proteins were pooled together and concentrated with Amicon® Ultra-15 (30 kDa, Millipore®) for RhIZ and Amicon® Ultra-15 (15 kDa) for RhIY, and washed with 900 volumes of buffer (50 mM Tris-CI, pH 7.7, 1 mM EDTA, 10 mM β-mercaptoethanol, 0.5 mM PMSF). The Amicon®-concentrated protein solutions were assayed using Bradford method (protein concentration was 8 mg/ml), then aliquoted and stored at -80°C. Intact protein masses were determined by mass spectrometry (Quattro Premier® XE, Micromass®) equipped with an electrospray ionization interface at the positive mode using direct infusion of purified proteins at 800 mg/L that were diluted and washed in 50 mM ammonium carbonate solution. The obtained spectra were integrated using the MaxEnt 1 option of MassLynx™ software. Intact masses of purified proteins were 31 ,933 and 42,648 Da positively identifying the proteins as His- tagged RhIY and RhIZ, respectively, both lacking their N-terminal fMet amino acid.
[00148] Clarified cell lysate of WT PA14 strain and of the mutants ΔΑΛ/Ζ and RhlYZ were prepared similarly from cells collected from a 25-ml fresh cultures in TSB broth that were then washed and sonicated in lysis buffer (50 mM Tris-CI, pH 7.7, 1 mM EDTA, 10 mM β-mercaptoethanol, 0.5 mM PMSF) (Davis et al., 2008) to be used freshly or kept at 4°C until use.
[00149] Synthesis of trans-2-decenoyl-CoA and chiral HPLC-MS/MS assay of hydratase.
[00150] Trans-2-decenoyl-Co was synthesized using the mixed anhydride method (Abdel- Mawgoud et al., 2013). An amount of 2-4 μg of pure enzyme and/or 20 μΙ of clarified cell lysates was used for the activity assays. Enzymatic reaction set up and enzyme product assay using chiral HPLC- MS/MS were conducted as described by Abdel-Mawgoud et al. (2013).
[00151] Chromatographic analysis of rhamnolipids, 3-hydroxy fatty acids and PHA.
[00152] RL were analyzed using a high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) as described previously (Deziel et al., 1999). 3-hydroxy fatty acids were analyzed using gas chromatography-mass spectrometry (GC/MS). PHA was analyzed by the GC/MS estimation of 3-hydroxy fatty acids pool as well as by estimation of the trans-sectional area of PHA granules as visualized by transmission electron microscope.
[00153] HPLC-MS analysis and identification of rhamnolipids. HPLC/MS analysis of RL was performed as reported previously (Deziel et al., 1999) with some modifications. RL containing culture broths were centrifuged at 10,000 g for 10 min to prepare cell free supernatants. An aliquot of 250 μί of such supernatant was kept at -20°C until the day of analysis. In HPLC vials, 25 μΙ of such supernatant
was diluted to 500 μΙ of acetonitrile:water (70:30) to which 2-heptyl-3-hydroxyquinoline-d4 (HHQ-d4) was added at 5.6 mg/L as internal standard (Dubeau et al., 2009). Samples were analyzed using an HPLC (Waters® 2795, Alliance® HT) equipped with a C8 reversed phase column (Eclipse® XDB-C8, particle size of 5 μιτι, and of 4.6 x 150 mm dimensions, Agilent®, USA). The detector was a triple tandem quadrupole mass spectrometer (q MS/MS, Quattro Premier® XE, Micromass®) equipped with an electrospray ionization interface. All rhamnolipids were detected and quantified in the negative ion mode generating [M-H]~ pseudomolecular ions. Data were processed by MassLynx® software with its QuanLynx® function (version 4.1 , Waters®).
