WO2010068953A2 - Green process and compositions for producing poly(5hv) and 5 carbon chemicals - Google Patents

Green process and compositions for producing poly(5hv) and 5 carbon chemicals Download PDF

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Publication number
WO2010068953A2
WO2010068953A2 PCT/US2009/067943 US2009067943W WO2010068953A2 WO 2010068953 A2 WO2010068953 A2 WO 2010068953A2 US 2009067943 W US2009067943 W US 2009067943W WO 2010068953 A2 WO2010068953 A2 WO 2010068953A2
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WIPO (PCT)
Prior art keywords
recombinant organism
lysine
glutarate
coa
organism
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PCT/US2009/067943
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French (fr)
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WO2010068953A3 (en
Inventor
William R. Farmer
Jeff Bickmeier
Chenfeng Lu
Dong-Eun Chang
Frank Skraly
Thomas Martin Ramseier
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Metabolix Inc.
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Application filed by Metabolix Inc. filed Critical Metabolix Inc.
Priority to EP09793403.8A priority Critical patent/EP2376622B1/en
Priority to KR1020177033765A priority patent/KR101886012B1/en
Priority to BRPI0922410-6A priority patent/BRPI0922410B1/en
Priority to ES09793403.8T priority patent/ES2639413T3/en
Priority to KR1020167004466A priority patent/KR20160025043A/en
Priority to JP2011540960A priority patent/JP5770099B2/en
Priority to CN2009801568724A priority patent/CN102317437B/en
Priority to MYPI2011700085A priority patent/MY183971A/en
Publication of WO2010068953A2 publication Critical patent/WO2010068953A2/en
Publication of WO2010068953A3 publication Critical patent/WO2010068953A3/en

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Definitions

  • the invention is generally related to transgenic organisms that produce polyhydroxyalkanoates and co-polymers thereof containing 5- hydroxyvalerate and chemical intermediates containing five carbon atoms (C5 chemicals).
  • PHAs are biodegradable plastics which can be used to make, without limitation, films (e.g., packaging films, agricultural films, mulch film), golf tees, caps and closures, agricultural supports and stakes, paper and board coatings (e.g., for cups, plates, boxes, etc), thermoformed products (e.g., trays, containers, yogurt pots, plant pots, noodle bowls, moldings, etc.), housings (e.g., for electronic items) bags (e.g., trash bags, grocery bags, food bags, compost bags, etc.), hygiene articles (e.g., diapers, feminine hygiene products, incontinence products, disposable wipes, etc.) and coatings for pelleted products (e.g., pelleted fertilizer, herbicides, pesticides, seeds, etc.).
  • films e.g., packaging films, agricultural films, mulch film
  • golf tees, caps and closures e.g., for cups, plates, boxes, etc
  • thermoformed products e
  • PHAs have also been used to develop biomedical devices including sutures, repair devices, repair patches, slings, cardiovascular patches, orthopedic pins, adhesion barriers, stents, guided tissue repair/regeneration devices, articular cartilage repair devices, nerve guides, tendon repair devices, bone marrow scaffolds, and wound dressings.
  • Polyhydroxyalkanoates can be produced by a fermentation process.
  • Existing fermentation methods for producing polyhydroxyalkanoates utilize wild-type or transgenic microorganisms cultured on specific substrates to produce the desired PHA polymer composition.
  • the polymers of interest are copolymers of the (D)-isomer of 3-hydroxybutyrate copolymerized with one other 3, 4 or 5-hydroxyacids. These copolymers are produced as granular inclusions inside the cells and are random copolymers.
  • the copolymer poly(3-hydroxybutyrate-co-5-hydroxyvalerate) (PHB5HV) and the homopolymer poly(5-hydroxyvalerate) (P5HV) are industrially useful as materials and plastics with the advantage that they are biodegradable and bioresorbable materials.
  • Suitable renewable resources include carbohydrate feedstocks available from agriculture including one or more feedstocks selected from: starch, sucrose, glucose, lactose, fructose, xylose, maltose, arabinose and amino acid feedstocks including lysine and proline.
  • a further object of the invention is to provide techniques and procedures to stably engineer recombinant organisms that synthesize PHAs containing 5 -hydroxy valerate either as sole constituent or as a co-monomer. It is another object of the invention to provide techniques and procedures to stably engineer recombinant organisms that synthesize 5 carbon chemicals such as 5-aminopentanoate (5AP), glutarate, and 1,5 pentanediol (PDO).
  • 5AP 5-aminopentanoate
  • glutarate glutarate
  • PDO pentanediol
  • PHAs polyhydroxyalkanoates
  • 5HV 5 -hydroxy valerate
  • PHAs polyhydroxyalkanoates
  • Certain recombinant hosts that produce 5 carbon chemicals such as 5 ⁇ aminopentanoate (5AP), 5HV, glutarate, and 1,5 pentanediol (PDO) are also provided.
  • 5AP 5 ⁇ aminopentanoate
  • 5HV glutarate
  • PDO pentanediol
  • One embodiment provides a recombinant host expressing genes encoding a polyhydroxyalkanoate (PHA) synthase and a 5-hydroxyvalerate- CoA (5HV-CoA) transferase or 5HV-CoA synthetase and at least one transgene encoding a heterologous enzyme involved in lysine catabolic pathways wherein the host produces a PHA polymer containing 5HV monomers when the organism is provided with a renewable carbon substrate selected from: lysine, starch, sucrose, glucose, lactose, frucrose, xylose, maltose, arabinose or combinations thereof and the level of 5HV monomer produced is higher than in the absence of expression of said transgene(s).
  • An exemplary host expresses one or more genes encoding lysine 2- monooxygenase, 5-aminopentanamidase, 5-aminopetanoate transaminase, glutarate semialdehyde reductase, 5 -hydroxy valerate CoA-transferase, and polyhydroxyalkanoate synthase to produce a PHA polymer containing 5HV monomers.
  • the host has deletions or mutations in genes encoding glutarate semialdehyde dehydrogenase and/or lysine exporter encoding genes.
  • Particularly suitable hosts also have the ability to overproduce lysine and are resistant to toxic lysine analogs, like S ⁇ (2-aminoethyl) cysteine.
  • one or more of the genes encoding PHA synthase, 5HV-CoA transferase or 5HV-CoA synthetase is also expressed from a transgene.
  • the recombinant organism is fed lysine in combination with one or more renewable carbon substrates selected from: starch, sucrose, glucose, lactose, frucrose, xylose, maltose, arabinose or combinations thereof such that the 5HV containing PHA polymer is produced and the polymer is recovered from the cells.
  • the recombinant organism is fed one or more renewable carbon substrates selected from: starch, sucrose, glucose, lactose, fructose, xylose, maltose, arabinose or combinations thereof such that the 5HV containing PHA polymer is produced and the polymer is recovered from the cells.
  • the polymers produced by the recombinant hosts can be homopolymers or copolymers of 5HV monomers.
  • a preferred copolymer is PHB5HV.
  • Other useful polymers produced by the recombinant hosts are the copolymers poly(3-hydroxypropionate-co-5-hydroxyvalerate) and poly(4- hydroxybutyrate-co-5-hydroxyvalerate) and the homopolymer P5HV.
  • the host can be prokaryotic or eukaryotic.
  • Preferred prokaryotic hosts are E. coli, Ralstonia eutropha, Alcaligenes lotus and C, glutamicum.
  • Recombinant hosts for producing PHA polymers from lysine, or one or more renewable carbon substrate selected from starch, sucrose, glucose, lactose, fructose, xylose, maltose, arabinose or or a combinations thereof are also provided.
  • An exemplary host expresses lysine 2-monooxygenase, 5- aminopentanamidase, 5-aminopetanoate transaminase, glutarate semialdehyde reductase, 5 -hydroxy valerate CoA-transferase, and polyhydroxyalkanoate synthase to produce a polymer including 5HV.
  • the polymer is produced using lysine and one or more renewable carbon substrate selected from starch, sucrose, glucose, lactose, fructose, xylose, maltose and arabinose as a feedstock.
  • the host has deletions in glutarate semialdehyde dehydrogenase and lysine export encoding genes.
  • 1,5-PentanedioI Production Another recombinant host is genetically engineered to overexpress 5- hydroxyvalerate CoA transferase, CoA dependent propionaldehyde dehydrogenase, and 1,3 -propanediol dehydrogenase to produce 1,5 pentanediol.
  • 1,5 pentaned ⁇ ol is produced using 5 -hydroxy valerate, lysine starch, sucrose, glucose, lactose, nestrose, xylose, maltose and arabinose alone or in combinations as feedstock.
  • the recombinant host has deletions in adhE, idhA, and ackA-pta and expresses lysine 2- monooxygenase, 5-amino ⁇ entanamidase, 5-aminopetanoate transaminase and one or more glutarate or succinate semialdehyde reductase encoding genes.
  • Particularly suitable hosts have the ability to overproduce lysine and are resistant to toxic lysine analogs, like S-(2-aminoethyl) cysteine.
  • the organism has a reduced or no glutarate semialdehyde dehydrogenase activity.
  • a method for producing 1,5-pentanediol from renewable carbon substrates where a recombinant organism is fed a renewable carbon substrate and 1,5-pentanediol is produced, secreted to the medium and recovered therefrom.
  • the invention provides 1,5-pentandiol produced from renewable resources. Glutaric Acid Production
  • Recombinant hosts for overproducing glutarate (glutaric acid) from lysine, or one or more renewable carbon substrate selected from starch, sucrose, glucose, lactose, fructose, xylose, maltose and arabinose, or a combination thereof are also provided.
  • An exemplary host expresses lysine 2-monooxygenase, 5-aminopentanamtdase, 5-aminopetanoate transaminase and one or more glutarate semialdehyde dehydrogenase encoding genes.
  • Particularly suitable hosts have the ability to overproduce lysine and are resistant to toxic lysine analogs, like S-(2-aminoethyl) cysteine,
  • a method for overproducing glutarate from renewable carbon substrates where a recombinant organism is fed a renewable carbon substrate selected from lysine, starch, sucrose, glucose, lactose, frucrose, xylose, maltose and arabinose, or combinations thereof and glutarate is overproduced, secreted to the medium and recovered therefrom.
  • a renewable carbon substrate selected from lysine, starch, sucrose, glucose, lactose, frucrose, xylose, maltose and arabinose, or combinations thereof.
  • FIG. 1 shows enzymes catalyzing the biosynthetic reactions: (1) lysine 2- monooxygenase, EC 1.13.12.2; (2) 5-aminopentanamidase (a.k.a.
  • Figure 3 is a schematic diagram showing biochemical pathways of an alternate route from L-lysine to 5-aminopentanoate via cadaverine and 5- aminopentanal.
  • Figure 4 is a schematic diagram showing biochemical pathways from
  • Figure 5 is a schematic diagram showing biochemical pathways from alpha-ketoglutarate to glutarate semialdehyde, a metabolic intermediate to produce 5 -hydroxy valerate and its derivatives, and glutarate.
  • Figure 6 is a schematic diagram showing biochemical pathways from oxaloacetate, an intermediate of the TCA cycle, to L-lysine.
  • Figure 7 is a schematic diagram showing biochemical pathways to 1,5 pentanediol.
  • Figure 8A is chromatogram showing time (minutes) versus total ion abundance of processed cell culture from strain 3291.
  • Figure 8B is an ion spectrum of processed cell culture from strain 3291 showing mass-to-charge ratio "m/z" versus its ion abundance (relative units).
  • PHA copolymer refers to a polymer composed of at least two different hydroxy alkanoic acid monomers.
  • PHA homopolymer refers to a polymer that is composed of a single hydroxyalkanoic acid monomer.
  • a "vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.
  • the vectors can be expression vectors.
  • an "expression vector” is a vector that includes one or more expression control sequences.
  • an "expression control sequence” is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence.
  • operably linked means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest.
  • transformed and transfected encompass the introduction of a nucleic acid into a cell by a number of techniques known in the art.
  • Plasmids are designated by a lower case “p” preceded and/or followed by capital letters and/or numbers. As used herein the term “heterologous” means from another host.
  • the other host can be the same or different species.
  • Recombinant organisms having enzymes for the biochemical pathways for production of 5 -hydroxy valerate containing polyhydroxyalkanoate polymers, and 5-carbon chemicals such as 5- aminopentanoate (5AP), 5-hydroxyvalerate (5HV), glutarate, and 1,5 pentanediol (PDO) are provided.
  • Prokaryotic or eukaryotic hosts are genetically engineered to express the enzymes needed to produce 5- hydroxy valerate, polymers thereof, or the disclosed 5 -carbon chemicals from renewable resource based feedstocks. The enzymatic pathways for producing the desired products are provided below.
  • 5-aminopentanoate 5-aminopentanoate can be produced in two enzymatic steps from L-lysine, an ⁇ -amino acid, with 5-aminopentanamide as the intermediate ( Figure 2).
  • the first of these enzymes, lysine 2-monooxygenase is the first enzymatic step in the lysine degradation pathway of various pseudomonad strains (Takeda and Hayaishi, J. Biol. Chem. 241 :2733-2736; (1966); Hayaishi, Bacteriol. Rev. 30:720-731 (1966); Reitz and Rodwell, J. Biol Chem. 245: 3091-3096 (1970); Flashner and Massey, J. Biol.
  • an alternate pathway can be utilized to convert L-iysine to 5 AP in three enzymatic reactions which include a lysine decarboxylase to produce cadaverine, a putrescine transaminase to form 5- aminopentenal, and a ⁇ -aminobutyraldehyde dehydrogenase to biosynthesize 5AP.
  • 5AP may also be produced from L-proline, instead of L-lysine, in two enzymatic reactions which include a proline racemase to biosynthesize D-proline and a proline reductase to form 5 AP.
  • Biosynthesis of another 5-carbon chemical, 5HV can occur from 5AP with 2 enzymatic steps as outlined in Figure 2.
  • 5AP is converted to glutarate semialdehyde by 5AP transaminase (Reitz and Rodwell, J. Biol. Chem. 245: 3091-3096 (1970)) and a gene from P. putida which was identified and named davT (Espinosa-Urgel and Ramos, Appl. Environ. Microbiol. 67:5219-5224 (2001)).
  • 5AP transaminase Reitz and Rodwell, J. Biol. Chem. 245: 3091-3096 (1970)
  • davT Espinosa-Urgel and Ramos, Appl. Environ. Microbiol. 67:5219-5224 (2001)
  • ATEG_00539 was discovered to efficiently catalyze this reaction and was hence renamed gsaR for glutarate semialdehyde reductase
  • thermophilic bacteria and lower fungi including yeasts lysine is synthesized via the ⁇ -aminoketoadipate pathway (Xu, Cell Biochem. Biophys. 46:43-64 (2006)) in which 2-ketoadipate is the fourth intermediate and therefore a potential precursor for C5 chemicals such as glutarate and 5HV, as well as for 5HV-containing PHA polymers.
  • This pathway starts from ⁇ -ketoglutarate and acetyl-CoA to biosynthesize 2- ketoadipate as shown in Figure 5.
  • Recombinant E. coli host strains expressing these four enzymes were shown to produce 2-ketoadipate (Andi et al, Biochem. 43:1 1790-1 1795 (2004); Jia et al., Biochem. J. 396:479-485 (2006); Miyazaki et al, J. Biol. Chem. 278:1864-1871 (2003)).
  • Ketoadipate may be converted by an ⁇ -ketoglutarate dehydrogenase to glutaryl-CoA due to the structural similarity of ⁇ -ketoglutarate and 2- ketoadipate.
  • Glutaryl-CoA may be converted by an succinic semialdehyde (SSA) dehydrogenase such as succinyl-CoA synthetase (SucD) (S ⁇ hling and Gottschalk J. Bacterid. 178:871-880 (1996)), again due to the structural similarity of succinyl-CoA and glutaryl-CoA.
  • SSA succinic semialdehyde
  • Glutarate is useful for the production of polymers such as polyesters, polyester polyols and polyamides.
  • the odd number of carbon atoms i.e. 5
  • 1,5-pentanediol is a common plasticizer and precursor to polyesters that is manufactured by hydrogenation of glutarate and its derivatives (Werle and Morawietz, "Alcohols, Polyhydr ⁇ c" in: Ulhnann's Encyclopedia of Industrial Chemistry: 2002, Wiley-VCH: Weinheim. DOI 10.1002/14356007.a01_305).
  • Biosynthesis of a homopolymer consisting of poly(5- hydroxyvalerate) (a.k.a. P(SHV)) PHA can proceed from 5HV via 5- hydroxyva ⁇ erate-CoA.
  • Two different enzymatic reactions may catalyze the first step, i.e. either by a CoA-transferase as described by Huisman et al. (U.S. Patent No. 7,229,804), S ⁇ hling and Goltschalk (J. Bacterid. 178:871- 880 (1996)), and Eikmanns and Buckel (Biol. Chem.
  • E. poIy(3-hydroxybufryrate-co-5-hydroxyva ⁇ erate) Biosynthesis of a copolymer including ⁇ oly(3-hydroxybutyrate-co-5- hydroxyvalerate) (a.k.a. P(3HB-co-5HV)) can occur by supplying 3- hydroxybutyryl-CoA (3HB-CoA) and 5-HV-CoA monomer precursor molecules.