[00154] HPLC separation of RLs was performed with a sample injection volume of 20 μΙ_ and mobile phase at a flow rate of 0.4 ml/min with a gradient of water (A):acetonitrile (B), both containing 2 mM ammonium acetate and adjusted to pH 8. The gradient was set according to the following profile: 0 min, 30% A + 70% B; 1 min, 25% A + 75% B; 10 min, 100% B; 16 min, 100% B; 17 min, 30% A + 70% B; 20 min, 30% A + 70% B. The mobile phase flow was split through a Valco® Tee with 10% of the flow introduced into the mass spectrometer. The mass spectrometer was in the negative ESI mode with capillary voltage set at 3.0 kV, cone voltage at 20 V, source and desolvation temperatures of 120 and 150°C respectively, cone and desolvation gas (N2) flow of 15 and 100 L/Hr, respectively. The scanning mass range was from 130 to 800 Da. Quantification was performed by comparing integration data of the pseudomolecular ion [M-H]~ peak areas of different RLs homologues with those of the internal standard using response factors previously determined through calibration curves made with pure mono-RL and pure di-RL. Collision-induced dissociation (CID) experiments for fragmentation of parent molecular ion were performed using argon as the collision gas at a pressure of 3.28 X 10-3 87 mBar, corresponding to a flow of 0.35 mL/min. The scanning mass range for daughter ions was from 100 to 530 Da for mono-RL and from 100 to 650 Da for di-RL. Collision energy was adjusted at 20 eV for both molecules.
[00155] GC/MS analysis of 3-hydroxy fatty acids and PHA. For the analysis of total 3-hydroxy fatty acids, 3 ml of whole culture broths grown in MSM-glycerol were collected in screw-capped glass tubes to which nonanoic acid (Sigma®, USA) was added as the internal standard at 15 mg/L. These broths were then extracted with 3 ml ethyl acetate and vortexed for 10 seconds. Two ml of the organic phase was collected in small glass vials and evaporated to dryness under a stream of nitrogen gas. Residues were derivatized by silylation using BSTFA (/V, 0-bis(trimethylsilyl) trifluoroacetamide); Supelco®, PA, USA) in a total reaction volume of 100 μΙ, composed of 25 μΙ BSTFA and 75 μΙ acetonitrile, each added freshly. The reaction was incubated at 70°C for 30 min, then cooled to room temperature and diluted to 1000 μΙ with acetonitrile prior to injection into the gas chromatograph (see below). This way, the final concentrations of 3-hydroxy fatty acids as well as internal standard are twice
higher than their original concentrations in the culture broths.
[00156] Similar steps were followed for the estimation of PHA (Costa et al., 2009), with some modifications. A triplicate of two batches of washed cell pellets recovered from 4 ml of 50-60 hours old cultures of P. aeruginosa in MSM-glycerol medium grown under rotation at 34°C were prepared. Cell pellets of first batch, intended for free intracellular fatty acid analysis, were suspended in 1 ml dhbO and extracted with 1 ml CHC while vortexing for 15 min. Cell pellets of the second batch, intended for analysis of total (free and hydrolyzed) fatty acids including PHA, were suspended in 1 ml 15% H2SO4 and similarly extracted with CHCI3 while heating at 100°C for 140 min to hydrolyze PHA. CHCI3 layers were collected and evaporated to dryness before derivatization by silylation as above. The values of free intracellular 3-hydroxyfatty acids obtained from the first batch (were 10-fold lower) were subtracted from those of the second batch to obtain the concentration of PHA. Analysis of the trimethylsilyl derivatives of the 3-hydroxyfatty acid was performed by gas chromatography/mass spectrometry (GC/MS) (Trace GC Ultra® - Polaris® Q, Thermo Fisher Scientific®, Waltham, MA) equipped with a ZB- 5MS (ZebronTM1 13) capillary column (30 m X 0.25 mm internal diameter, 0.25 μΜ film thickness, Phenomenex®) using the electron impact ionization mode. The GC program started at an oven temperature of 70°C that was increased at 30°C/min to 110°C and held at 1 10°C for 5 min, then increased at 20°C/min to 165°C and held at 165°C for 5 min, then increased at 30°C/min to 310°C and held for 0.5 min with an overall oven run time of 19.42 min. The sample injection volume was 1 μΙ_. The Polaris Q MS was operated in full scan positive ion mode covering the mass range of 70-400 with a total scan time of 0.41 sec. MS scan start time was at 6.6 min and the ion source temperature was at 250°C. The injector and detector (ion source) temperatures were 200°C and 250°C, respectively. Helium was used as the carrier gas at a constant flow rate of 1.1 ml/min. Quantification was performed by comparing integration data of Μ·+ 122 fragment ions resulting from the electron impact of the trimethylsilyl esters of 3-hydroxy fatty acids with those of the internal standard using response factors previously determined through calibration curves made with pure standards. The experiments were conducted in triplicate.
[00157] Electron microscopy for PHA granules visualization and measurement. A volume of 1 ml of culture of cells grown in MSM-glycerol was sampled after 50 hours (Οϋβοο = 3.0) of incubation at 34°C under rotation. Cells, recovered by centrifugation at 5,000 x g for 5 minutes, were fixed using 2.5% gluteraldehyde in phosphate buffer saline (PBS) for 1 hour then washed three times with 3% sucrose in PBS and kept at 4°C overnight. Staining was performed with 1 .3% osmium tetroxide in collidine buffer. Fixed stained material was dehydrated in solutions with progressively increasing concentrations of acetone (25-100%). The material was kept overnight in a 1 :1 volume mixture of Spurr resin and acetone, then immersed twice, each 1 hour, in a bath of Spurr mixtures. The block containing fixed cells
was cut into small pieces, placed in BEEM capsules, filled with Spurr resin and held at room temperature for 16 hours and then polymerized at 60°C for 20-30 hours. Ultrathin sections (70-100 nm thick) were examined by a Hitachi H-7100 electron microscope with an accelerating voltage of 75 kV. Estimation of PHA production was made using the photos obtained from the electron microscope and the area measurement tools of Adobe Photoshop® (version CS3 extended).
Example 2: Inhibition of β-oxidation decreases RLs production
[00158] The implication of β-oxidation on RLs biosynthesis in P. aeruginosa was investigated using specific β-oxidation inhibitors. This was conducted in a mineral salts medium (MSM) using glycerol as sole carbon source and not a fatty acid source naturally demanding β-oxidation for assimilation and growth. The β-oxidation inhibitors tested were acrylic, 4-pentenoic and 2- bromooctanoic acids. Strikingly, at this carbon source condition (glycerol), 2-bromooctanoic acid (2-Br- Cs) reduced RLs production by 50% and the lipid precursor HAA by 80% (FIG. 8A), whereas the other inhibitors had no effect (FIG. 8B).
[00159] To explore the specificity of 2-Br-Cs in RLs inhibition, the dose-dependency of 2-Br-Cs- mediated inhibition of RLs production was examined. Moreover, the chain length specificity of this inhibitory activity was also studied using shorter (2-Br-C4 and 2-ΒΓ-ΟΘ) and longer (2-Br-Ci2) homologues at equivalent molar concentrations. The results show that 2-Br-Cs inhibits RLs and HAA production in a dose-dependent manner (FIG. 1A). Surprisingly, 2-ΒΓ-ΟΘ (5 mM), not previously reported as a β-oxidation inhibitor, also inhibits RLs production to the same extent as 2-Br-Cs (5 mM) (FIG. 1 B). The other congeners, 2-Br-C4 and 2-Br-Ci2, caused considerable growth inhibition down to 3 mM.
[00160] Besides acting as an inhibitor of β-oxidation (Raaka and Lowenstein, 1979), 2-Br-Cs was previously hypothesized to inhibit PhaG, which transacylates the FAS II intermediate R-3-hydroxyacyl- ACP to the CoA counterpart for polyhydroxyalkanoates (PHA) biosynthesis (Lee et al., 2001). To rule out the hypothesis that PhaG product could act as precursor for RL biosynthesis and that the inhibition of RLs production by 2-Br-Cs is caused by its effect on PhaG (FIG. 9A), RL production was tested in a phaG knockout as well as a transposon insertion phaG mutant. Neither of the two mutants affects RL production (FIG. 9B), excluding a role of PhaG in the 2-Br-Cs-mediated reduction in RL production.
Example 3: Contribution of β-oxidation as source of lipid precursors for RLs
[00161] To further evaluate the relative contribution of β-oxidation versus FASII in RL synthesis, PA14 were cultivated in MSM using dodecanoic-12,12,12-d3 acid as sole carbon source. Implication of
either pathway was traced and estimated via examination of the pattern and extent of labeling of the HAA (C10-C10) moiety of RLs produced. If RLs are labeled with 3 extra Daltons per chain, this would entail that decanoic-12, 12,12-d3 intermediate of β-oxidation is incorporated as the lipid chains of RLs (FIG. 10A). Alternatively, if de novo fatty acid synthesis (FASH) is predominantly implicated, produced RLs would be mostly unlabeled as its chains would be de novo synthesized from the more abundant unlabeled acetyl-CoA (five unlabeled versus one labeled) units released after complete β-oxidation of dodecanoic-12,12,12-d3 acid (FIG. 10A).