  • 3HB-CoA can be biosynthesized from acetyl-CoA via 2 enzymatic steps: (i.) a ⁇ -ketoacyl-CoA thiolase reaction that converts acetyl-CoA to acetoacetyl-CoA (Nishimura et al., J. Biol. Chem. 116:21-27 (1978)) using suitable genes such as, but not limited to, bktB from Ralstonia eutropha (Slater et al 5 J. Bacteriol.
  • Figure 6 outlines the biosynthetic pathway of lysine metabolism in E. coii. Dotted and solid lines in the center of the diagram indicate allosteric feedback inhibition and transcriptional repression by L-lysine, respectively, which provide targets for genetic modifications necessary to increase L- Iy sine production in recombinant host cells such as E. coii.
  • Figure 7 provides an overview of the production of 1,5-pentanediol (PDO) from 5HV.
  • 5HV may be converted to 5HV-CoA by a CoA- transferase as described by Huisman et al. (U.S. Patent No. 7,229,804), S ⁇ hling and Gottschalk (J. Bacterid. 178:871-880 (1996)), and Eikmanns and Buckel (Biol Chem. Hoppe-Seyler 371 :1077-1082 (1990)), or by a CoA-synthetase as described by van Beilen et al. (Molec, Microbiol. 6:3121- 3136 (1992)).
  • 5HV may be converted to 5-hydroxypentenal by a propionaldehyde dehydrogenase or alcohol dehydrogenase such as pduP from Salmonella typhimurium (Leal, Arch. Microbiol. 180:353-361 (2003)) or eutE from K coii (Skraly, WO Patent No. 2004/024876).
  • 5- Hydroxypentenal may be converted to PDO by 1,3 -propanediol dehydrogenase or alcohol dehydrogenase such as dhaT from Klebsiella pneumoniae (Tong et al., Appl. Environ. Microbiol. 57(12):3541-3546 (1991)).
  • a copolymer containing 5-hydroxyvalerate-co-hydroxyproprionate can be produced from 5HV.
  • E. coii Kl 2 strains that were either fadlt (repressed fatty acid degradation (FAD)) oxfadK (constitutive FAD) are transfected with nucleic acids expressing polyhydroxyalkanoate and 5- hydroxyvalerate CoA transferase.
  • FAD reduced fatty acid degradation
  • oxfadK constitutive FAD
  • Preferred E. coii strains include MG 1655 and LS5218 (Spratt et al., J. Bacteriol 146(3): 1166- 1169 (1981)).
  • GC-FID analysis indicated that LS5218 [ ⁇ MS93] produced 6.4%dcw PHA, with a polymer composition of 52% 5HV and 48% 3HP.
  • MG1655 [pMS93] made 63.1%dcw PHA that consisted only of 5HV.
  • GC-MS analysis of LS5218 [pMS93] confirmed the presence of 3HP in the polymer sample.
  • the active FAD system in LS5218 is able to synthesize 3HP from NaSHV.
  • Transgenic organisms for producing polyhydroxyalkanoates and 5 carbon chemicals are produced using conventional techniques known in the art.
  • Table IA Genes in microbial host strains producing 5HV-containing PHA and 5 -carbon chemicals.
  • Suitable homologues for the DavB protein (lysine 2- monooxygenase, from Pseudomonas putida KT2440, EC No, 1.13.12.2, which acts on L-Iy sine to produce 5-amino ⁇ entanamide; protein ace. no. BAG54787 (Revelles et al., J Bacteriol. 187:7500-10 (2005))).
  • Nitrilase/cyanide YP 001265763 hydratase and apolipoprotein N- acyltransferase
  • Nitrilase/cyanide YP 001666657 hydratase and apolipoprotein N- Protein Name Protein Accession No.
  • Suitable homologues for the DavT protein (5-amino ⁇ entanoate transaminase, from Pseudomonas putida KT2440, EC No. 2.6.1.48, which acts on 5-aminopentanoate to produce glutarate semialdehyde; Protein ace, no. AAK97868 (Espinosa-Urgel and Ramos, Appl. Environ. Microbiol. 67 (H) 5 5219-5224 (2001))).
  • GsaR At2 protein giutarate semialdehyde reductase, from Aspergillus terreus NIH2624, EC No. 1 ,1.1.61, which acts on giutarate semialdehyde (or succinic semialdehyde) to produce 5-hydroxyvalerate (or 4-hydroxybutyrate); Protein ace. no. XPJ)01210625 (Birren, The Broad Institute Genome Sequencing Platform, direct submission to NCBI))).
  • GsaR At2 protein giutarate semialdehyde reductase, from Aspergillus terreus NIH2624, EC No. 1 ,1.1.61, which acts on giutarate semialdehyde (or succinic semialdehyde) to produce 5-hydroxyvalerate (or 4-hydroxybutyrate); Protein ace. no. XPJ)01210625 (Birren, The Broad Institute Genome Sequencing Platform, direct submission to NCBI))
  • GsaR At protein glutarate semialdehyde reductase, from Arabidopsis thaliana, EC No. 1.1.1.61 , which acts on glutarate semialdehyde (or succinic semialdehyde) to produce 5- hydroxyvalerate (or 4-hydroxybutyrate); Protein ace. no. AAK94781 (Breit Regen et al., J. Biol. Chem. 278 (42), 41552-41556 (2003))).
  • AIkK protein Suitable homologues for AIkK protein (Acyl-CoA synthetase, from Pseudomonas oleovorans; EC No. 6.2.1.3, which acts on 5- hydroxyvalerate to produce 5-hydroxyvaleryl-CoA; protein ace. no. Q00594 (van Beilen et al., MoI. Microbiol. 6 (2I) 5 3121-3136 (1992))).
  • PhaC protein polyhydroxyalkanoate synthase, from Ralstonia eutropha, EC No. 2.3.1.n, which acts on (R)-3- hydroxybutyryl-CoA + [(R)-3-hydroxybutanoate]n to produce [(R)-3- hydroxybutanoate](n+l) + CoA; Protein ace. no. YP_725940 (Peoples and Sinskey, J. Biol. Chem. 264:15298-15303 (1989))).
  • PhaE protein PhaE subunit of PhaEC PHA synthase, from Thiocapsa pfenigii, which acts on (R)-3- hydroxybutyryl-CoA + [(R)-3-hydroxybutanoate]n to produce [(R)-3- hydroxybutanoate](n+l) + CoA).
  • PhaC protein PhaC subunit of PhaEC PHA synthase, from Thiocapsa pfenigii, which acts on (R)-3- hydroxybutyryl-CoA + [(R)-3-hydroxybutanoate]n to produce [(R)-3- hydroxybutanoate](n+l) -f CoA).
  • Suitable homologues for the BktB (PhaA) protein ( ⁇ -ketoacyl- CoA thiolase, from Ralstonia eutropha Hl 6, EC No. 2.3.1.9, which acts on acetyl-CoA to produce acetoacetyl-CoA; Protein ace. no. CAJ92573 (Peoples & Sinskey, J Biol Chem. 1989 Sep 15;264(26): 15293-7. Pohlmann et al, Nature Biotech 24 (10), 1257-1262 (2006))).
  • PhaB protein acetoacetyl-CoA reductase, from Bacillus megaterium, EC. No. 1.1.1.36, which acts on acetoacetyl-CoA to produce (R)-3-hydroxybutyryl-CoA; Protein ace. no. AAD05259 (McCool & Cannon, J. Bacteriol. 183 (14), 4235-4243 (2001))).
  • Suitable homologues for the DavD protein (glutarate- semialdehyde dehydrogenase, from Pseudomonas putida KT2440, EC No. 1.2.1,20, which acts on glutarate senaialdehyde to produce glutarate; Protein ace. no. NP_742381 (Espinosa-Urgel and Ramos, Appl. Environ. Microbiol.
  • Suitable homologues for the GabD protein succinate semialdehyde dehydrogenase, NADP+-de ⁇ endent, from Escherichia coli str. K-12 substr. MG1655, EC No. 1.2.1.20, which acts on glutarate semialdehyde (or succinic semialdehyde) to produce glutarate (or succinate); Protein ace. no. NP_417147 (Riley et al, Nucleic Acids Res. 34 (1), 1-9 (2006))).
  • succinate-semialdehyde ZP_05433422 dehydrogenase I succinate-semialdehyde YP_001744810 dehydrogenase (NAD(PX+)) hypothetical protein ZPJB838O93 CH292JM137 succinate-semialdehyde YP_002638371 dehydrogenase succinate-semialdehyde YPJ)01333939 dehydrogenase I succinate-semialdehyde NPJ742381 dehydrogenase I succinate-semialdehyde YP_002932123 dehydrogenase [NADP+] (ssdh) succinic semialdehyde YPJ)01951927 dehydrogenase succinate semialdehyde YP 298405 dehydrogenase
  • Suitable homologues for the PduP protein (CoA-dependent propionaldehyde dehydrogenase, from Salmonella typhimurium LT2, EC No. 1.2.1.3, which acts on 5 -Hydroxy valery 1-CoA to produce 5- Hydroxypentanal; Protein ace. no. NP_460996 (Leal et al. s Arch. Microbiol. 180:353-361 (2003), McClelland et al., Nature 413: 852-856 (2001))).
  • Suitable homologues for the EutE protein (predicted aldehyde dehydrogenase, ethanolamine utilization protein, from Escherichia coli str. K-12 substr. MG1655, EC No. 1.2.1.3, which acts on 5-hydroxyvaleryl- CoAtp produce 5-hydroxypentanal, protein ace. no, NP_416950 (Riley et al., Nucleic Acids Res. 34 (1), 1-9 (2006))).
  • Suitable homologues for the ArgO (YggA) protein arginine export protein, from Escherichia coli str. K- 12 substr. MG 1655, which acts on L-lysine (cytoplasm) and produces L-lysine (outside); Protein ace. no. NP_417398 (Nandineni and Gowrishankar, J. Bacteriol. 186:3539-3546 (2004))).
  • LysE protein lysine efflux permease, from Corynebacterium glutamicum ATCC 13032, which acts on L- lysine (cytoplasm) to produce L-lysine (outside); Protein ace, no. NP_600485 (Tokyo Research Laboratories, Kogyo Co. Ltd., Japan, direct submission to NCBI)),
  • LysP protein (LysP lysine APC transporter, from Escherichia coli str. K- 12 substr. MGl 655, which acts on L-lysine (outside) and produces L-lysine (cytoplasm); Protein ace. no. NP_416661 (Steffes et al., J Bacteriol 174(10): 3242-9 (1992))).
  • LdcC protein lysine decarboxylase 2, from Escherichia coli str. K- 12 sub str. MG 1655, which acts on Lysine to produce Cadaverine; Protein ace. no. NP_414728 (Riley et al., Nucleic Acids Res. 34 (1), 1-9 (2006))).
  • Lysine decarboxylase ZPJ 4621086 lysine decarboxylase 1 YP_003294813 lysine decarboxylase 1 YPJ59739
  • YjeK protein lysine 2,3- aminomutase, from Escherichia coli str. K-12 sub sir. MGl 655, which acts on L-lysine to produce (R)-b-Lysine; Protein ace. no. NP_418570 (Riley et al., Nucleic Acids Res. 34 (I) 5 1-9 (2006))).
  • One embodiment provides a transgenic or recombinant organism for producing P(5HV) or other PHAs containing 5HV monomers .
  • the organism can be prokaryotic or eukaryotic. Suitable prokaryotes include but are not limited to bacteria, for example E. coli.
  • Organisms or cells that can be modified for producing 5HV containing PHA biopolymers, 5-aminopentanoate (5 AP), 5-hydroxyvalerate (5HV), glutarate, and 1,5-pentanediol (PDO) include eukaryotes and prokaryotes. Suitable prokaryotes include bacteria. A number of bacteria can be genetically engineered to produce polyhydroxyalkanoates. Examples include E.
  • E. coli Alcaligenes lotus, Alcaligenese eutrophus, Azotobacter, Pseudomonas putida, Ralstonia eutropha, Salmonella, Klebsiella, Corynebacterium glutamicum, Rhodoccocus, and Brevibacterium lactofermentum.
  • Additional prokaryotes include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli.
  • E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31 ,446); E. coli Xl 776 (ATCC 31.537); (ATCC 27,325) and K5772 (ATCC 53,635).
  • R. eutropha is an example of an organism which produces PHAs naturally.
  • E. coli and C. glutamicum are examples of organisms where it would be necessary to introduce transgenes which encode the enzymes for PHA production.
  • 5-aminopentanoate 5AP
  • 5- hydroxyvalerate 5HV
  • glutarate glutarate
  • 1,5-pentanediol where they are not polymerized to 5-hydroxvalerate containg PHAs but secreted into the growth medium then it is useful to use industrial microorganisms which are used to manufacture lysine.
  • Corynebacterium glutamicum including its subspecies Brevibacterium flavum, Brevibacterium lactofermentum, Corynebacterium lilium > Corynebacterium efficiem and Brevibacterium divaricatum, which has been used for the industrial production of L-lysine (Microbiol.
  • Monogr.5:39-70 (2007) is an example of the microorganisms can be developed for the production of glutarate, 1,5-pentandiol, 5-hydroxyvalerate, and other products that can be produced from lysine via lysine degradation pathways described in this invention.
  • Engineered E. coli have also been used for lysine production. The procedures to obtain C.
  • glutamicum strains for the production of L-lysine such as random mutagenesis and subsequent selection of the mutant that resistant to toxic lysine analogues, like S-(2-aminoethyl) cysteine, and introduction of mutant alleles of the gene targets such as lysC and horn which encode aspartate kinase and homoserine dehydrogenase, respectively, are well -established and has been described (Curr. Opin. Microbiol. 9:268-274 (2007)). Aspartate kinase is subject to a feedback inhibition by threonine and lysine, and the release of this enzyme from the feedback inhibition is regarded as one of the key features to develop a lysine producer strains.
  • lysine exporter such as LysE in C. glutamicum. Mutagenesis of Iy sE gene of L-lysine producing C. glutamicum strains will prevent the excretion of lysine from the cytoplasm and thus increase the yield of products via the pathway devised to convert lysine to the products.
  • Methods for constructing C glutamicum strains for the production of PHAs such as PHB are also known in the art (Jo, S-J et. Al., 2006. J. Bioscience and Bioengineering 102: 233-236).
  • Suitable eukaryotic organisms or cells include fungi such as filamentous fungi or yeast. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. 2. Methods for Generating Transgenic Organisms i. Extrachromosal Transfection
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including chemical transformation such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran- mediated transfection,, lipofection, or electroporation.
  • Conventional transformation techniques are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001. Transformations into yeast are typically carried out according to the method of Van Solingen et al. J Bad, 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sd. (USA), 76:3829 (1979).
  • Typical integration mechanisms include homologous recombination using linearized DNA in recBC or recD strains followed by Pl transduction (Miller 1992, A Short Course in Bacterial Genetics: A Laboratory Manual & Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor laboratory Press, Cold Spring Harbor, N. Y.) special plasmids (Hamilton et al., J Bacteriol. 171 :4617 (1989); Metcalf et al., Plasmid 35: 1 (1996); U.S. Pat. No. 5,470,727 to
  • Expression of the genes of interest for chromosomal integration can be achieved by including a transcription-activating sequence (promoter) in the DNA construct to be integrated.
  • promoter transcription-activating sequence
  • Site-directed, homologous recombination can be combined with amplification of expression of the genes of interest, as described by U.S. Pat. No. 5,000,000 to Ingram et al.
  • Chromosomal integration can also be achieved by the methods of Datsenko and Wanner (Proc. Natl. Acad. Sci. U S A. 97:6640-6645 (2000)), and as used in Example 7, below.
  • a series of expression cassettes have been developed for inserting heterologous genes into bacterial chromosomes.
  • cassettes are based on the transposon delivery systems described by Herrero et al., J Bacterial. 172:6557-67 (1990); de Lorenzo et al., J Bacterid. 172:6568 (1990). Although these systems specify RP4-mediated conjugal transfer and use only transposon TnIO and Tn5, any combination of transposon ends and delivery system could be adapted for the technology described, resulting in sustained and homogeneous PHA production.
  • a promoterless antibiotic resistance (abr) gene is cloned in the polylinker of a suitable plasmid such as pUC18NotI or pUCl ⁇ Sfil so that the major part of the polylinker is upstream of abr;
  • pha genes, genes encoding a GABA transaminase, a succinic semi-aldehyde reductase, and a 4-hydroxybutyrate-CoA transferase are subsequently cloned upstream of and in the same orientation as the abr gene;
  • ⁇ hepha-abr cassette is excised as a Noil or Avrl ⁇ fragment (Avrll recognizes the Sfil site in pUClSSfi ⁇ ) and cloned in the corresponding sites of any plasmid like those from the pUT- or pLOF-series; (4) the resulting plasmids are maintained in E.
  • selective medium for the host e.g., naladixic acid when the host is naladixic acid resistant
  • the cassette e.g., chloramphenicol, kanamycin, tetracyclin, mercury chloride, bialaphos.
  • the resulting pha integrants are screened on minimal medium in the presence of glucose for
  • the insertion of the PHA genes is selected based on a marker present in the vector and integrated strains producing the desired level of PHA are detected by screening for PHA production.