[00162] Strikingly, the RLs produced, which consisted solely of mono-RLs, were almost exclusively (> 99%) deuterium-labeled with 88% having an extra mass of 6 Da, ([M-H]~ m/z 509) and 12% with an extra mass of 3 Da, ([M-H]~ m/z 506). Tandem MS/MS fragmentation of m/z 509 confirmed that the extra 6 Da were distributed equally, 3 Da per chain (FIG. 10B) and thus corresponding to Rha- Cio(d3)-Cio-(d3). This clearly demonstrates that β-oxidation is the main supplier of the lipid precursor of RLs when fatty acids are the sole carbon source.
Example 4: β-oxidation is directly linked to RLs pathway [00163] Using perdeuterooctadecanoic acid (Ci8-d3s) and glucose as co-substrate, Zhang et al. (2012) suggested that the fatty acid substrates of RLs are shortened via β-oxidation to the Cs chain length after which one C2-elongation step via FASII takes place before diversion of corresponding FAS II Cio-intermediate to RLs biosynthesis (Zhang et al., 2012). However, our isotope tracing experiments using dodecanoic-12,12,12-d3 acid are suggesting instead a direct link between β-oxidation and RL biosynthesis. We thus verified the contribution of the hypothesis Zhang et al. in the supply of RLs lipid precursors relative to β-oxidation. We fed PA14 with pandeuterododecanoic acid (C12-CI23) as sole carbon source. If β-oxidation is solely implicated, each 3-hydroxydecanoate chain of RLs would be labeled with an extra 17 Da (FIG. 11 A). On the other hand, if FAS II is implicated according to the model of Zhang et al., the fatty acid chain of RL would bear extra 16 Da (67%) or 15 Da (33%) as the main species (FIG. 11 B).
[00164] Interestingly, using C12-CI23 as the sole carbon source generated of 3-hydroxydecanoate bearing +17 Da as the species of the highest abundance, while those bearing +16 Da and +15 Da only represented 43% and 26% of the intensity of the +17 Da species, respectively. The abundances of the +16 Da and +15 Da species relative to each other were 62% and 38% respectively, close to theoretical prediction mentioned above (FIG. 12A). These results demonstrate that, when using dodecanoic acid as sole carbon source, the majority (-60%) of the RLs lipidic precursors is directly supplied from β-
oxidation without resorting to a bypass through FASH.
[00165] To further investigate the hypothesis of Zhang et al., C12- J23 (3.75 g/L) was provided together with glucose (10 g/L) as co-carbon sources. Unexpectedly, due to a possible hydrogen- deuterium isotopic negative metabolic effect, glucose was preferentially utilized resulting in the formation of almost exclusively unlabeled 3-hydroxydecanoate (-95%) (FIG. 12B). To overcome this effect, the bacterium were fed with an excess of glucose-13C6 (15 g/L) (83 mM) together with dodecanoate-12,12,12-d3 (30 mM) or tetradecanoate-14,14,14-d3 (30 mM). Under these conditions, if β- oxidation is solely involved, the lipid chains of RLs would bear an extra 3 Da each. However, if FASII is implicated in the C2-elongation step (mostly with labeled 13C2) of the Cs-d3 intermediate according to the Zhang et al. hypothesis, the lipid chains of RLs would bear an extra 5 Da instead. The 2 extra Da in this case arises from the FASII-mediated condensation of 13C2-labelled malonate units mostly originating from glucose-13C6. Strikingly, under these conditions 3-hydroxydecanoate predominantly bore an extra 3 Da only, and minor amounts with extra 4 Da or 5 Da labeling (FIG. 12C, D).
[00166] Taken together, these results indicate that, whether fatty acids are provided alone or as co-carbon sources with glucose, β-oxidation remains the main direct supplier of lipid precursors for RLs biosynthesis.
Example 5: Enoyl-CoA intermediates of β-oxidation are diverted to RLs biosynthesis
[00167] Involvement of β-oxidation implicates that one of its intermediates, namely, trans-2-enoyl- CoA, S-3-hydroxyacyl-CoA or 3-ketoacyl-CoA, is converted via an enoyl-CoA hydratase, an epimerase, or a 3-ketoacyl-CoA reductase, respectively, into R-3-hydroxyfatty acids, the ultimate lipid precursor of RLs. To figure out this β-oxidation intermediate, RLs production was examined in mutants defective in enzymes carrying out these conversions.