  • the pha genes may have, but do not need, endogenous transcription sequences, such as upstream activating sequences, RNA polymerase binding site, and/or operator sequences, ⁇ f the pha genes do not have such sequences, the described approach is limited to the use of vectors like the pUT series in which transcription can proceed through the insertion sequences. This limitation is due to the inability of RNA polymerase to read through the TnIO flanking regions of the pLOF plasmids.
  • the abr gene may carry its own expression sequences if so desired.
  • the construct may be designed such that an essential gene serves as selective marker when the host strain has a mutation in the corresponding wild-type gene. Examples of genes useful for this purpose are generally known in the art. Different constructs can be integrated into one host, either subsequently or simultaneously, as long as both constructs carry different marker genes. Using multiple integration events, /?/z ⁇ genes can be integrated separately, e.g., the PHB polymerase gene is integrated first as aphaC-cat cassette, followed by integration of the thiolase and reductase genes as aphaAB-kan cassette. Alternatively, one cassette may contain all pha genes whereas another cassette contains only some pha genes required to produce a desired PHA polymer.
  • a transposon integration vector such as pJMSl 1 (Panke et al. App ⁇ . Enviro, Microbiol 64: 748-751) may be used such that the selectable marker can be excised from the chromosome of the integrated strain. This is useful for a number of reasons including providing a mechanism to insert multiple transposon constructs using the same marker gene by excising the marker following each insertion event.
  • Th&pha genes may be derived from different sources and combined in a single organism, or from the same source.
  • Reductase-encoding genes have been isolated from A. latus, R, eutropha (Peoples & Sinskey, J Biol. Chem. 264(26): 15298-303 (1989); Acinetobacter sp. (Schembri, et al., J Bacteriol. 177(15):4501-7 (1995)), C. vinosum (Liebergesell & Steinbuchel, Eur. J. Biochem. 209(1): 135-50 (1992)), P. acidophila, P. denitriflcans (Yabutani, et al., FEMS Microbiol. Lett. 133 (l-2):85-90 (1995)), R.
  • U.S. Patent No. 7,229,804 discloses transgenic organisms that produce P4HB using the 4HbD gene that encodes the 4- hydroxybutyrate dehydrogenase from C kluyveri (Sohling and Gottschalk, J Bacteriol 178, 871 880 (1996)). 4hbD requires NADH. Preferred reductases include, but are not limited to, those that do not require NADH.
  • Exemplary reductases include AKR7A5 from Mus musculus (Genlnfo Identifier :27659727 )(Hinshelwood, A. et al. FEBS Letters 523:213-218 (2002), GHBDH from Arabidopsis thaliana (GI: 145338934) (Breitnch, K. et al. J. Biol Chem. 278:41552-41556, ATEGJ)0539 from Aspergillus fwew (GI: 115491994).
  • CoA Transferase and CoA Synthetase Suitable CoA transferases (EC 2.8.3.n) include but are not limited to orfZ from C. kluyveri.
  • PHA polymerase-encoding genes have been isolated from Aeromonas caviae (Fukui & Doi, J Bacteriol. 179(15):4821-30 (1997)), A. latus, R. eutropha (Peoples & Sinskey, J Biol. Chem. 264(26): 15298-303 (1989); Acinetobacter (Schembri, et al., J Bacteriol. 177(15):4501-7 (1995)), C vinosum (Liebergesell & Steinbuchel, Eur. J. Biochem. 209(1): 135-50 (1992)), Methylobacterium extorquens (Valentin & Steinbuchel, Appl. Microbiol.
  • Rhizobium etli (Cevallos, et al, J Bacteriol 178(6): 1646-54 (1996)), R. meliloti (Tombolini, et al., Microbiology 141 (Pt 10):2553-59 (1995)), Rhodococcus ruber (Pieper & Steinbuchel, FEMS Microbiol Lett. 96(l):73-80 (1992)), Rhodospir ⁇ lum rubrum (Hustede, et al., FEMS Microbiol. Lett.
  • Rhodobacter sphaeroides Steinbuchel, et al., FEMS Microbiol. Rev. 9(2-4):217-30 (1992); Hustede, et al., Biotechnol Lett. 15:709-14 (1993)), Synechocystis sp. (Kaneko, DNA Res. 3(3): 109-36 (1996)), T. violaceae (Liebergesell & Steinbuchel, Appl. Microbiol. Biotechnol. 38(4: 493-501 (1993)), and Z ramigera (GenBank Ace. No. U66242).
  • genes with significant homology to the phaB gene encoding acetoacetyl CoA reductase have been isolated from several organisms, including Azospirillum brasiliense (NCBI Accession Nos. X64772, X52913) Rhizobium sp. (NCBI Accession Nos. U53327, Y00604), E. coli (NCBI Accession No. D90745), Vibrio harveyi (NCBI Accession No.U39441), H. influenzae (NCBI Accession No.U32701), B. subtilis (NCBI Accession No.
  • bacteria are transformed or transfected with one or more nucleic acid constructs encoding the genes necessary for producing 5-aminopentanoate (5AP), 5-hydroxyvalerate (5HV), glutarate, and 1,5-pentanediol (PDO) and polymers thereof from renewable resources, IV.
  • the disclosed transgenic organisms can be used to produce C5 chemicals such as 5-arnino ⁇ entanoate (5AP), 5-hydroxyvalerate (5HV), glutarate, and 1,5-pentanediol (PDO) as well as PHA biopolymers comprising 5HV monomers.
  • the recombinant organism expressing the appropriate transgenes(s), optionally including having genes encoding competing patheways inactiviated or deleted are grown on a renewable fermentation substrate.
  • the substrate is selected from carbohydrates, lysine, proline vegetable oils, fatty acids or combinations thereof.
  • a useful combination for some embodiments would be a mixture of glucose and lysine.
  • Another suitable combination would be sucrose and lysine.
  • the feedstock comprises predominantly one substrate, for example glucose or sucrose.
  • Suitable carbohydrate substrates comprise one or more sugars selected from glucose, fructose, xylose, arabinose, sucrose, lactose and maltose.
  • the recombinant organism is grown on the renewable substrate until the desired end product accumulates in the growth medium at which time the cells are removed by flocculation, settling, centrifugation or filtration and the product is recoverd from the cell-free medium by standard procedures.
  • Such procedures are known in the art for the recovery of other fermentation produed acids and diols such as lactic acid, succinic acid, 3-hydroxypropionic acid, 1,3 -propanediol and 1,4- butanediol.
  • the recombinant organisms can be used for the fermentation production of 5HV containing PHA biopolymers including the homopolymer P5HV and 5HV containing PHA copolymers by culturing the organisms in the presence of renewable carbons sources such as glucose, lysine, etc. and other substrates selected to provide a desired polymer or copolymer.
  • renewable carbons sources such as glucose, lysine, etc.
  • the recombinant organsims are grown on the substrates until the PHA polymers have accumulated inside the cells at which point the PHA polymers are extracted from the cells by methods known to those skilled in the art.
  • 5HV containing PHA polymers obtained from the organisms can be used in a wide range of industrial plastics applications such as for films, fibers, foams, injection molded goods, blow molded bottles, paper coating and the like. They can also be used for biomedical applications including tissue augmentation, to produce heart valves, as sutures, and to produce vascular grafts.
  • Exemplary co-polymers include, but are not limited to, PHB5HV and p(3 -hydroxypropionate-co- 5 -hydroxy valerate) p(4-hydroxy butyrate-co-5 - hydroxy valerate).
  • the recombinant organisms can be genetically engineered to include additional genes such as beta-ketothiolase and acetoacetyl-CoA reductase as needed for the production of copolymers.
  • Na5HV sodium 5 -hydroxy valerate
  • E. coli strains expressing both a CoA transferase or CoA synthetase and a PHA synthase, in order to determine if the fed 5HV monomer would be accepted and incorporated directly into P(SHV) homopolymer.
  • NaSHV was synthesized via base hydrolysis of ⁇ -valero lactone (DVL). This procedure involved adding 0,1 mol NaOH to 50 mL methanol with stirring until dissolved. To this, 0.1 mol DVL was added with vigorous stirring.
  • the resulting precipitate was dried, dissolved in water, pH-adjusted to 8.5, and filter- sterilized with a 0.2 ⁇ M filter. Analysis of the salt solution was performed on a Waters Alliance HPLC in order to confirm that all of the DVL had been saponified,
  • CoA transferase/synthetase and PHA synthase genes were tested in order to find the best combination suitable for P(SHV) production.
  • the CoA transferases/synthetases tested were C. kluyveri orfZ and P. oleovorans alkK, and the PHA synthases were R. eutrophaphaC and T. pfenigii phaEC (Table IA). All strains used in this experiment were derived from MGl 655 (Jensen, J. Bacteriol, 175(11):3401 ⁇ 3407 (1993)).
  • Plasmid construction Four expression plasmids (pFS92, pMS96 ; pMS93, and pMS102), each containing different combinations of CoA transferase/synthetase and PHA synthase, were constructed as described in the following paragraphs. Plasmid construction
  • Plasmid ⁇ FS30 was constructed by digesting pAET41 (Peoples and Sinskey, J. Biol Chem. 264(26): 15298-15303 (1989)) with Xmal and Stul in order to remove a fragment containing R. eutropha phaC and its native promoter (P /?e ).
  • Plasmid pAET41 is a pUC18 vector (Accession No. L08752) that contains a chromosomal fragment from R. eutropha Hl 6 encompassing the phaC gene.
  • Plasmid pFS16 (Skraly and Peoples, U. S. Patent No.
  • Plasmid pFS92 was made in a multi step process.
  • T. pfenigii phaE was amplified from pMON25893 (Reiser et al., Appl. Microbiol, Biotechnol 53(2):209-218 (2000)) with primers FS-E5' and FS-E3', which contain engineered EcoRI and Acc65I restriction sites.
  • Thep/z ⁇ E PCR product was then digested with restriction enzymes EcoRI and Acc65I and ligated to similarly digested pTrcN (Gerngross et al,, Biochemistry 33:931 1- 9320 (1994)) to form pFS89.
  • the primer sequence for FS-E5' is (5')- GGAATTCAGGAGGTTTTTATGAACGATACGGCCAACAAGACCAGC (SEQ ID NO:1) and the primer sequence for FS-E3' is (5')-
  • T. pfenigii phaC was amplified from pMON25894 (Reiser et al., Appl, Microbiol. Biotechnol. 53(2):209-218 (2000)) with primers FS-C5' and FS-C3', which contain engineered Acc65I and BamHI restriction sites.
  • the phaC PCR product was then digested with restriction enzymes Acc65I and BamHI and ligated to similarly digested pTrcN to form pFS90.
  • the primer sequence for FS-C5' is (5>
  • pFS91 contained the phaEC operon fusion under the ? Trc promoter.
  • pFS92 was created by ligating a MIuI- BamHI fragment from pFS91 containing phaEC to pFS16 that had been digested with MIuI and BamHI. Plasmid pFS92 contains the phaEC-orfZ operon fusion under the P ⁇ > c promoter.
  • Plasmid pMS96 was made by cloning alkK (see Table IA) into pFS91 downstream of phaEC, First, alkK was PCR amplified from P. oleovor ⁇ ns genomic DNA using primers K5-1 and K3-1, which were engineered to incorporate BamHI sites onto the ends of the PCR product.
  • the sequence for primer K5-1 is (5')-
  • Plasmids pMS93 and pMS102 were constructed by first digesting pACYC 177 (Accession #X06402) with BspHI in order to remove the k ⁇ n marker and ligating it into the unique BspHI site of ⁇ FS30 to form pFS73, which contains ph ⁇ C-orfZ under the control of tandem promoters VT K and ? Re .
  • the P /?e promoter region was removed by replacing the EcoRI-BspEI fragment of ⁇ FS73 containing both ⁇ P Re and 837 bp from the 5' end of the ph ⁇ C CDS with an EcoRI-BspEI fragment from ⁇ KAS4 (Peoples et al., U.S. Patent No.
  • Plasmid pMS74 was then digested with Spel and Sbfl and then made blunt- ended via Klenow fill-in and the alkK PCR fragment ligated to the blunted pMS74 backbone to form pMS92.
  • Plasmid pMS92 thus contains XhsphaC- alkK operon fusion under the control of tandem promoters ⁇ T K and ⁇ & ,
  • the P & , promoter region was removed by replacing the EcoRI- BspEI fragment of pMS92 containing both P & , and 837 bp from the 5' end of the phaC CDS with an EcoRI-BspEI fragment from ⁇ MS93 that contained only the 837 bp from the 5' end of phaC.
  • This resulting plasmid which contains phaC-alkK under only the Pr re promoter, was designated pMS102.
  • Plasmid pTrcN-A.eut-AlkK was created by first PCR-amplifying alkK from P. oleovorans genomic DNA using primers Posynrbs.c (5 ! - GGAATTCAGGAGGTTTTTATGTTAGGTCAGATGATGCGTAATCAG) (SEQ ID NO:9) and Posynrbs.r (5'-
  • Plasmids pFS92, pMS96, pMS93, and pMS102 were individually transformed into MGl 655 (Jensen, J Bacterid. 175(11):3401-3407 (1993)) to create four plasmid-bearing strains that contained different combinations of CoA transferase/synthetase (orfZ or alkK) and PHA synthase iphaC or phaEC). These strains were grown in 250 niL shake flasks to characterize P(5HV) homopolymer production, as described in the following section. Media, growth conditions, and testing for production of P(SHV) homopolymer in shake flask cultures
  • Each plasmid-bearing MG 1655 strain was grown overnight in a test tube containing 3 mL LB (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (2001)) supplemented with appropriate antibiotic at 37 0 C with 250 rpm shaking.
  • Antibiotics appropriate for each strain are as follows: 100 ⁇ g/mL ampicillin was added to overnight cultures of pFS92- and pMS96-bea ⁇ ng MGl 655 strains; and 50 ⁇ g/mL Km was added to overnight cultures of pMS93- and ⁇ MS102-bearing MG1655 strains.
  • the production medium consists of Ix E2 minimal salts solution containing 10 g/L glucose, 2.5 g/L LB, 10 g/L NaSHV 5 2mM MgSO 4 , and Ix Trace Salts Solution.
  • 50x E2 stock solution consists of 1.275 M NaNH 4 HPO 4 -4H 2 O, 1.643 M K 2 HPO 4 , and 1.36 M KH 2 PO 4 .
  • 100Ox stock Trace Salts Solution is prepared by adding per 1 L of 1.5 N HCL: 50 g FeSO 4 -7H 2 0, 11 g ZnSO 4 -7H 2 O, 2.5 g MnSO 4 -4H 2 O, 5 g CuSO 4 -5H 2 O, 0.5 g (NH 4 ) 6 Mo 7 0 24 -4H 2 0, 0.1 g Na 2 B 4 O 7 , and 10 g CaCl 2 -2H 2 O.
  • the resuspended cultures were transferred to 250 mL shake flasks and incubated at 3O 0 C for 24 to 72 hours with shaking. At the end of the experiment, cultures were spun down at 4150 rpm, washed once with distilled water, frozen at -8O 0 C for at least 30 minutes, and lyophilized overnight. The next day, a measured amount of lyophilized cell pellet was added to a glass tube, followed by 3 mL of butanolysis reagent that consists of an equal volume mixture of 99.9% n-butanol and 4.0 N HCl in dioxane with 2 mg/mL diphenylmethane as internal standard.
  • GC-FID gas chromatography- fiame ionization detection
  • the quantity of P(SHV) homopolymer in the cell pellet was determined by comparing against standard curves that were made by adding defined amounts of DVL in separate butanolysis reactions. Three DVL standards ranging from 2-6 mg were used to create the standard curves.
  • Table 2 shows that all constructs were able to generate P(5HV). However, MGl 655 [pMS93] generated significantly more P(5HV) than any of the other strains demonstrating that the optimal gene combination to polymerize P(5HV) is phaC and orfZ.
  • strain used in this example was MBX2641, which is an MG 1655 derivative that contains an operon consisting of R eutropha H16 bktB-B megaterium phaB-kan randomly integrated into the chromosome
  • strain Sl7-l ⁇ pir (Miller and Mekalanos, J Bacteriol 170(6):2575-2583 (1988)) containing pCJ022 (described below) was mated with MBX1987, which is a nalidixic acid-resistant mutant of MGl 655, using a protocol taken from the literature (De Lorenzo and Timmis, Methods Enzymol 235:386-405 (1994)).
  • MBX1987 carrying the bktB-phaB-kan cassette in the chromosome were selected on LB plates containing 30 ⁇ g/mL nalidixic acid (Nl) and 50 ⁇ g/mL kanamycin (Km).
  • Nl nalidixic acid
  • Km kanamycin
  • One such integrant displaying a N1 R Km R phenotype was saved as MBX2079.
  • phaEC-cat was randomly integrated into the chromosome of MBX2079 by mating with an S ⁇ 7 - ⁇ pir strain carrying the integration vector pUT-C16 ⁇ cat (described below).
  • Integrants of MBX2079 carrying the phaEC-cat cassette in the chromosome were selected on LB plates containing 25 ⁇ g/mL chloramphenicol (Cm).
  • Cm chloramphenicol
  • Several integrants possessing the N1 R Km R Cm R phenotype were pooled and subjected to nitrosoguanidine mutagenesis (Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory (1972)), with selection on LB plates containing 100 ⁇ g/mL Cm.
  • One mutant was isolated and designated MBX21 14.