[00168] A survey of the P. aeruginosa PA14 genome (Winsor et al., 2011 ) for genes that might code for such enzymes revealed fourteen loci predicted to encode enoyl-CoA hydratases (ECH) (Table 3), while no predicted epimerases or 3-ketoacyl-CoA reductases were found. The polyhydroxyalkanoate (PHA)-related ECH, PhaJ1 -4 (Tsuge et al., 2003), was also included in the survey. RLs productions in transposon (Tn) or knockout (KO) mutants of 12 of these 14 ECH-coding loci were tested. Transposon mutants of phaJ2 and phaJ4 had no negative effect on RLs production (FIG. 13A). Interestingly, three putative enoyl-CoA hydratases/isomerases (ECH/I) mutants, PA14_40980 (Tn), PA14_54640 (Tn), and PA14_54660 (KO) were associated with remarkable 35%, 56% and 54% reductions in RLs productions, respectively (FIG. 13B). Because of their impact on RL synthesis, they were respectively named RhIK,
RhIY and RhIZ.
Table 3: Predicted enoyl-CoA hydratases (crotonases) expressing genes and orthologues oi phaJ genes having crotonoase like activity in P. aeruginosa PA14 and the availability of their transposon
mutants.
PA14 locus tag PA01 locus tag Transposon mutant available (Liberati et al.,
2006)
PA14 17850 PA3591 No
PA14J 9740 PA3426 Yes
PA14 26690 PA2890 Yes
PA14_27360 PA2841 Yes
PA14 28310 PA2767 Yes
PA14_40980 PA1821 Yes
PA14 41950 PA1748 No
PA14_43440 PA1629 Yes
PA14_48200 PA1240 Yes
PA14 51 1 10 PA1021 Yes
PA14_54640 PA0745 Yes
PA14 54660 PA0744 No**
PA14_56250 PA4330 Yes
PA14 65840 PA4980 No
PA14 21310 PA3302* No
PA14_51 160 PA101 8* Yes
PA14 63290 PA4788* No
PA14 1 1910 PA401 5* Yes
* PA3302, PA1 018, PA4788 and PA4015 are reported in Pseudomonas aeruginosa as PHA-related hydratases; PhaJ 1 , PhaJ2, PhaJ3 and PhaJ4, respectively.
** A knockout deletion mutant of this gene was prepared in this study.
[00169] RhIY and RhIZ were selected for further studies, as their corresponding mutants were the most affected in RL production. These two genes are arranged in a single computationally predicted operon with RhIZ downstream to RhIY (Winsor et al., 2011 ). To exclude the possibility that the RL phenotype associated with the RhIY transposon mutant (Tn) is ultimately caused by a polar effect on its downstream gene RhIZ, a double RhIYZ knockout mutant (KO) was constructed and its RLs production was compared to that of the simple RhIZ knockout mutant. In addition, RhIZ was expressed in trans from a multicopy plasmid in both mutants. Interestingly, the double knockout did not cause further decrease in RLs production, and complementation with RhIZ only partially complemented RLs production in this double mutant (FIG. 2A). Moreover, overexpression of RhIZ in the WT did not increase RLs production (FIG. 13C). Collectively, these results demonstrate a direct role for RhIZ in RLs production and, also that RhIY cooperates with RhIZ to attain native level of RLs production.
Example 6: The ECH/I RhIY and RhIZ ar main suppliers of 3-hydroxydecanoate/octanoate pools
[00170] To experimentally confirm the putative ECH/I role assigned to RhIY and RhIZ
production of the 3-hydroxyalkanoate precursors of RLs, the total intra- and extracellular pool of 3-OH- Cio±2 was compared in the mutants and WT (FIG. 2B). In agreement with their putative function, both the single LRhlZ and more markedly the double LRhlYZ knockouts presented a reduction of 3-OH- Cio±2, more pronouncedly of 3-OH-Cio species followed by 3-OH-Cs then 3-OH-Ci2 (FIG. 2B). This significant decrease in 3-hydroxy fatty acids precursors correlates with the RLs reduction in these mutants (FIG. 2A). Moreover, these results demonstrate a metabolic role for RhIYiRhIZ in the biosynthesis of 3-OH-alkanoates in P. aeruginosa in general. Again, RhIZ complementation in the double mutant only partially restored the WT level of 3-hydroxyfatty acid, supporting the hypothesis that RhIY cooperates with RhIZ to attain the WT level of 3-hydroxyfatty acids precursors and hence RLs. Example 7: RhIZ is an R-specific ECH/I
[00171] It was hypothesized that RhIZ is an R-specific ECH/I (R-ECH/I) converting a 2-decenoyl- CoA intermediate of β-oxidation into the corresponding R-3-hydroxydecanoyl-CoA precursor of RL (FIG. 9A). This was verified in vitro using purified RhIZ (FIG. 3) and trans-2-decenoyl-CoA as substrate. The relative proportion of the R- and S-stereoisomers of 3-hydroxydecanoyl-CoA (3-OH-Cio-CoA) produced was estimated using a chiral HPLC-MS/MS method.
[00172] Purified RhIZ alone did not catalyze the formation of 3-hydroxydecanoate from trans-2- decenoate. However, clarified cell lysate of WT PA14 strain did convert trans-2-decenoate to 3- hydroxydecanoate at 0.53 mg/L (SD: ± 0.016) where the R-isomer was produced at 0.3 mg/L (SD: ± 0.015) and the S-isomer at 0.23 mg/L (SD: ± 0.012), i.e. at nearly racemic mixture of enantiomers (FIGs. 4 and 3B). Interestingly, combination of purified RhIZ with a clarified WT cell lysate caused a nearly 1 .5-fold increase in the total yield of 3-hydroxydecanoate (0.75 mg/L, SD: ± 0.003) of which the R-isomer (0.61 mg/L, SD: ± 0.004) was at least 4-fold more abundant than the S-isomer (0.14 mg/L, SD: ± 0.007) (FIGs. 4 and 3A). This demonstrates that RhIZ is indeed an R-ECH/I, but that a factor present in the WT cell lysate is involved in its activity. It was hypothesized that this factor is a protein since heat treatment of the clarified cell lysate abolished RhIZ activity with no any 3-hydroxydecanoate produced. According to the previous data (FIG. 2), this heat-sensitive factor is likely RhIY. To verify this, purified RhIZ was combined with the clarified cell lysates of t RhlYZ or t RhlZ. Consistent with the hypothesis that the heat-sensitive factor is RhIY, the R-3-OH-Cio enrichment was conserved when purified RhIZ was combined with the lysate of t RhiZ (FIG. 3C, D), whereas, no such enrichment was observed when this purified RhIZ was combined with the lysate of the double mutant t RhlYZ (FIG. 10E, F).
[00173] The dependency of RhIZ on RhIY suggested a sort of interaction or complex formation. This was verified by a pull-down experiment using purified hexahistidine-tagged RhIZ (RWZ-His6) incubated with the clarified total cell lysate of WT. RWZ-His6 pulled RhIY from the lysate (FIG. 5), entailing a stable RhIY-RhIZ physical interaction between these two proteins. [00174] Taken together, these results indicate that RhIZ is an R-specific ECH/I that is functional in vivo in the presence of RhIY with which it forms a stable complex.
Example 8: Rh/Z-associated R-specific enrichment is inhibited by 2-bromooctanoate
[00175] As shown above, treatment with 2-Br-Cs or inactivation of RhIZ decreases RLs production. In order to verify if RhIZ is one of the targets of this β-oxidation inhibitor, 3 mM of 2-Br-Cs was directly added to ECH assay buffer containing RhIZ in combination with clarified lysate of WT. Under these conditions, the RWZ-associated R-specific enrichment was not inhibited (FIG. 4). However, because previous reports suggested that the active form of this inhibitor could be one of its metabolites (Raaka and Lowenstein, 1979), ECH assays were conducted using clarified lysate of WT cells cultivated in the presence of 2-Br-Cs (5 mM) and compared this to the control lysate of cells cultivated in the absence of the inhibitor, both in combination with purified RhIZ. Using lysate of cells cultivated in the presence of 2-Br-Cs, supposed to contain the active metabolite of this inhibitor, the RWZ-mediated enrichment of the R-isomer was lost and its abundance approached that when using the control lysate alone (FIG. 4). This indicates that RhIZ is at least partially inhibited by a 2-bromooctanoate-derived metabolite. Example 9: R-3-hydroxyacyl-CoA pool is shared between RLs and polyhydroxyalkanoates pathways
[00176] Because R-3-hydroxyacyl-CoA are also the precursors of polyhydroxyalkanoates (PHA) (Madison and Huisman, 1999; Wang et al., 2012), the hypothesis that the lipid precursors supplied by RhIYZ is shared by PHA and RLs was tested. The impact of RhIYZ double mutation on the production of PHA was first examined (FIG. 6A). Second, PHA production was directly assessed by transmission electron microscopy using the percentage cross-sectional area of intracellular PHA granules (FIGs. 6B, D). A consistent 25% reduction in PHA was found in the RhIYZ mutant (FIGs. 6A, B) entailing a shared precursor pool. Third, it was verified whether this pool is competitively shared between PHA and RLs by studying the effect of blocking PHA biosynthesis on RLs production. To do so, most of PHA-biosynthetic genes, namely t phaC1DC2DFI {hpha), were knocked out. This mutation resulted in an increase in mono-RL (Rha-Cio-Cio) accompanied with a small decrease in di-RL (Rha-Rha-Cio-Cio) that together
resulted in a 13% increase in total RLs compared to the WT (FIG. 6C). This is explained by the accumulation of the shared lipid precursor pool in the PHA-negative mutant that becomes more available for RL biosynthesis. Assuming a fixed rhamnose pool, the consequent high lipid to rhamnose precursors availability, led to this remarkable doubling in mono-RL congener of high lipid to rhamnose content at the expense of di-RL having lower lipid to rhamnose content. It thus appears that the fixed rhamnose precursor pool limited a more striking increase in total RL.