  • MBX2641 was created by raising a Pl lysate on MBX21 14 and transducing into MGl 655 as described by Miller (Experiments in Molecular Genetics, Cold Spring Harbor Laboratory (1972)) using Km R as selection.
  • One such transductant was saved and designated MBX2641, which is MGl 655 with the bktB-phaB-kan gene cassette randomly integrated into the genome.
  • Plasmids pFS92 (see Example 1), and pJB84 (construction described below) were individually transformed into MBX2641 that contains bktB- phaB-kan randomly integrated into the chromosome.
  • Plasmid construction Plasmid pCJ022 was made by creating a mini-TnJ integration vector containing bktB-phaB upstream of a kan marker. To do this, the bktB-phaB operon was assembled on pSE380 (Invitrogen, Carlsbad, CA) by first PCR- amplifying bktB with primers MS069 and MS070 from pMON25765 (Slater et al, J. Bacteriol 180(8): 1979- 1987 (1998)). The sequence for primer MS069 is (5>
  • GGTGGATCCTTAAGAGGAGGTTTTTATGACGCGTGAAGTGGTAGT GG (SEQ ID NO: 13) and the sequence for primer MS070 is (5> GGTGCTAGCTCAGATACGCTCGAAGATGGCG (SEQ ID NO: 14).
  • the resulting PCR fragment was digested with BamHI and Nhel and ligated to ⁇ SE380 that had been cut with the same enzymes.
  • the resulting plasmid was designated pSE380-bktB.
  • phaB was PCR-amplified from pGMIO (McCool and Cannon, J Bacterial 181(2):585-592 (1999)) with primers MS071 and MS072.
  • the sequence for primer MS071 is (5')-
  • GGTCCTAGGTTAAGAGGAGGTTTTTATGACAACATTACAAGGTAA AG (SEQ ID NO: 15) T and the sequence for primer MS072 is (5> GGTGCGGCCGCTTACATGTATAAGCCGCCGTTAAT (SEQ ID NO; 16).
  • the resulting PCR fragment was digested with Avrll and Notl and ligated to pSE38O ⁇ bktB that had been treated with the same enzymes.
  • the resulting plasmid was designated pSE380-bktBphaB19.
  • pTrcN-fam (a derivative of pTrcN that had the bla gene replaced with the lean gene) by digesting pSE380- bklBphaB19 with EcoRI and Spel and ligating the fragment containing bktB- phaB to pTrcN-A ⁇ w that had been cut with EcoRI and Xbal.
  • the resulting plasmid was designated pMSl 15.
  • the bktB-phaB operon was then transferred from pMSl 15 to the pBSLl 18 integration vector (Alexeyev et al., Can. J. Microbiol.
  • Plasmid pUT-C16cat was made by removing phaEC from pFS91 with EcoRI and Xbal and blunting the sticky ends using Klenow.
  • Integration vector pUT-cat (De Lorenzo and Timmis, Methods Enzymol. 235:386-405 (1994)) was digested with Avrl ⁇ , blunted using Klenow fill-in, and then ligated to the phaEC fragment. After verifying that phaEC was in the same orientation as the downstream cat marker, the plasmid was designated pUT- Cl ⁇ cat.
  • Plasmid pJB84 was constructed by PCR-amplifying the aacC (Gm R ) marker from pBSL202 (Alexeyev et al., Can. J, Microbiol 41 : 1053-1055 (1995)) using primers JB 123b and JB 124a, which were engineered to include BspHI sites, on the 5' flanking ends.
  • the sequence for primer JB 123b is (5 >TT ATTTCATGAACCCTCGAATTGACGCGCTC (SEQ ID NO: 17) and the sequence for primer JB 124a is (5')- TTATTTCATGAGCTTATCGATACCGTCGACC (SEQ ID NO: 18).
  • MBX2641 strains carrying plasmids pFS92 (described in Example 1) or pJB84 were grown and prepared for GC-FID analysis as described in Example 1.
  • MBX2641 [pJB84] made more copolymer (69.5%dcw) and incorporated more 5HV into copolymer (82.3%PHA) as shown in Table 3.
  • EXAMPLE 3 Tunable 5HV monomer composition in P(3HB-co-5HV) copolymer and effect on material properties
  • the copolymer composition was modulated by altering the amounts of NaSHV and glucose added to the production medium. Alternate ways to accomplish this include (1) feeding different amounts of L-Iy sine to the growth medium to a recombinant cell that can produce 5HV from L-lysine as is shown in Example 6, or (2) deregulating L-lysine pathway genes in recombinant cells that can produce 5HV from glucose via L-lysine as shown in Example 9 and 10.
  • strain MBX2641 [ ⁇ FS30] was used that expresses the enzymes for the 3HB pathway ⁇ ktB and phaB) in addition to the CoA- transferase (or ⁇ ) and PHA synthase iphaC).
  • Parallel cultures of MBX2641 [ ⁇ FS30] were grown in either decreasing concentrations of glucose (10, 5, 1, 0.5, 0.1, 0 g/L) or NaSHV (10, 5, 1, 0.5, 0.I 5 0 g/L) and analyzed for polymer content as described in Example 1.
  • Table 4 shows that various amounts of 5HV can be incorporated into the P(3HB-co-5HV) copolymer. Table 4. Effect of co-feed on 5HV incorporation into P(3HB-co-5HV)
  • fatty acid degradation (FAD) system of E coli could break down 5HV to acetyl-CoA and 3-hydroxypropkmyl-CoA (3HP-CoA) 5 plasmid pMS93 (expressing phaC-orfZ from a P trc promoter; see Example 1) was transformed into E coli Kl 2 strains that were either fadR + , atoC + (repressed FAD) ovfadR ' , atoC comt (derepressed FAD) MGl 655 and LS5218 (Spratt et al, J Bacterial 146(3) 1166-1169 (1981)) were used as the fadR*, atoC* said fadR " , atoC consi strains, respectively.
  • FAD fatty acid degradation
  • MG1655 [pMS93] and LS5218 [pMS93] were tested by feeding NaSHV in shake flasks as described in Example 1 GC-FID and GC-Mass Spectroscopy (MS) analysis demonstrated that LS5218 [pMS93] produced P(5HV-co-3HP), whereas MG1655 [pMS93] did not (Table 6). This shows that active fatty acid degradation will produce 3HP from 5HV. Table 6. Incorporation of 3HP into P(5HV-co-3HP)
  • the pathway devised to convert L-lysine to P(SHV) is schematically diagrammed in Figure 2B and requires six heterologous genes to be cloned and expressed in E. coli: P. putida davB, P. putida davA, P. putida davT, A, thaliana gsaR / n, C. kluyveri orfZ, and R. eutropha phaC (see Table 1 A).
  • a cloning strategy was designed such that the davBAT genes would be cloned into pACYC184 (Chang and Cohen, J. Bacterid.
  • the multiple cloning site of plasmid pSE380 was PCR-amplified with primers JB134 (5'- TGAGCGGATAACAATTTCAC) (SEQ ID NO: 19) and JBl 35 (5'- AATAACACGTCAACGCAAAAAGGCCATCCGT) (SEQ ID NO:20).
  • JB134 5'- TGAGCGGATAACAATTTCAC
  • JBl 35 5'- AATAACACGTCAACGCAAAAAGGCCATCCGT
  • Plasmid pJB91 was constructed in a three step process.
  • the davBA genes from P. putida were PCR-amplified from a genomic DNA preparation using primers JBl 36 and JBl 37, which were engineered to incorporate Ndel and BsrGI restriction sites, respectively, on the 5' and 3' ends of the davBA PCR product,
  • the sequence for primer JBl 36 is (5')- TTTTTCATATGAGGAGGTTTTTATGAACAAGAAGAACCGCCA (SEQ ID NO:21) and the sequence for primer JB137 is (5>
  • TTTTTTGTACATCAGCCTTTACGCAGGTGCA SEQ ID NO:22.
  • the resulting PCR product was digested with Ndel and BsrGI and ligated to pJB78 that had been treated with the same enzymes, to give plasmid pJB79.
  • the davT gene from P, putida was PCR-amplified from genomic DNA using primers JB 138 and JB 139, which were engineered to incorporate Spel and ApaLI restriction sites, respectively, on the 5' and 3' ends of the davT PCR product.
  • primer JB138 The sequence for primer JB138 is (5')- TATATACTAGTAGGAGGATAATATGAGCAAAACCAACGAATC (SEQ ID NO:23) and the sequence for primer JB 139 is (5 * )- TTTTTGTGCACTCAGGCGATTTCAGCGAAGC (SEQ ID NO:24).
  • the resulting PCR product was digested with Spel and ApaL ⁇ and ligated to pJB79 that had been digested with the same enzymes, thus creating plasmid pJB80.
  • the ompA promoter was PCR-amplif ⁇ ed from E.
  • Plasmid pJB91 was constructed by digesting a davBA PCR product created with primers JB 136 and JB 137 (as described above) with DraIII and ligating the 507 bp fragment to ⁇ JB82 that had been digested with the same enzyme, thus creating plasmid pJB91.
  • Plasmid ⁇ JB80 contains the davBAT ap&tm under the constitutive ?, et promoter, while plasmid pJB91 contains the same operon under the strong T? O mpA promoter.
  • Plasmid pMZ28 was constructed by digesting plasmid pJ31:7950, which is a construct created by DNA 2.0 (Menlo Park, CA) and contained gsaR At that is codon-optimized for expression in E. coli Kl 2, with BsrGI. The resulting fragment containing gsaR ⁇ was ligated to pFS30 that had also been cut with BsrGI. After verifying that the orientation of gsaR A t was in the same direction as phaC-orfZ by restriction enzyme digestion, the resulting plasmid was designated pMZ28.
  • Example 1 The recipes for E2 salts and Trace Salts Solution are given in Example 1. Shake flask growth conditions and the analysis protocol for PHA content are as described in Example 1. Table 7 shows that eight-fold more P(SHV) was produced after introduction of the davBAT opsmn.
  • genes comprising the pathway in this example include: P putid ⁇ d ⁇ vB, P putid ⁇ d ⁇ vA, P putid ⁇ d ⁇ vT, C kluyveri orfZ, R. eutroph ⁇ ph ⁇ C, and either A th ⁇ li ⁇ n ⁇ gs ⁇ R ⁇ t or A terreus gsaR ⁇ (see Table IA).
  • Plasmid pJB90 which contains an alternate pathway consisting of the gsaR A t2, phaC, and orfZ genes, was created in the following manner.
  • primer JB 145 The sequence for primer JB 145 is (5')- TTTTTAGATCTAGGAGGTTTTTATGCTGCGTGCTGCTTCTCG (SEQ ID NO;25) and the sequence of primer JB146 is (5')- TTTTTAGATCTTTAGCGGAAATAGTTTGGAC (SEQ ID NO:26).
  • the resulting PCR fragment was digested with BgIII and ligated into the corresponding site of pJB84 to create pJB90.
  • Strains MBX2641 [ ⁇ JB78, pJB84], MBX2641 [pJB91, pMZ28], and MBX2641 [pJB91, pJB90] were grown in shake flasks and analyzed for PHA content and composition as described in Example 1 and 2, in order to characterize the production of P(3HB-co-5HV) from L-lysine and glucose.
  • MBX2641 [pJB78, pPB84], MBX2641 [pJB91, pMZ28], and MBX2641 [ ⁇ JB91, pJB90) were inoculated in test tubes containing 3 mL LB supplemented with 25 ⁇ g/mL chloramphenicol and 100 ⁇ g/mL ampicillin and grown overnight at 37 0 C with 250 rpm shaking. The next day, 0.5 mL of each overnight culture was used to inoculate a shake flask containing 50 mL of fresh LB supplemented with the same antibiotic and grown at 37 0 C with 250 rpm shaking.
  • 0.1 raM IPTG was added to the liquid cultures, and at 5 hours, the cultures were spun down at 4150 rpm (Sorvall Legend RT benchtop centrifuge) and resuspended in 50 mL of production medium that consisted of Ix E2 minimal salts solution containing 10 g/L glucose, 2.5 g/L LB, 10 g/L L-lysine, 2mM MgSO 4 , Ix Trace Salts Solution, and 0.1 mM IPTG.
  • the recipes for E2 salts and Trace Salts Solution are given in Example 1.
  • strain MBX2641 [pJB78 5 pJB84] which does not have the genes that convert L-lysine to 5HV, was unable to produce 5HV from L-lysine and produced only P(3HB) homopolymer.
  • Strains MBX2641 [pJB91, ⁇ MZ28] and MBX2641 [pJB91, pJB90] both of which contain the entire pathway from L-lysine to 5HV-CoA, incorporated 5HV to 2.5%wt and 5%wt, respectively, of the total copolymer. This demonstrates that the davBAT&nd gsaR genes need to be expressed in order to produce 5HV- containing copolymers from L-lysine.
  • EXAMPLE 7 Improved biosynthesis of 5HV-eontaining PHA polymers from L-lysine Due to the fact that 5HV was incorporated at an unexpectedly low level, the existence of a competing pathway was considered.
  • MGl 655 [pJB91] expressing the davBAT genes from the plasmid was grown in LB medium containing 25 mg/L chloramphenicol at 30 0 C with shaking for 6h. Aliquots of 25 ⁇ L mid- log phase cultures were inoculated into 475 ⁇ L EO minimum medium and incubated at 30 0 C with shaking at 250 rpm for 48 h.
  • the EO minimum medium consisted of 10 g/L glucose, 4 g/L lysine, 58 mM K 2 HPO 4 , 27 mM KH 2 PO 4 , 2 mM MgSO 4 , 25 ⁇ g/mL chloramphenicol, 0.1 mM IPTG, and trace elements.
  • Glutarate present in the supernatant, was measured by GC- MS as outlined below: supernatants from fermentation broth were obtained by centrifugation and 1 ⁇ L of the sample was pipetted to a fresh Eppendorf tube containing 1 ⁇ L of 1 g/L 4-aminobutyrate (GABA) as internal standard.
  • GABA 4-aminobutyrate
  • Samples were dried in a Labconco centrivap and resuspended in 100 ⁇ L acetonitrile (ACN): N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) 1 :1 solution by sonication for 3 h Samples were then derivatized at 65°C for 30 min, centrifuged to remove insoluble material and supernatants injected into an Agilent 5975 GC-MS equipped with a HP-5ms column using the following acquisition parameters: carrier gas Helium flow rate 2 ml/min, scan mode m/z 65-700, solvent delay for 3.5 min, oven program: 15O 0 C for 2 min, then ramp up to 280 0 C at 3°C/min, ion source temperature 230°C, the quadrupole mass filter temperature 150°C.
  • ACN acetonitrile
  • MTBSTFA N-(t-butyldimethylsilyl)-N
  • 0.6 g/L glutarate was produced from L-lysine when the davBA T operon was overexpressed in MG 1655.
  • the davB ⁇ T operon expresses genes encoding enzymes that convert L-lysine to GSA.
  • Glutarate may be produced by an endogenous E. coli gene whose encoded enzyme can oxidize GSA to glutarate.
  • Single null gabD and ynel mutants were constructed by the Red/ET Recombineering method described by Datsenko and Wanner (Proc. Natl, Acad. Sci. USA. 97:6640-6645 (2000)).
  • the process of deleting gabD from the chromosome of MG 1655 involved the replacement of gabD with an FRT-flanked kan marker via PCR-mediated homologous recombination.
  • the FRT-flanked kan marker was PCR-arnplified from plasmid pKD4 (Datsenko and Warmer, Proc. Natl. Acad. ScL USA.
  • This marker was PCR- amplified from plasmid pKD4 using primers MS220 5'- GCAAGAGTAAATCTGCGTATCTTCATACCATGACTCATAAAGGAG ATACCCCGGTGTAGGCTGGAGCTGCTTC (SEQ ID NO:29) and MS217 5'-
  • GCCACTTTCTACTCCTGGACCGCAGGTCTGAAAAGACCTGCGAGT ATATCAGAGCTG SEQ ID NO:32.
  • Successful replacement of ywlwith FRT-Ar ⁇ rc-FRT was achieved.
  • the kctn marker was then removed as described in Datsenko and Wanner (Proc. Natl. Acad. Sci. U S A. 97:6640- 6645 (2000)).
  • the MG1655 AgabD: :FRT- ⁇ w-FRT, ⁇ j/ne/::FRT was constructed by Pl-mediated transduction from MG ⁇ 655AgabD::FKT-kcm-FRT to MG1655 ⁇ yr ⁇ e/::FRT, and the remaining kan marker was further removed using the same method as described above.
  • the resulting strain MGl 655 AgabD: :FRT, Ayne ⁇ . :FRT was transformed with pJB91 and analyzed for glutarate production from L-lysine in an experiment analogous to the one described above with MG1655 [pJB91] expressing the davBAT genes from the plasmid.
  • MBX2855 was constructed by transforming plasmids pJB91 and pJB90 into strain MBX2641. This strain has all the genes to produce P(3HB- co-SHV) from glucose and L-lysine.
  • MG 1655 AgabDv.FKT, ⁇ ywe/::FRT was Pl -transduced with donor strain MBX21 14 that conferred PHB producing capabilities as described in Example 2.
  • This strain was further transformed with pJB90 and pJB91, which expressed the L-lysine to P(5HV) pathway genes as described in 5 Examples 6 and 5, respectively.