[00177] The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.
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Claims
1 . A method for decreasing the production of rhamnolipids (RLs) by a microorganism, said method comprising inhibiting the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I) in said microorganism.
2. A method for attenuating the virulence of a rhamnolipid-producing microorganism, said method comprising inhibiting the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I) in said rhamnolipid-producing microorganism.
3. The method of claim 1 or 2, wherein said inhibiting comprises contacting said microorganism with an inhibitor of said one or more R-specific ECH/I.
4. The method of claim 1 or 2, wherein said inhibitor is an inactive or dominant-negative form of said one or more R-specific ECH/I.
5. The method of claim 1 or 2, wherein said inhibitor is an RNA interference agent targeting a nucleic acid encoding said one or more ECH/I.
6. The method of claim 5, wherein said RNA interference agent is a shRNA or siRNA.
7. The method of any one of claims 1 to 6, wherein said one or more R-specific ECH/I comprise a sequence having at least 50% identity with the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:6.
8. The method of claim 7, wherein said one or more R-specific ECH/I comprise a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:6.
9. The method of claim 8, wherein said one or more R-specific ECH/I comprise the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:6.
10. The method of any one of claims 1 to 9, wherein said method is an in vitro method.
1 1. The method of any one of claims 1 to 9, wherein said method is an in vivo method.
12. A method for increasing the production of rhamnolipids (RLs) by a microorganism, said method comprising increasing the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I) in said microorganism.
13. The method of claim 12, wherein said method comprises introducing into said microorganism one or more nucleic acids encoding said one or more R-specific ECH/I.
14. The method of claim 12 or 13, wherein said method further comprises introducing one or more nucleic acids encoding one or more polypeptides involved in the RL biosynthetic pathway.
15. The method of claim 14, wherein said one or more polypeptides involved in the RL biosynthetic pathway are rhamnosyl-transferases.
16. The method of claim 15, wherein said encoding one or more rhamnosyl-transferases are RhIB and/or RhIC.
17. The method of any one of claims 13 to 16, wherein said one or more nucleic acids encodes the R-specific ECH/I defined in any one of claims 7 to 9.
18. The method of claim 17, wherein said one or more nucleic acids comprise a sequence having at least 50% identity with the nucleotide sequence of SEQ ID NO:1 , SEQ ID NO:3 and/or SEQ ID NO:5.
19. The method of claim 18, wherein said one or more nucleic acids comprise a sequence having at least 90% identity with the nucleotide sequence of SEQ ID NO:1 , SEQ ID NO:3 and/or SEQ ID NO:5.
20. The method of claim 19, wherein said one or more nucleic acids comprise the nucleotide sequence of SEQ ID NO:1 , SEQ ID NO:3 and/or SEQ ID NO:5.
21. A method for determining whether an agent may be useful for (i) inhibiting the production of rhamnolipids (RLs) by a microorganism and/or (ii) attenuating the virulence of a rhamnolipid-producing microorganism, said method comprising determining whether said agent inhibits the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I).
22. The method of claim 21 , comprising (a) contacting said agent with said one or more R-specific ECH/I and (b) determining whether said agent inhibits the expression and/or activity of said one or more R-specific ECH/I, wherein the inhibition of said expression and/or activity is indicative that said agent may be useful for (i) inhibiting the production of RLs by a microorganism and/or (ii) attenuating the virulence of a rhamnolipid-producing microorganism.
23. A method for determining whether an agent may be useful for increasing the production of rhamnolipids (RLs) by a microorganism, said method comprising determining whether said agent
increases the expression and/or activity of one or more R-specific enoyl-CoA hydratases/isomerases (ECH/I).
24. The method of claim 23, comprising (a) contacting said agent with said one or more R-specific ECH/I and (b) determining whether said agent increases the expression and/or activity of said one or more R-specific ECH/I, wherein the increase of said expression and/or activity is indicative that said agent may be useful for increasing the production of rhamnolipids (RLs) by a microorganism.
25. The method of any one of claims 21 to 24, wherein said method is a cell-free method.
26. The method of any one of claims 21 to 24, wherein said one or more R-specific ECH/I are expressed in a cell.
27. The method of any one of claims 21 to 26, wherein said one or more R-specific ECH/I comprise a sequence having at least 50% identity with the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:6.
28. The method of claim 27, wherein said one or more R-specific ECH/I comprise a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:6.
29. The method of claim 28, wherein said one or more R-specific ECH/I comprise the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:6.
30. The method of any one of claims 1 to 29, wherein said microorganism is of the Pseudomonas genus.
31. The method of claim 30, wherein said microorganism is Pseudomonas aeruginosa.
32. Use of an inhibitor of R-specific enoyl-CoA hydratases/isomerases (ECH/I) for decreasing the production of rhamnolipids (RLs) by a microorganism.
33. Use of an inhibitor of R-specific enoyl-CoA hydratases/isomerases (ECH/I) for the preparation of a medicament for decreasing the production of rhamnolipids (RLs) by a microorganism.
34. Use of an inhibitor of R-specific enoyl-CoA hydratases/isomerases (ECH/I) for attenuating the virulence of a rhamnolipid-producing microorganism.
35. Use of an inhibitor of R-specific enoyl-CoA hydratases/isomerases (ECH/I) for the preparation of a medicament attenuating the virulence of a rhamnolipid-producing microorganism.
36. The use of any one of claims 31 to 35, wherein said inhibitor is the inhibitor defined in any one of claims 4 to 6.
37. The use of any one of claims 32 to 36, wherein said one or more R-specific ECH/I comprise a sequence having at least 50% identity with the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:6.
38. The use of claim 37, wherein said one or more R-specific ECH/I comprise a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:6.
39. The use of claim 38, wherein said one or more R-specific ECH/I comprise the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:6.
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WO2017150849A1 (en) * | 2016-03-03 | 2017-09-08 | 아이진 주식회사 | Attenuated pseudomonas aeruginosa improved in productivity and safety and vaccine composition comprising same |
US9884883B2 (en) | 2015-01-12 | 2018-02-06 | Logos Technologies, Llc | Production of rhamnolipid compositions |
US10829507B2 (en) | 2017-02-06 | 2020-11-10 | Stepan Company | Decolorization of concentrated rhamnolipid composition |
-
2014
- 2014-11-05 CA CA2934340A patent/CA2934340A1/en not_active Abandoned
- 2014-11-05 WO PCT/CA2014/051060 patent/WO2015085411A1/en active Application Filing
Non-Patent Citations (2)
Title |
---|
ABDEL-MAWGOUD, A.M. ET AL.: "A stereospecific pathway diverts beta-oxidation intermediates to the biosynthesis of rhamnolipid biosurfactants''.", CHEMISTRY & BIOLOGY, vol. 21, 16 January 2014 (2014-01-16), pages 156 - 164 * |
ABDEL-MAWGOUD, A.M. ET AL.: "Rhamnolipids: diversity of structures, microbial origins and roles''.", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY., vol. 86, 2010, pages 1323 - 1336, XP019800002 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9884883B2 (en) | 2015-01-12 | 2018-02-06 | Logos Technologies, Llc | Production of rhamnolipid compositions |
WO2017150849A1 (en) * | 2016-03-03 | 2017-09-08 | 아이진 주식회사 | Attenuated pseudomonas aeruginosa improved in productivity and safety and vaccine composition comprising same |
US10829507B2 (en) | 2017-02-06 | 2020-11-10 | Stepan Company | Decolorization of concentrated rhamnolipid composition |
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CA2934340A1 (en) | 2015-06-18 |
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