  • the resulting strain was designated as MBX3378 and has all the genes to produce P(3HB-co-5HV) from glucose and L-lysine but unlike MBX2855 has both the gabD and ynel genes removed from the genome.
  • a shake-plate fermentation was conducted using strain MBX2855 0 and its GSA dehydrogenase-deficient counterpart MBX3378. Cells were incubated and analyzed under the same conditions as described above (shaking at 300 rpm at 30 0 C).
  • the EO minimum medium consisted of 10 g/L glucose, 2 g/L L-lysine, 58 niM K 2 HPO 4 , 27 mM KH 2 PO 4 , 2 mM MgSO 4 , 25 ⁇ g/mL chloramphenicol, 5 ⁇ g/mL gentamicin, 0.1 mM IPTG, and trace 5 elements as described in a previous example.
  • EXAMPLE 8 Biosynthesis of L-lysine from glucose 5 Allosteric feedback regulation occurs in the L-lysine pathway through the genes lysC and dap A. Therefore, this control needs to be eliminated in order to enable increased L-lysine production from glucose. The procedure to do this is well-established and has been described for both genes (Kojima et al., U.S. Patent No. 6,040,160). E. coli mutants possessing deregulated lysC and dap A can be obtained first by deleting metL and thrA from E. coli.
  • LysC, MetL and ThrA are isozymes that all catalyze the same aspartate kinase reaction, so it will be necessary to eliminate the latter two before mutations in lysC can be positively selected.
  • NTG N-methyl-N'-nitro-N- nitrosoguanidine
  • the resulting mutant pool would then be grown in a minimal medium containing S-2-aminoethylcysteine (AEC), a non- metabolizable analog of L-lysine, in order to put pressure on lysC and dap A.
  • AEC S-2-aminoethylcysteine
  • P(SHV) was produced from glucose as the sole carbon source in an E. coli strain capable of synthesizing the 5HV-CoA monomers from glucose and incorporating them into PHA.
  • a strain was constructed that expressed not only the genes required to produce P(5HV) homopolymer from L-lysine, but which also expressed a mutated dapA gene called da ⁇ A ⁇ r that encodes a L-lysine feedback-resistant dihydrodipicolinate synthase.
  • da ⁇ A ⁇ r mutated dapA gene
  • the primers DE082 and DE083 are reverse complementary and were designed to introduce the cytosine to thymine base change at the 352 th nucleotide residue.
  • the two DNA fragments from the first round of PCRs were fused by SOE-PCR using primers DE081 and DE084.
  • the resulting PCR product was digested with Xbal and Sad and ligated to pDE031 that had been digested with Spel and Sad, thus creating plasmid pDE035.
  • Plasmid pDE031, containing a synthetic constitutive promoter (F Syn d was constructed by digesting a synthesized 63 bp double-stranded DNA fragment (5'-
  • TTTTTCTAGATTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCA CTAGTGTTTAAACCCCC (SEQ ID NO:37) with Xbal and Pmel and ligated into the same restriction enzyme sites of a pBluescript II SK(+) plasmid (Stratagene, La Jolla, CA) that was previously engineered to contain these sites.
  • the ⁇ syfl rdapA Ci52 ⁇ gene construct in pDE035 was digested with Xhol and Pmel, which was followed by ligation with the plasmid pJB90 (described in Example 6) that had been digested with BsrGI, blunted with Mung Bean nuclease, and digested a second time with Xhol to generate plasmid pJG22 that expressed phaC ⁇ g$aR ⁇ r orfl operon from the P hc promoter and the dapA 03521 gene from the P sy ,,i promoter.
  • Experimental results Plasmid ⁇ JG22 was transformed along with plasmids pJB91
  • strain MBX3342 (MGl 655 ⁇ gabDy.FRT AyneI:: ⁇ KT) into form strain MBX3342 [pJB91 , pJG22].
  • the medium consisted of 15 g/L glucose, 52 mM NaNH 4 HPO 4 , 66 niM K 2 HPO 4 , 54 mM KH 2 PO 4 , 2 mM MgSO 4 , 0.1 mM IPTG, and trace elements as described above.
  • pACYC184 empty vector control pJG22: ⁇ frc -phaC-gsaR Ar orfZ
  • the btkB-phaB-kan genes from strain MBX2114 were Pl -transduced into MBX3342 generating strain MBX3344.
  • MBX3344 was transformed with plasmids pJB91 and pJG22 to create strain MBX3344 [pJB91, pJG22],
  • the plasmids pSE380 and pACYC184 5 the empty vectors used to construct pJG22 and pJB91, respectively, were also transformed into MBX3344 to create a negative control strain, MBX3344 [pSE38O, pACYC184].
  • strains were incubated for 48 h shaking at 300 rpm at 30°C in 2x E2 medium and analyzed as described in the earlier examples.
  • the medium consisted of 15 g/L glucose, 52 mM NaNH 4 HPO 4 , 66 mM K 2 HPO 4 , 54 mM KH 2 PO 4 , 2 mM MgSO 4 , 0.1 mM IPTG, and trace elements as described above.
  • strain MBX3824 (W31 10 AgabD::FRT AyneliiFRT AcadA::FRT AldcCr.FR ⁇ AargOv.FRT bktB-phaB-kan) was tested as the host strain to produce P(3HB-co-5HV) copolymer from glucose.
  • the competing pathways that may divert L-lysine away from the 5HV-CoA co-monomer were removed from the E. coli genome.
  • the first competing pathway may convert L-lysine to cadaverine and consists of two L-lysine decarboxylase enzymes (EC number 4.1.1.18) encoded by cadA (Meng and Bennett, J. Bacterid. 174(8):2659- 2669 (1992); EcoCyc accession number: EG10131) and ldcC (see Table IA; Yamamoto et al., Genes Genet. Syst. 72(3):167-72 (1997); EcoCyc accession number: G6094).
  • a second competing pathway may export L-lysine out of the microbial cell.
  • the L-lysine export protein has been identified as LysE (see Table IA; Vrljic et al., MoI. Microbiol. 22 (5): 815-826 (1996)).
  • LysE the L-lysine export protein
  • Table IA the L-lysine export protein
  • BLAST the L-lysine exporter genes
  • Psi-BLAST searches using C. glutamicum LysE as the query were conducted.
  • Six proteins were found to be targets for removal from the E. coli genome in order to prevent L-lysine export outside of the cell. They include: (1) ArgO (a.k.a. YggA, Nandineni and Gowrishankar, J.
  • ArgO appeared to be the most likely candidate to export L-lysine out of E. coli cells based on its lowest e-value of 2e-22 in BLASTP searches using LysE from C glutamicum as the query, the closest clustered with LysE in a Neighbor Join Tree after ClustalX (Thompson et al,, Nucleic Acids Res. 25: 4876-4882 (1997)) with C glutamicum LysE and the six E.
  • Another competing pathway may convert L-lysine to (R)- ⁇ -lysine which is catalyzed by lysine 2,3-aminomutase (EC number 5.4.3.-) encoded by yjeK of E. coli (EcoCyc accession number: G7836; Behshad et al, Biochemistry 45(42): 12639-46 (2006)).
  • GTGTTTTCTTATTACTTTCAAGGTCTTGCACTTGGGGCGGCTATGG TGTAGGCTGGAGCTGCTTC (SEQ ID NO:42) and DEl 07 (5'- CTAACTGAACAAGGCTTGTGCATGAGCAATACCGTCTCTCGCCAG ATGGGAATTAGCCATGGTCC) (SEQ ID NO:43).
  • the W31 10 AgabD: :FRT Ayneh:FRT AcadA::FRT AldcC.-.FRT AargOv.FRT was constructed by iterative Pl -mediated transductions of AgabDvJFR ⁇ -kan-FRT, AyneI::FRT- kan-FRT, AcadA::FR7-kan-FR ⁇ , AldcC::FRT-cat-FRT, AcadAv.FRT-kan- FRT cassettes into strain W3110 (Bachmann, Bacterial Rev., 36 (4):525-557 (1972)), which was followed by removal of kan or cat markers after each Pl -mediated transduction as described in earlier examples.
  • the resulting strain MBX3818 was Pl -transduced with donor strain MBX2114 to finish the construction of MBX3824.
  • the plasmids pJG22 and ⁇ JB91 were transformed into MBX3824 and the resulting strain MBX3824 [pJG22, ⁇ JB91] was tested for the production of P(3HB-co-5HV) copolymer along with MBX3344 [ ⁇ JG22, ⁇ JB91].
  • These strains were incubated for 48 h shaking at 300 rpm at 30 0 C in 1.5x E2 medium and analyzed as described in the earlier examples.
  • the medium consisted of 15 g/L glucose, 39 mM NaNH 4 HPO 4 , 49.5 mM K 2 HPO 4 , 40.5 mM KH 2 PO 4 , 2 mM MgSO 4 , 0.1 mM IPTG, and trace elements as described above.
  • the culture media were supplemented with 25 ⁇ g/mL chloramphenicol and 5 ⁇ g/mL gentamicin. Table 12 shows that various strains with different genetic backgrounds have the ability to produce P(3HB-co-5HV) from glucose with different compositions of 5HV in the polymer.
  • removing competing 0 pathways that divert carbon away from 5HV-CoA such as removing the L- lysine export protein argO or the two lysine decarboxylase genes cadA and idcC, increase 5HV incorporation into PHA.
  • Na4HB for use as substrate was prepared analogous to the method described for preparation of NaSHV in Example 1 with use of ⁇ - butyrolactone (GBL) in place of DVL.
  • the culture conditions used for copolymer production was also the same as that described in Example 1 with the difference that 4 g/L of Na4HB was added to the production medium.
  • analysis of polymer content of the MG 1655 [pMS93] culture proceeded as described in Example 1 except that a standard curve for determining 4HB content was made using GBL standards in addition to the standard curve made for determining the 5HV content.
  • the three strains were grown in EO minimum medium containing 2 g/L 5-aminopentanoate as described earlier and glutarate was measured by a GC-MS method from culture supernatants.
  • the incubation method and conditions were the same as described in Example 7.
  • MG1655 ⁇ gabD [pJB91] did not accumulate any detectable glutarate.
  • the dehydrogenase encoded by gab D has the major activity towards GSA. Therefore, if high amounts of glutarate is to be produced, production hosts need to express gabD or homologues (see Table IP) thereof.
  • gabD or homologues see Table IP
  • MGl 655AyneI [pJB91] accumulated slightly lower amounts of glutarate from 5-aminopentanoate as compared to MG1655 [pJB91], 0.75 g/L versus 1.0 g/L glutarate, respectively, the dehydrogenase encoded by ynel also has moderate activity towards GSA.
  • overexpression of ynelo ⁇ homologues (see Table IQ) thereof may also yield high amounts of glutarate from GSA, L-lysine or glucose.
  • the two best GabD homologues present in Corynebacterium glutamicum include (1 ) the NAD-dependent aldehyde dehydrogenase (Accession No. NP_599302) and (2) the hypothetical protein cgR_0068 (Accession No. YP_001136931). Unexpectedly, these two C. glutamicum proteins were also identified as the two closest homologues to E. coli Ynel. Next, glutarate was produced from glucose.
  • plasmid pDE033 which contains the L-lysine feedback resistant dapA Ci52 ⁇ gene was constructed as follows: the product of SOE-PCR for the fabrication of dapA C352T gene described in Example 9 was digested with EcoRI and Sad, followed by ligation with ⁇ SE380 that had been digested with the same enzymes, thus creating plasmid pDE033.
  • the dapA Q5 ⁇ gene in pDE033 is under the IPTG-inducible promoter P,, c .
  • Plasmids pDEO33 and pJB91 (described above) were transformed into MG 1655 strain to create strain MG 1655 [pDE033, pJB91].
  • the strains MG1655 and MG1655 [pDEO33, pJB91] were incubated for 48 h with shaking at 300 rpm at 3O 0 C in a medium that contained 25 g/L glucose, 16 g/L (NH 4 ) 2 SO 3 , 1 g/L KH 2 PO 4 , 1 g/L MgSO 4 , 2 g/L yeast extract, 30 g/L CaCO 3 , 0.1 mM IPTG, and trace elements as described above.
  • the culture media for MG1655 [pDE033, pJB91] was supplemented with 100 ⁇ g/mL ampicillin and 25 ⁇ g/mL chloramphenicol. Glutarate was measured as described in Example 7.
  • Strain MBX3017 (LS5218 AadhE:: ⁇ RT, A!dhA::FRT, AackA- ptav.f KT) and K- 12 strain MGl 655 were used as a host strain to assess if 1 ,5-pentanediol (PDO) could be accumulated and secreted into the medium.
  • PDO 1 ,5-pentanediol
  • the CoA-dependent propionaldehyde dehydrogenase encoding pduP from S. typhimurium was amplified by primers JRG47 and JRG48.
  • the 1,3 -propanediol dehydrogenase encoding dhaT from Klebsiella pneumoniae was amplified with primers JRG49 and JRG50.
  • the two genes were fused together by SOE-PCR using primers JRG47 and JRG50.
  • Strain MBX3017 or MG1655 harboring pFS16 or pSE380 and pJGlO or pJB78 were used for PDO studies. Strains were grown under oxygen limited conditions in 5HV- containing E2 medium for 40 h. The medium consisted of: 10g/L glucose, 2g/L 5HV, 26 niM NaNH 4 HPO 4 , 33 mM K 2 HPO 4 , 27 mM KH 2 PO 4 , 2 niM MgSO 4 , 25 ⁇ g/mL chloramphenicol, 100 ⁇ g/mL ampicillin, 0.1 mM IPTG, and trace elements as described above.
  • the acetonitrile solution was centrifuged to remove insoluble material, and the supernatant was injected into an Agilent 5975 GC-MS equipped with a DB-225ms column using the following acquisition parameters: carrier gas Helium flow rate 1 ml/min, scan mode m/z 30-400, oven program: 4O 0 C for 2 min, then ramp up to 220 0 C at 10°C/min, ion source temperature 230 0 C, the quadrupole mass filter temperature 150 0 C.
  • Gene ID 002 Nucleotide Sequence: Arabidopsis thaliana glutarate semialdehyde reductase gene gsaR At

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Abstract

Recombinant hosts for producing polyhydroxyalkanoates and methods of producing polyhydroxyalkanoates from renewable carbon substrates are provided. Certain recombinant hosts that produce 5 carbon chemicals such as 5-aminoρentanoate (5AP), 5 -hydroxy valerate (5HV), glutarate, and 1,5 pentanediol (PDO) are also provided. One embodiment provides a recombinant host expressing a gene encoding a heterologous enzyme selected from the group consisting of a polyhydroxyalkanoate synthase and a 5-hydroxyvalerate-CoA (5HV-CoA) transferase, wherein the host produces a polymer containing 5-hydroxyvalerate. Preferably, the host expresses both a polyhydroxyalkanoate synthase and a 5HV-CoA transferase. The host can be prokaryotic or eukaryotic. A preferred prokaryotic host is E. coli. The polymers produced by the recombinant hosts can be homopolymers or copolymers of 5-hydroxyvalerate. A preferred copolymer is poly(3-hydroxybutyrate-co-5-hydroxyvalerate).

Description

GREEN PROCESS AND COMPOSITIONS FOR PRODUCING
POLY(SHV) AND 5 CARBON CHEMICALS
FIELD OF THE INVENTION The invention is generally related to transgenic organisms that produce polyhydroxyalkanoates and co-polymers thereof containing 5- hydroxyvalerate and chemical intermediates containing five carbon atoms (C5 chemicals).
CROSS REFERENCE TO RELATED APPLICATION This application claims benefit of and priority to US Provisional
Patent Application No, 61/122,250 filed on December 12, 2008, and where permissible is incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION Polyhydroxyalkanoates (PHAs) are biodegradable plastics which can be used to make, without limitation, films (e.g., packaging films, agricultural films, mulch film), golf tees, caps and closures, agricultural supports and stakes, paper and board coatings (e.g., for cups, plates, boxes, etc), thermoformed products (e.g., trays, containers, yogurt pots, plant pots, noodle bowls, moldings, etc.), housings (e.g., for electronic items) bags (e.g., trash bags, grocery bags, food bags, compost bags, etc.), hygiene articles (e.g., diapers, feminine hygiene products, incontinence products, disposable wipes, etc.) and coatings for pelleted products (e.g., pelleted fertilizer, herbicides, pesticides, seeds, etc.). PHAs have also been used to develop biomedical devices including sutures, repair devices, repair patches, slings, cardiovascular patches, orthopedic pins, adhesion barriers, stents, guided tissue repair/regeneration devices, articular cartilage repair devices, nerve guides, tendon repair devices, bone marrow scaffolds, and wound dressings.
Polyhydroxyalkanoates can be produced by a fermentation process. Existing fermentation methods for producing polyhydroxyalkanoates utilize wild-type or transgenic microorganisms cultured on specific substrates to produce the desired PHA polymer composition. In many cases the polymers of interest are copolymers of the (D)-isomer of 3-hydroxybutyrate copolymerized with one other 3, 4 or 5-hydroxyacids. These copolymers are produced as granular inclusions inside the cells and are random copolymers. The copolymer poly(3-hydroxybutyrate-co-5-hydroxyvalerate) (PHB5HV) and the homopolymer poly(5-hydroxyvalerate) (P5HV) are industrially useful as materials and plastics with the advantage that they are biodegradable and bioresorbable materials. To date these materials have been produced by feeding petroleum derived 5-carbon substrates like 5- hydroxy valeric acid (5HV) or 1 ,5-pentanediol to a microorganism which has the capability to metabolize these substrates to the activated monomer 5HV- Coenzyme A and polymerize it by the action of a PHA polymerase to form the PHB5HV or P5HV polymers, PHB5HV and P5HV polymers produced by these methods are only partly made from renewable resources and expensive due to the high cost of the 5-carbon petroleum substrates. It is highly desirable to use non-petroleum renewable carbon substrates as feedstock for the production of PHV5HV and P5HV polymers both to lower cost and to provide materials that are made entirely from renewable resources. It is also desirable to develop processes for the production of these polymers which reduce the production of greenhouse gasses. Suitable renewable resources include carbohydrate feedstocks available from agriculture including one or more feedstocks selected from: starch, sucrose, glucose, lactose, fructose, xylose, maltose, arabinose and amino acid feedstocks including lysine and proline.
Therefore, it is an object of the invention to provide recombinant organisms and processes whereby genes can be introduced in wild-type or genetically engineered polyhydroxyalkanoate producers to create new strains that synthesize monomers, such as 5 -hydroxy valerate, that are produced from substrates that are not derived from petroleum.
A further object of the invention is to provide techniques and procedures to stably engineer recombinant organisms that synthesize PHAs containing 5 -hydroxy valerate either as sole constituent or as a co-monomer. It is another object of the invention to provide techniques and procedures to stably engineer recombinant organisms that synthesize 5 carbon chemicals such as 5-aminopentanoate (5AP), glutarate, and 1,5 pentanediol (PDO). SUMMARY OF THE INVENTION
5HV Containing PHA Biopolymers
Recombinant hosts for producing polyhydroxyalkanoates (PHAs) comprising 5 -hydroxy valerate (5HV) monomers and methods of producing PHAs comprising 5HV monomers from renewable carbon substrates are provided. Certain recombinant hosts that produce 5 carbon chemicals such as 5~aminopentanoate (5AP), 5HV, glutarate, and 1,5 pentanediol (PDO) are also provided. One embodiment provides a recombinant host expressing genes encoding a polyhydroxyalkanoate (PHA) synthase and a 5-hydroxyvalerate- CoA (5HV-CoA) transferase or 5HV-CoA synthetase and at least one transgene encoding a heterologous enzyme involved in lysine catabolic pathways wherein the host produces a PHA polymer containing 5HV monomers when the organism is provided with a renewable carbon substrate selected from: lysine, starch, sucrose, glucose, lactose, frucrose, xylose, maltose, arabinose or combinations thereof and the level of 5HV monomer produced is higher than in the absence of expression of said transgene(s). An exemplary host expresses one or more genes encoding lysine 2- monooxygenase, 5-aminopentanamidase, 5-aminopetanoate transaminase, glutarate semialdehyde reductase, 5 -hydroxy valerate CoA-transferase, and polyhydroxyalkanoate synthase to produce a PHA polymer containing 5HV monomers. Preferably the host has deletions or mutations in genes encoding glutarate semialdehyde dehydrogenase and/or lysine exporter encoding genes. Particularly suitable hosts also have the ability to overproduce lysine and are resistant to toxic lysine analogs, like S~(2-aminoethyl) cysteine.
In a further embodiment one or more of the genes encoding PHA synthase, 5HV-CoA transferase or 5HV-CoA synthetase is also expressed from a transgene. In another embodiment the recombinant organism is fed lysine in combination with one or more renewable carbon substrates selected from: starch, sucrose, glucose, lactose, frucrose, xylose, maltose, arabinose or combinations thereof such that the 5HV containing PHA polymer is produced and the polymer is recovered from the cells. In another embodiment the recombinant organism is fed one or more renewable carbon substrates selected from: starch, sucrose, glucose, lactose, fructose, xylose, maltose, arabinose or combinations thereof such that the 5HV containing PHA polymer is produced and the polymer is recovered from the cells.
The polymers produced by the recombinant hosts can be homopolymers or copolymers of 5HV monomers. A preferred copolymer is PHB5HV. Other useful polymers produced by the recombinant hosts are the copolymers poly(3-hydroxypropionate-co-5-hydroxyvalerate) and poly(4- hydroxybutyrate-co-5-hydroxyvalerate) and the homopolymer P5HV.
The host can be prokaryotic or eukaryotic. Preferred prokaryotic hosts are E. coli, Ralstonia eutropha, Alcaligenes lotus and C, glutamicum. Recombinant hosts for producing PHA polymers from lysine, or one or more renewable carbon substrate selected from starch, sucrose, glucose, lactose, fructose, xylose, maltose, arabinose or or a combinations thereof are also provided. An exemplary host expresses lysine 2-monooxygenase, 5- aminopentanamidase, 5-aminopetanoate transaminase, glutarate semialdehyde reductase, 5 -hydroxy valerate CoA-transferase, and polyhydroxyalkanoate synthase to produce a polymer including 5HV. The polymer is produced using lysine and one or more renewable carbon substrate selected from starch, sucrose, glucose, lactose, fructose, xylose, maltose and arabinose as a feedstock. Preferably the host has deletions in glutarate semialdehyde dehydrogenase and lysine export encoding genes.
1,5-PentanedioI Production Another recombinant host is genetically engineered to overexpress 5- hydroxyvalerate CoA transferase, CoA dependent propionaldehyde dehydrogenase, and 1,3 -propanediol dehydrogenase to produce 1,5 pentanediol. 1,5 pentanedϊol is produced using 5 -hydroxy valerate, lysine starch, sucrose, glucose, lactose, trucrose, xylose, maltose and arabinose alone or in combinations as feedstock. Preferably the recombinant host has deletions in adhE, idhA, and ackA-pta and expresses lysine 2- monooxygenase, 5-aminoρentanamidase, 5-aminopetanoate transaminase and one or more glutarate or succinate semialdehyde reductase encoding genes. Particularly suitable hosts have the ability to overproduce lysine and are resistant to toxic lysine analogs, like S-(2-aminoethyl) cysteine. Preferably, the organism has a reduced or no glutarate semialdehyde dehydrogenase activity.
A method for producing 1,5-pentanediol from renewable carbon substrates is provided where a recombinant organism is fed a renewable carbon substrate and 1,5-pentanediol is produced, secreted to the medium and recovered therefrom.
In another embodiment the invention provides 1,5-pentandiol produced from renewable resources. Glutaric Acid Production
Recombinant hosts for overproducing glutarate (glutaric acid) from lysine, or one or more renewable carbon substrate selected from starch, sucrose, glucose, lactose, fructose, xylose, maltose and arabinose, or a combination thereof are also provided. An exemplary host expresses lysine 2-monooxygenase, 5-aminopentanamtdase, 5-aminopetanoate transaminase and one or more glutarate semialdehyde dehydrogenase encoding genes. Particularly suitable hosts have the ability to overproduce lysine and are resistant to toxic lysine analogs, like S-(2-aminoethyl) cysteine,
A method for overproducing glutarate from renewable carbon substrates is provided where a recombinant organism is fed a renewable carbon substrate selected from lysine, starch, sucrose, glucose, lactose, frucrose, xylose, maltose and arabinose, or combinations thereof and glutarate is overproduced, secreted to the medium and recovered therefrom. BRIEF DESCRIPTIONS OF THE DRAWINGS Figure 1 is a schematic diagram showing the various 5 -carbon molecules that can be produced biologically from renewable resources in some instances these renewable resource based molecules can be interconverted using standard chemistry and used to make polymers etc. by chemical polymerization processes. Figure 2A is a schematic diagram showing biochemical pathways to
5 -hydroxy valerate containing polyhydroxyalkanoate polymers, and 5-carbon chemicals such as 5-aminopentanoate (5-APO), glutarate, δ-valero lactone (DVL) andl,5 pentanediol. Also shown are competing metabolic pathways that may have to be removed or the activities reduced (as indicated by a cross (X)) to achieve optimal carbon flux to the desired products listed above. Figure 2 B shows enzymes catalyzing the biosynthetic reactions: (1) lysine 2- monooxygenase, EC 1.13.12.2; (2) 5-aminopentanamidase (a.k.a. δ- aminovaleramidase), EC 3.5.1.30; (3) 5-aminopentanoate transaminase (a.k.a. δ-aminovalerate transaminase), EC 2.6.1.48; (4) succinate semialdehyde reductase (a.k.a. 5-oxopentanoate reductase), EC 1.1.1.61 ; (5) CoA-transferase, EC 2.8.3.n; (6) Acyl-CoA synthetase, EC 6.2.1.3; (7) PHA synthase, EC 2.3. Ln; (8) β-ketoacyl-CoA thiolase, EC 2.3.1.9; (9) acetoacetyl-CoA reductase, EC 1.1.1.36; (10) glutarate-semialdehyde dehydrogenase, EC 1.2.1.20.
Figure 3 is a schematic diagram showing biochemical pathways of an alternate route from L-lysine to 5-aminopentanoate via cadaverine and 5- aminopentanal. Figure 4 is a schematic diagram showing biochemical pathways from
L-proline to 5-aminopentanoate.
Figure 5 is a schematic diagram showing biochemical pathways from alpha-ketoglutarate to glutarate semialdehyde, a metabolic intermediate to produce 5 -hydroxy valerate and its derivatives, and glutarate. Figure 6 is a schematic diagram showing biochemical pathways from oxaloacetate, an intermediate of the TCA cycle, to L-lysine.
Figure 7 is a schematic diagram showing biochemical pathways to 1,5 pentanediol.
Figure 8A is chromatogram showing time (minutes) versus total ion abundance of processed cell culture from strain 3291. Figure 8B is an ion spectrum of processed cell culture from strain 3291 showing mass-to-charge ratio "m/z" versus its ion abundance (relative units).
DETAILED DESCRIPTION OF THE INVENTION I. Definitions A number of terms used herein are defined and clarified in the following section.
The term "PHA copolymer" refers to a polymer composed of at least two different hydroxy alkanoic acid monomers. The term "PHA homopolymer" refers to a polymer that is composed of a single hydroxyalkanoic acid monomer.
As used herein, a "vector" is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. The vectors can be expression vectors.
As used herein, an "expression vector" is a vector that includes one or more expression control sequences.
As used herein, an "expression control sequence" is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence.
As used herein, "operably linked" means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest. As used herein, "transformed" and "transfected" encompass the introduction of a nucleic acid into a cell by a number of techniques known in the art.
As used herein "overproduced" means that the particular compound is produced at a higher quantity in the engineered organism as compared to the non-engineered organism.
As used herein the terms "renewable feedstock", "renewable carbon substrate" and "renewable substrate" are all used interchangeably.
"Plasmids" are designated by a lower case "p" preceded and/or followed by capital letters and/or numbers. As used herein the term "heterologous" means from another host.
The other host can be the same or different species.
II. Metabolic Pathways for Producing Poϊyhydroxyaϊkanoates and 5 Carbon Chemicals
Recombinant organisms having enzymes for the biochemical pathways for production of 5 -hydroxy valerate containing polyhydroxyalkanoate polymers, and 5-carbon chemicals such as 5- aminopentanoate (5AP), 5-hydroxyvalerate (5HV), glutarate, and 1,5 pentanediol (PDO) are provided. Prokaryotic or eukaryotic hosts are genetically engineered to express the enzymes needed to produce 5- hydroxy valerate, polymers thereof, or the disclosed 5 -carbon chemicals from renewable resource based feedstocks. The enzymatic pathways for producing the desired products are provided below.
A. 5-aminopentanoate 5-aminopentanoate (5AP) can be produced in two enzymatic steps from L-lysine, an α-amino acid, with 5-aminopentanamide as the intermediate (Figure 2). The first of these enzymes, lysine 2-monooxygenase is the first enzymatic step in the lysine degradation pathway of various pseudomonad strains (Takeda and Hayaishi, J. Biol. Chem. 241 :2733-2736; (1966); Hayaishi, Bacteriol. Rev. 30:720-731 (1966); Reitz and Rodwell, J. Biol Chem. 245: 3091-3096 (1970); Flashner and Massey, J. Biol. Chem. 249: 2579-2586 (1974)). The gene encoding lysine 2-monooxygenase was identified in Pseudomonas putida and called davB (Revelles et al., J. Bacteriol. 187:7500-7510 (2005)). The second enzymatic step converts 5- aminopentanamide to 5AP and is catalyzed by 5-aminopentanamidase (Reitz et al., Anal. Biochem. 28:269-272 (1969); Reitz and Rodwell, J. Biol. Chem. 245: 3091-3096 (1970)), which is encoded by davA in P. putida (Revelles et al., J. Bacteriol. 187:7500-7510 (2005)).
As shown in Figure 3, an alternate pathway can be utilized to convert L-iysine to 5 AP in three enzymatic reactions which include a lysine decarboxylase to produce cadaverine, a putrescine transaminase to form 5- aminopentenal, and a γ-aminobutyraldehyde dehydrogenase to biosynthesize 5AP.
As outlined in Figure 4, 5AP may also be produced from L-proline, instead of L-lysine, in two enzymatic reactions which include a proline racemase to biosynthesize D-proline and a proline reductase to form 5 AP. B. 5-hydroxyvalerate
Biosynthesis of another 5-carbon chemical, 5HV, can occur from 5AP with 2 enzymatic steps as outlined in Figure 2. 5AP is converted to glutarate semialdehyde by 5AP transaminase (Reitz and Rodwell, J. Biol. Chem. 245: 3091-3096 (1970)) and a gene from P. putida which was identified and named davT (Espinosa-Urgel and Ramos, Appl. Environ. Microbiol. 67:5219-5224 (2001)). As outlined in Example 7, several recombinant semialdehyde reductase genes were cloned and tested to investigate which encoded enzyme efficiently converted glutarate semialdehyde to 5HV. The hypothetical protein ATEG_00539 was discovered to efficiently catalyze this reaction and was hence renamed gsaR for glutarate semialdehyde reductase.
In some thermophilic bacteria and lower fungi including yeasts, lysine is synthesized via the α-aminoketoadipate pathway (Xu, Cell Biochem. Biophys. 46:43-64 (2006)) in which 2-ketoadipate is the fourth intermediate and therefore a potential precursor for C5 chemicals such as glutarate and 5HV, as well as for 5HV-containing PHA polymers. This pathway starts from α-ketoglutarate and acetyl-CoA to biosynthesize 2- ketoadipate as shown in Figure 5. Recombinant E. coli host strains expressing these four enzymes were shown to produce 2-ketoadipate (Andi et al, Biochem. 43:1 1790-1 1795 (2004); Jia et al., Biochem. J. 396:479-485 (2006); Miyazaki et al, J. Biol. Chem. 278:1864-1871 (2003)). 2-
Ketoadipate may be converted by an α-ketoglutarate dehydrogenase to glutaryl-CoA due to the structural similarity of α-ketoglutarate and 2- ketoadipate. Glutaryl-CoA may be converted by an succinic semialdehyde (SSA) dehydrogenase such as succinyl-CoA synthetase (SucD) (Sδhling and Gottschalk J. Bacterid. 178:871-880 (1996)), again due to the structural similarity of succinyl-CoA and glutaryl-CoA. C. glutarate
Biosynthesis of the 5-carbon chemical, glutarate, proceeds from glutarate semialdehyde via a dehydrogenase reaction (Ischihara et al, J. Biochem. (Tokyo) 49:154-157 (1961); Reitz and Rodwell, J. Biol. Chem. 245: 3091-3096 (1970)) as outlined in Figure 2. The davD gene was identified to encode such a glutarate semialdehyde dehydrogenase activity in P. putida (Espinosa-Urgel and Ramos, Appl. Environ. Microbiol. 67:5219- 5224 (2001); Revelles et al., J. Bacterid. 186:3439-3446 (2004)). Glutarate is useful for the production of polymers such as polyesters, polyester polyols and polyamides. The odd number of carbon atoms (i.e. 5) is useful in for example decreasing polymer elasticity. In addition, 1,5-pentanediol is a common plasticizer and precursor to polyesters that is manufactured by hydrogenation of glutarate and its derivatives (Werle and Morawietz, "Alcohols, Polyhydrϊc" in: Ulhnann's Encyclopedia of Industrial Chemistry: 2002, Wiley-VCH: Weinheim. DOI 10.1002/14356007.a01_305).
D, poly(5-hydroxyvalerate)
Biosynthesis of a homopolymer consisting of poly(5- hydroxyvalerate) (a.k.a. P(SHV)) PHA can proceed from 5HV via 5- hydroxyvaϊerate-CoA. Two different enzymatic reactions may catalyze the first step, i.e. either by a CoA-transferase as described by Huisman et al. (U.S. Patent No. 7,229,804), Sδhling and Goltschalk (J. Bacterid. 178:871- 880 (1996)), and Eikmanns and Buckel (Biol. Chem. Hoppe-Seylβr 371 :1077- 1082 (1990)), or by a CoA-synthetase as described by van Beilen et al. (Molec. Microbiol. 6:3121-3136 (1992)). Polymerization of 5- hydroxyvalerate-CoA may be catalyzed by PHA polymerase such as encoded by the Ralstonia eutropha phaCl (Peoples and Sinskey, J. Biol. Chem. 264:15298-15303 (1989)). Alternatively, the PhaC3/C5 synthase fusion protein may be employed as described by Huisman et al. (U. S . Patent No. 6,316,262).
E. poIy(3-hydroxybufryrate-co-5-hydroxyvaϊerate) Biosynthesis of a copolymer including ρoly(3-hydroxybutyrate-co-5- hydroxyvalerate) (a.k.a. P(3HB-co-5HV)) can occur by supplying 3- hydroxybutyryl-CoA (3HB-CoA) and 5-HV-CoA monomer precursor molecules. As outlined in Figure 2, 3HB-CoA can be biosynthesized from acetyl-CoA via 2 enzymatic steps: (i.) a β-ketoacyl-CoA thiolase reaction that converts acetyl-CoA to acetoacetyl-CoA (Nishimura et al., J. Biol. Chem. 116:21-27 (1978)) using suitable genes such as, but not limited to, bktB from Ralstonia eutropha (Slater et al5 J. Bacteriol. 180(8):1979-1987 (1998)) and (ii.) an acetoacetyl-CoA reductase reaction that converts acetoacetyl-CoA to 3HB-CoA (Fukui et al., Biochim. Biophys. Acta 917:365-371 (1987)) using suitable genes such as, but not limited to, phaB from Bacillus megaterium (McCool and Cannon, J. Bacteriol. 181(2):585- 592 (1999)). As outlined above, PHA co-polymer can by synthesized by various PHA synthases. F. Lysine
Figure 6 outlines the biosynthetic pathway of lysine metabolism in E. coii. Dotted and solid lines in the center of the diagram indicate allosteric feedback inhibition and transcriptional repression by L-lysine, respectively, which provide targets for genetic modifications necessary to increase L- Iy sine production in recombinant host cells such as E. coii.
G. 1,5-penlanediol
Figure 7 provides an overview of the production of 1,5-pentanediol (PDO) from 5HV. 5HV may be converted to 5HV-CoA by a CoA- transferase as described by Huisman et al. (U.S. Patent No. 7,229,804), Sδhling and Gottschalk (J. Bacterid. 178:871-880 (1996)), and Eikmanns and Buckel (Biol Chem. Hoppe-Seyler 371 :1077-1082 (1990)), or by a CoA-synthetase as described by van Beilen et al. (Molec, Microbiol. 6:3121- 3136 (1992)). 5HV may be converted to 5-hydroxypentenal by a propionaldehyde dehydrogenase or alcohol dehydrogenase such as pduP from Salmonella typhimurium (Leal, Arch. Microbiol. 180:353-361 (2003)) or eutE from K coii (Skraly, WO Patent No. 2004/024876). 5- Hydroxypentenal may be converted to PDO by 1,3 -propanediol dehydrogenase or alcohol dehydrogenase such as dhaT from Klebsiella pneumoniae (Tong et al., Appl. Environ. Microbiol. 57(12):3541-3546 (1991)).
H. S-hydroxyvalerate-co-S-hydroxypropionate A copolymer containing 5-hydroxyvalerate-co-hydroxyproprionate can be produced from 5HV. E. coii Kl 2 strains that were either fadlt (repressed fatty acid degradation (FAD)) oxfadK (constitutive FAD) are transfected with nucleic acids expressing polyhydroxyalkanoate and 5- hydroxyvalerate CoA transferase. Preferred E. coii strains include MG 1655 and LS5218 (Spratt et al., J. Bacteriol 146(3): 1166- 1169 (1981)). As shown in Table 6, GC-FID analysis indicated that LS5218 [ρMS93] produced 6.4%dcw PHA, with a polymer composition of 52% 5HV and 48% 3HP. MG1655 [pMS93], on the other hand, made 63.1%dcw PHA that consisted only of 5HV. Furthermore, GC-MS analysis of LS5218 [pMS93] confirmed the presence of 3HP in the polymer sample. Thus, the active FAD system in LS5218 is able to synthesize 3HP from NaSHV. III. Production of Transgenic Organisms for Producing Polyhydroxyalkanoates and 5 Carbon Chemicals
Transgenic organisms for producing polyhydroxyalkanoates and 5 carbon chemicals are produced using conventional techniques known in the art.
A. Genes for Producing Transgenic P(SHV) Producers
The genes cloned and/or assessed for host strains producing 5HV- containing PHA and 5 -carbon chemicals are presented below in Table IA, along with the appropriate Enzyme Commission number (EC number) and references. As discussed further below, some genes were synthesized for codon optimization while others were cloned via PCR from the genomic DNA of the native or wild-type organism.
Table IA. Genes in microbial host strains producing 5HV-containing PHA and 5 -carbon chemicals.
Figure imgf000014_0001
Figure imgf000015_0001
Other proteins capable of catalyzing the reactions listed in Table IA can be discovered by consulting the scientific literature, patents or by BLAST searches against e.g. nucleotide or protein database at NCBI (www.ncbi.nlm.nih.gov/). Synthetic genes can then be created to provide an easy path from sequence databases to physical DNA. Such synthetic genes are designed and fabricated from the ground up, using codons to enhance heterologous protein expression, optimizing characteristics needed for the expression system and host. Companies such as e.g. DNA 2.0 (Menlo Park, CA 94025, USA) will provide such routine service. Proteins that may catalyze the biochemical reactions listed in Table IA are provided in Tables IB -IAA.
Table IB. Suitable homologues for the DavB protein (lysine 2- monooxygenase, from Pseudomonas putida KT2440, EC No, 1.13.12.2, which acts on L-Iy sine to produce 5-aminoρentanamide; protein ace. no. BAG54787 (Revelles et al., J Bacteriol. 187:7500-10 (2005))).
Protein Name Protein Accession
No. amine oxidase YPJΪ01265764 amine oxidase YPJKM666658 amine oxidase YPJ)01751665 amino oxidase YP_606177 amine oxidase, flavin- YP_262728 containing tryptophan 2- YP_350882 monooxygenase tryptophan 2- ZP_04590895 monooxygenase tryptophan 2- NP_790366 monooxygenase tryptophan 2- ZP 02189967 monooxygenase
Table 1C. Suitable homologues for the DavA protein (5- aminopentanamidase, from Pseudomonas putida KT2440, EC No. 3.5.1.30, which acts on 5-aminopentanamide to produce 5-aminopentanoate, protein ace. no. BAG54788 (Revelles et al., J Bacteriol. 187:7500-10 (2005))).
Protein Name Protein Accession
No.
Nitrilase/cyanide YP 001265763 hydratase and apolipoprotein N- acyltransferase Nitrilase/cyanide YP 001666657 hydratase and apolipoprotein N- Protein Name Protein Accession No. acyltransferase Nitrilase/cyanide YP 001751666 hydratase and apolipoprotein N- acyltransferase amidohydrolase YP_606176 carbon-nitrogen family NPJ790365 hydrolase carbon-nitrogen family ZPJH590894 hydrolase putative hydrolase YPJ)02875091 Nitrilase/cyanide YP 350883 hydratase and apolipoprotein N- acyltransferase nitrilase YP 703491
Table ID. Suitable homologues for the DavT protein (5-aminoρentanoate transaminase, from Pseudomonas putida KT2440, EC No. 2.6.1.48, which acts on 5-aminopentanoate to produce glutarate semialdehyde; Protein ace, no. AAK97868 (Espinosa-Urgel and Ramos, Appl. Environ. Microbiol. 67 (H)5 5219-5224 (2001))).
Protein Name Protein Accession No.
4-aminobutyrate YPJ788435 aminotransferase
4-aminobutyrate YP_002294190 aminotransferase
4-aminobutyrate YPJ45921 aminotransferase
4-aminobutyrate YPJ)02801747 transaminase
4-aminobutyrate YPJX) 1333938 aminotransferase
4-aminobutyrate NPJ790151 aminotransferase
4-aminobutyrate NP_417148 aminotransferase, PLP- dependent
4-aminobutyrate YP_311652 aminotransferase
4-aminobutyrate YP 257332 aminotransferase Table IE. Suitable homologues for the GabT protein (4-aminobutyrate transaminase, from Escherichia coli str. K- 12 substr. MGl 655, EC No. 2.6.1.48 (or EC No. 2.6.1.19), which acts on 5-aminopentanoate (or 4 aminobutyrate) to produce giutarate semialdehyde (succinic semialdehyde); Protein Ace. No. NP_417148 (Riley et al, Nucleic Acids Res. 34 (1), 1-9 (2006))).
Protein Name Protein Accession
No.
4-aminobutyrate ZPJ35433421 aminotransferase
4-aminobutyrate YPJ)02381614 aminotransferase hypothetical protein ZP_03838094
CIT292_04138
4-aminobutyrate YP_001333938 aminotransferase
4-aminobutyrate NP_461718 aminotransferase
4-aminobutyrate NP^248957 aminotransferase
4-aminobutyrate YP_964435 aminotransferase
4-aminobutyrate YP_982853 aminotransferase
4-aminobutyrate YP 583770 aminotransferase Table IF. Suitable homologues for the GsaRAt2 protein (giutarate semialdehyde reductase, from Aspergillus terreus NIH2624, EC No. 1 ,1.1.61, which acts on giutarate semialdehyde (or succinic semialdehyde) to produce 5-hydroxyvalerate (or 4-hydroxybutyrate); Protein ace. no. XPJ)01210625 (Birren, The Broad Institute Genome Sequencing Platform, direct submission to NCBI))).
Protein Name Protein Accession
No. afiatoxin B 1 -aldehyde CBF8901 1 reductase GliO-like, putative afiatoxin B 1 -aldehyde XP 752707 reductase GHO- like afiatoxin Bl -aldehyde reductase, putative afiatoxin B 1 -aldehyde XP 001264422 reductase GliO-like, putative afiatoxin B 1 aldehyde reductase XP 002375825 Protein Name Protein Accession
No. hypothetical protein EEH21318 An08g06440 aflatoxin B 1 aldehyde reductase member, putative
XP 001392759
EER27170
Chain A, Mouse 2C91 _A
Succinic Semialdehyde
Reductase, Akr7a5
Chain A, Structure Of 2BP1_A
The Aflatoxin
Aldehyde Reductase In
Complex with NADPH
Table IG. Suitable homologues for the GsaRAt protein (glutarate semialdehyde reductase, from Arabidopsis thaliana, EC No. 1.1.1.61 , which acts on glutarate semialdehyde (or succinic semialdehyde) to produce 5- hydroxyvalerate (or 4-hydroxybutyrate); Protein ace. no. AAK94781 (Breitkreuz et al., J. Biol. Chem. 278 (42), 41552-41556 (2003))).
Protein Name Protein Accession No. hypothetical protein XP_0022ό6252 isoform 1 predicted protein XP_p02320548 Os02g0562700 NP_001047154 succinic semialdehyde BAG16485 reductase isofoml unknown ACU22717 hypothetical protein XP 002452295 SORBIDRAFT J)4gO2 3180
NAD-dependent 4- AAC41425 hydroxybutyrate dehydrogenase 1, 3 -propanediol ZP_00945634 dehydrogenase NAD-dependent A- AAA92348 hydroxybutyrate dehydrogenase NAD-dependent 4- NP 348201 Protein Name Protein Accession
No. hydroxybutyrate dehydrogenase
Table IH. Suitable homologues for lhe OrfZ protein (CoA-transferase, from Clostridium Jduyveri DSM 555, EC No. 2,8.3.n, which acts on 5- hydroxyvalerate to produce 5-hydroxyvaleryl-CoA; protein ace. no. AAA92344 (Huisman et al., U.S. Patent No. 7,229,804; Sohling and Gottschalk, J. Bacterid. 178:871-880 (1996))).
Protein Name Protein Accession No. acetyl-CoA ZP_05395303 hydrolase/transferase acetyl-CoA YPJ)01309226 hydrolase/transferase
4-hydroxybutyrate ZP_05618453 coenzyme A transferase
4-Hydroxybutyrate CAB60036
CoA-transferase
4-hydroxybutyrate NP_904965
CoA-transferase
4-hydroxybutyrate ZP_05427217
CoA-transferase acetyl-CoA YP_002430388 hydrolase/transferase acetyl-CoA YP_001433830 hydrolase/transferase acetyl-CoA YP 002509648 hydrolase/transferase
Table II. Suitable homologues for AIkK protein (Acyl-CoA synthetase, from Pseudomonas oleovorans; EC No. 6.2.1.3, which acts on 5- hydroxyvalerate to produce 5-hydroxyvaleryl-CoA; protein ace. no. Q00594 (van Beilen et al., MoI. Microbiol. 6 (2I)5 3121-3136 (1992))).
Protein Name Protein Accession No.
AMP-dependent YPJ)01185941 synthetase and ligase medium-chain acyl- ABO21016 CoA synthetase acyl-CoA synthetase CAB69080 medium-chain acyl- ZP_06063626 CoA synthetase acyl-CoA synthetase YP_523641 AMP-dependent ZP 03542412 synthetase and ligase Protein Name Protein Accession No. acyl-CoA synthetase YPJ726019 medium-chain-fatty- ZP_06016304 acid-CoA ligase medium-chain-fatty- ZP 02145453 acid--CoA ligase
Table IJ. Suitable homologues for the PhaC protein (polyhydroxyalkanoate synthase, from Ralstonia eutropha, EC No. 2.3.1.n, which acts on (R)-3- hydroxybutyryl-CoA + [(R)-3-hydroxybutanoate]n to produce [(R)-3- hydroxybutanoate](n+l) + CoA; Protein ace. no. YP_725940 (Peoples and Sinskey, J. Biol. Chem. 264:15298-15303 (1989))).
Protein Name Protein Accession
No. polyhydroxyalkanoic YP_002005374 acid synthase
PHB synthase BAB96552
PhaC AAF23364
Polyhydroxyalkanoate AAC83658 synthase protein PhaC polyhydroxybutyrate AAL17611 synthase poly(R)- YPJ302890098 hydroxyalkanoic acid synthase, class I poly-beta- YP 159697 hydroxybutyrate polymerase
PHB synthase CAC41638
PHB synthase YP 001100197
Table IK. Suitable homologues for the PhaE protein (PhaE subunit of PhaEC PHA synthase, from Thiocapsa pfenigii, which acts on (R)-3- hydroxybutyryl-CoA + [(R)-3-hydroxybutanoate]n to produce [(R)-3- hydroxybutanoate](n+l) + CoA).
Protein Name Protein Accession
No. orf2 5( to phbC AAC60429
Uncharacterized 40.5 P45372 kDa protein in phbC- phbA intergenic region hypothetical protein 2 S29275
Uncharacterized 41.3 P45367
IdDa protein in phbC- phbA intergenic region poly(R> ZP_04775558 Protein Name Protein Accession No. hydroxyalkanoic acid synthase, class III,
PhaE subunit hypothetical protein YPJ)02513584
Tgr7_1513
PHA synthase subunit AAG30260
PhaE
PoIy(R)- YP 865086 hydroxyalkanoic acid synthase, class III,
PhaE subunit
PHA synthase BAE20054
PHA synthase subunit ABK60192
E
Table IL. Suitable homologues for the PhaC protein (PhaC subunit of PhaEC PHA synthase, from Thiocapsa pfenigii, which acts on (R)-3- hydroxybutyryl-CoA + [(R)-3-hydroxybutanoate]n to produce [(R)-3- hydroxybutanoate](n+l) -f CoA).
Protein Name Protein Accession No. poly(R)- ZP_04774202 hydroxyalkanoic acid synthase, class III,
PhaC subunit
Poly-beta- P45366 hydroxybutyrate polymerase
PHA synthase subunit AAG30259
PhaC poly(R)- YP.J65087 hydroxyalkanoic acid synthase, class III,
PhaC subunit poly (3-hydroxybutyric YP_200860 acid) synthase poly(3- YP 001660017 hydroxyalkanoate) synthase
PhaC AAL76316 poly(3- NP 440750 hydroxyalkanoate) synthase poly(R)- YP 003151887 hydroxyalkanoic acid synthase, class III,
PhaC subunit Protein Name Protein Accession
No. poly(R)- YP 001374365 hydroxyalkanoic acid synthase, class IIIf
PhaC subunit
Table IM. Suitable homologues for the BktB (PhaA) protein (β-ketoacyl- CoA thiolase, from Ralstonia eutropha Hl 6, EC No. 2.3.1.9, which acts on acetyl-CoA to produce acetoacetyl-CoA; Protein ace. no. CAJ92573 (Peoples & Sinskey, J Biol Chem. 1989 Sep 15;264(26): 15293-7. Pohlmann et al, Nature Biotech 24 (10), 1257-1262 (2006))).
Protein Name Protein Accession No. acetyl-CoA YP_002005375 acetyltransferase acetyl-CoA YP_558680 acetyltransferase acetyl-CoA BAB96553 acetyltransferase acetyl-CoA YP_001021062 acetyltransferase beta-ketothiolase BAA33156 acetyl-CoA YP_523809 acetyltransferase acetyl-CoA NP_250691 acetyltransferase beta-ketothiolase YP_ 002802211 acetyl-CoA YP_001919890 acetyltransferase
Table IN. Suitable homologues for the PhaB protein (acetoacetyl-CoA reductase, from Bacillus megaterium, EC. No. 1.1.1.36, which acts on acetoacetyl-CoA to produce (R)-3-hydroxybutyryl-CoA; Protein ace. no. AAD05259 (McCool & Cannon, J. Bacteriol. 183 (14), 4235-4243 (2001))).
Protein Name Protein Accession No acetoacety 1-Co A NP_831099 reductase
3-ketoacyl-(acyl- YP_645133 carrier-protein) reductase
3-ketoacyl-(acyl- ZP_01126888 carrier-protein) reductase hypothetical protein YP_518893
DSY2660
3-ketoacyl-(acyl- YPJ)Ol 865860 Protein Name Protein Accession No. carrier-protein) reductase
3-ketoacyl-(acyl- YP 001658404 carrier-protein) reductase
3-oxoacyl-(acyl- YP 002949207 carrier-protein) reductase
3 -oxoacyl-(acyl-carrier NP 371755 protein) reductase
3-ketoacyl-(acyl- YP 001385912 carrier-protein) reductase
Table 1O. Suitable homologues for the DavD protein (glutarate- semialdehyde dehydrogenase, from Pseudomonas putida KT2440, EC No. 1.2.1,20, which acts on glutarate senaialdehyde to produce glutarate; Protein ace. no. NP_742381 (Espinosa-Urgel and Ramos, Appl. Environ. Microbiol.
67 (11), 5219-5224 (2001))).
Protein Name Protein Accession No. succinate- semialdehyde YP_605978 dehydrogenase I succinate-semialdehyde YP_345920 dehydrogenase I succinate-semialdehyde NP_417147 dehydrogenase I, NADP-dependent predicted aldehyde NP_416042 dehydrogenase succinate-semialdehyde NP_461717 dehydrogenase I Aldehyde YP_002801982 dehydrogenase succinic semialdehyde YP_002827846 dehydrogenase succinic semialdehyde YPJ)01228901 dehydrogenase succinate- semialdehyde YP 841054 dehydrogenase (NADP+) Table IP. Suitable homologues for the GabD protein (succinate semialdehyde dehydrogenase, NADP+-deρendent, from Escherichia coli str. K-12 substr. MG1655, EC No. 1.2.1.20, which acts on glutarate semialdehyde (or succinic semialdehyde) to produce glutarate (or succinate); Protein ace. no. NP_417147 (Riley et al, Nucleic Acids Res. 34 (1), 1-9 (2006))).
Protein Name Protein Accession
No. succinate-semialdehyde ZP_05433422 dehydrogenase I succinate-semialdehyde YP_001744810 dehydrogenase (NAD(PX+)) hypothetical protein ZPJB838O93 CH292JM137 succinate-semialdehyde YP_002638371 dehydrogenase succinate-semialdehyde YPJ)01333939 dehydrogenase I succinate-semialdehyde NPJ742381 dehydrogenase I succinate-semialdehyde YP_002932123 dehydrogenase [NADP+] (ssdh) succinic semialdehyde YPJ)01951927 dehydrogenase succinate semialdehyde YP 298405 dehydrogenase
Table IQ. Suitable homologues for the Ynel (Sad) protein (succinate semialdehyde dehydrogenase, NAD+-dependent, from Escherichia coli str. K-12 substr. MG1655, EC No. 1.2.1.24, which acts on glutarate semialdehyde (succinic semialdehyde) to produce glutarate (succinate); Protein ace, no. NP_416042 (Fuhrer et al., J Bacterid. 2007 Nov;189(22):8073-8. Dennis and Valentin, U.S. Patent No. 6,1 17,658))).
Protein Name Protein Accession
No. succinate NP_805238 semialdehyde dehydrogenase putative aldehyde YP _002919404 dehydrogenase aldehyde NP_745295 dehydrogenase aldehyde ZPJB269266 dehydrogenase aldehyde ZP_05726943 dehydrogenase Protein Name Protein Accession
No. aldehyde YPJX) 1906721 dehydrogenase hypothetical protein BAFO 1627 aldehyde ZP_03739186 dehydrogenase succϊnate- NP 637690 semialdehyde dehydrogenase
Table IR. Suitable homologues for the PduP protein (CoA-dependent propionaldehyde dehydrogenase, from Salmonella typhimurium LT2, EC No. 1.2.1.3, which acts on 5 -Hydroxy valery 1-CoA to produce 5- Hydroxypentanal; Protein ace. no. NP_460996 (Leal et al.s Arch. Microbiol. 180:353-361 (2003), McClelland et al., Nature 413: 852-856 (2001))).
Protein Name Protein Accession No. ethanolamine YP_002216136 utilization protein EutE
PduP ZP_04562531 propanediol utilization YP_002236771 protein ethanolamine NP_756394 utilization protein EutE aldehyde YP_961823 dehydrogenase possible aldehyde ZP_04969437 dehydrogenase
Ethanolamine ZP_04637794 utilization protein eutE
Aldehyde ZP_05373182
Dehydrogenase ethanolamine YP 002350444 utilization protein EutE
Table IS. Suitable homologues for the EutE protein (predicted aldehyde dehydrogenase, ethanolamine utilization protein, from Escherichia coli str. K-12 substr. MG1655, EC No. 1.2.1.3, which acts on 5-hydroxyvaleryl- CoAtp produce 5-hydroxypentanal, protein ace. no, NP_416950 (Riley et al., Nucleic Acids Res. 34 (1), 1-9 (2006))).
Protein Name Protein Accession No. hypothetical protein YP 001586756
SPAB_00490 ethanolamine YP __ 001336429 utilization protein probable ethanolamine ZP_01222600 Protein Name Protein Accession No. utilization protein
(EutE) putative aldehyde ZP_O33376O0 dehydrogenase ethanolamine ZPJ)4573939 utilization protein eutE
CoA-dependent ZP_00232619 propionaldehyde dehydrogenase
Aldehyde YP_003261430
Dehydrogenase
EutE YP_311399 hypothetical protein YP_001451939
CKO_00340 ethanolamine YP 066775 utilization protein
(EutE)
Table IT. Suitable homologues for the DhaT protein (1,3-propanediol dehydrogenase, from Klebsiella pneumoniae 342, EC No. 1.1.1,202, which acts on 5-hydroxypentanal to produce 1,5-pentanediol; Protein ace. no. YP_002236499 (Fouts et al., PLoS Genet. 4 (7), E1000141 (2008))).
Protein Name Protein Accession No.
1 , 3-propanediol ABD74004 dehydrogenase
1, 3 -propanediol YP_698334 dehydrogenase alcohol dehydrogenase YP_001211060
1 j 3-propanediol YPJ796272 dehydrogenase iron-containing alcohol YP_003192340 dehydrogenase conserved hypothetical ZP_06063679 protein hypothetical protein ZP_01723545
BB14905J2250
EutG protein ZP_02497862
1, 3-proρanediol AAXl 2915 dehydrogenase
1, 3-propanediol AAM54730 dehydrogenase Table IU. Suitable homologues for the EutG protein (predicted alcohol dehydrogenase in ethanolamine utilization, from Escherichia coli str. K- 12 substr. W3110, EC. No. 1.1.1.202, which acts on 5-hydroxypentanalto produce 1,5-pentanediol; Protein ace. no. AP_0O3038 (Riley et al, Nucleic Acids Res. 34 (1), 1-9 (2006))).
Protein Name Protein Accession No. ethanolamine YPJ)01881244 utilization protein EutG hypothetical protein YPJX) 1451941
CKOJ)0342 hypothetical protein YPJ)Ol 586758
SPAB_00492 putative transport YP_002920649 protein in ethanolamine utilization putative alchohol ZP_03365534 dehydrogenase
Ethanolamine ZPJ)2156849 utilization protein eutG
Ethanolamine ZPJW637792 utilization protein eutG eutG AAA80211
Iron-containing alcohol NP_ό34793 dehydrogenase ethanolamine ZP 06015246 utilization protein EutG
Table IV. Suitable homologues for the ArgO (YggA) protein (arginine export protein, from Escherichia coli str. K- 12 substr. MG 1655, which acts on L-lysine (cytoplasm) and produces L-lysine (outside); Protein ace. no. NP_417398 (Nandineni and Gowrishankar, J. Bacteriol. 186:3539-3546 (2004))).
Protein Name Protein Accession No. arginine exporter YP_409404 protein unnamed protein CAA32607 product arginine exporter ZPJ)4560344 protein arginine exporter NP_461982 protein arginine exporter YPJ)01336979 protein
Arginine exporter YP_003211829 protein argO
Arginine exporter YP J)02649944 Protein Name Protein Accession No. protein
Arginine exporter ZP_04613777 protein argO arginine exporter NP_930824 protein arginine exporter ZPJ) 1988459 protein ArgO
Table IW. Suitable homologues for the Ly sE protein (lysine efflux permease, from Corynebacterium glutamicum ATCC 13032, which acts on L- lysine (cytoplasm) to produce L-lysine (outside); Protein ace, no. NP_600485 (Tokyo Research Laboratories, Kogyo Co. Ltd., Japan, direct submission to NCBI)),
Protein Name Protein Accession
No.
Lysine exporter protein Q8RQM4 arginine exporter ZPJH835056 protein ArgO lysine exporter protein ZP_0393395 lysine exporter protein ZP_03931790
L-lysine exporter YP_002958101 lysine exporter protein ZPJ)5123881 amino acid transporter NP_794117
LysE arginine exporter ZP_03832031 protein hypothetical protein ACI78466
ECs3794
LysE family transporter NP 353947
Table IX. Suitable homologues for the LysP protein (LysP lysine APC transporter, from Escherichia coli str. K- 12 substr. MGl 655, which acts on L-lysine (outside) and produces L-lysine (cytoplasm); Protein ace. no. NP_416661 (Steffes et al., J Bacteriol 174(10): 3242-9 (1992))).
Protein Name Protein Accession No lysine transporter YP_002383360 hypothetical protein ZP_03837490
CIT292J)3450 lysine transporter YP_001336242
Lysine-specific YP_003211189 permease lysine transporter ZPJ)3383884 lysine transporter NP_930088
Lysine-specific ZP_04623004 permease Protein Name Protein Accession No. amino acid permease- YP 001565334 associated region ϊysine-specific YP 002008821 permease; amino acid- polyarøine- organocation (APC) superfamily amino acid permease- YP 776515 associated region
Table IY. Suitable homologues for the CadA protein (lysine decarboxylase I5 from Escherichia coli str. K- 12 substr. W3110, which acts on Lysine to produce Cadaverine; Protein ace. no. AP_004633 (Riley et al., Nucleic Acids Res. 34 (1), 1-9 (2006))).
Protein Name Protein Accession
No.
Lysine decarboxylase ZP_06166000 hypothetical protein YPJ)01569398 SARI_00317 Orn/Lys/Arg YP 002932309 decarboxylase family, putative
Arginine / lysine / ZP_06179259 ornithine decarboxylase lysine decarboxylase 1 YP_205440 Lysine decarboxylase ZP_04636370 lysine decarboxylase 2 ZP_04559973 lysine decarboxylase 2, YP_002396273 constitutive Lysine decarboxylase ZP_04617787 lysine decarboxylase, YP 855733 constitutive
Table IZ. Suitable homologues for the LdcC protein (lysine decarboxylase 2, from Escherichia coli str. K- 12 sub str. MG 1655, which acts on Lysine to produce Cadaverine; Protein ace. no. NP_414728 (Riley et al., Nucleic Acids Res. 34 (1), 1-9 (2006))).
Protein Name Protein Accession No. lysine decarboxylase 2 NPJ706131
hypothetical protein YP_001454701
CKOJ)3180 lysine decarboxylase, ZP_02686615 Protein Name Protein
Accession No. constitutive lysine decarboxylase, YPJ)02240326 constitutive
Lysine decarboxylase, YP_003209178 constitutive
Lysine decarboxylase YPJXJ2647915
Lysine decarboxylase ZPJ)4621086 lysine decarboxylase 1 YP_003294813 lysine decarboxylase 1 YPJ59739
Orn/Lys/Arg YP 002931768 decarboxylase family, putative
Table IAA. Suitable homologues for the YjeK protein (lysine 2,3- aminomutase, from Escherichia coli str. K-12 sub sir. MGl 655, which acts on L-lysine to produce (R)-b-Lysine; Protein ace. no. NP_418570 (Riley et al., Nucleic Acids Res. 34 (I)5 1-9 (2006))).
Protein Name Protein Accession No. hypothetical protein YP_691589 SFV_4304 putative lysine YP_002385208 aminomutase conserved hypothetical ZPJJ4559561 protein
KamA family protein YP_002240896 putative aminomutase NP_463197 hypothetical protein YPJ)01436296 ESAJ)Ol 56 lysine 2, 3- YP 003019317 aminomutase YodO family protein hypothetical protein YP_003294390 ETAE_0333 Uncharacterized kamA ZP_04617468 family protein yjeK lysine 2, 3- YP 002157135 aminomutase
One embodiment provides a transgenic or recombinant organism for producing P(5HV) or other PHAs containing 5HV monomers . The organism can be prokaryotic or eukaryotic. Suitable prokaryotes include but are not limited to bacteria, for example E. coli. B. Methods and Materials for Producing Recombinant Organisms or Cells
1. Organisms or Cells to be Modified
Organisms or cells that can be modified for producing 5HV containing PHA biopolymers, 5-aminopentanoate (5 AP), 5-hydroxyvalerate (5HV), glutarate, and 1,5-pentanediol (PDO) include eukaryotes and prokaryotes. Suitable prokaryotes include bacteria. A number of bacteria can be genetically engineered to produce polyhydroxyalkanoates. Examples include E. coli, Alcaligenes lotus, Alcaligenese eutrophus, Azotobacter, Pseudomonas putida, Ralstonia eutropha, Salmonella, Klebsiella, Corynebacterium glutamicum, Rhodoccocus, and Brevibacterium lactofermentum. Additional prokaryotes include,, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31 ,446); E. coli Xl 776 (ATCC 31.537); (ATCC 27,325) and K5772 (ATCC 53,635).
These include organisms that already produce polyhydroxyalkanoates, modified to utilize alternative substrates or incorporate additional monomers, or to increase production, and organisms that do not produce polyhydroxyalkanoates, but which express none to some of the enzymes required for production of polyhydroxyalkanoates. R. eutropha is an example of an organism which produces PHAs naturally. E. coli and C. glutamicum are examples of organisms where it would be necessary to introduce transgenes which encode the enzymes for PHA production.
For the production of the C5 chemicals 5-aminopentanoate (5AP), 5- hydroxyvalerate (5HV), glutarate, and 1,5-pentanediol where they are not polymerized to 5-hydroxvalerate containg PHAs but secreted into the growth medium then it is useful to use industrial microorganisms which are used to manufacture lysine. Corynebacterium glutamicum, including its subspecies Brevibacterium flavum, Brevibacterium lactofermentum, Corynebacterium lilium> Corynebacterium efficiem and Brevibacterium divaricatum, which has been used for the industrial production of L-lysine (Microbiol. Monogr.5:39-70 (2007)), is an example of the microorganisms can be developed for the production of glutarate, 1,5-pentandiol, 5-hydroxyvalerate, and other products that can be produced from lysine via lysine degradation pathways described in this invention. Engineered E. coli have also been used for lysine production. The procedures to obtain C. glutamicum strains for the production of L-lysine such as random mutagenesis and subsequent selection of the mutant that resistant to toxic lysine analogues, like S-(2-aminoethyl) cysteine, and introduction of mutant alleles of the gene targets such as lysC and horn which encode aspartate kinase and homoserine dehydrogenase, respectively, are well -established and has been described (Curr. Opin. Microbiol. 9:268-274 (2007)). Aspartate kinase is subject to a feedback inhibition by threonine and lysine, and the release of this enzyme from the feedback inhibition is regarded as one of the key features to develop a lysine producer strains. Another target of engineering for the development of the strains capable to produce chemicals derived from lysine degradation pathways is the lysine exporter such as LysE in C. glutamicum. Mutagenesis of Iy sE gene of L-lysine producing C. glutamicum strains will prevent the excretion of lysine from the cytoplasm and thus increase the yield of products via the pathway devised to convert lysine to the products. Methods for constructing C glutamicum strains for the production of PHAs such as PHB are also known in the art (Jo, S-J et. Al., 2006. J. Bioscience and Bioengineering 102: 233-236).
Suitable eukaryotic organisms or cells include fungi such as filamentous fungi or yeast. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. 2. Methods for Generating Transgenic Organisms i. Extrachromosal Transfection
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including chemical transformation such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran- mediated transfection,, lipofection, or electroporation. Conventional transformation techniques are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001. Transformations into yeast are typically carried out according to the method of Van Solingen et al. J Bad, 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sd. (USA), 76:3829 (1979). ii. Chromosomal Integration
Methods for incorporating engineered gene constructs into the chromosomal DNA of gram negative and gram positive bacterial cells are well known to those skilled in the art. Typical integration mechanisms include homologous recombination using linearized DNA in recBC or recD strains followed by Pl transduction (Miller 1992, A Short Course in Bacterial Genetics: A Laboratory Manual & Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor laboratory Press, Cold Spring Harbor, N. Y.) special plasmids (Hamilton et al., J Bacteriol. 171 :4617 (1989); Metcalf et al., Plasmid 35: 1 (1996); U.S. Pat. No. 5,470,727 to
Mascarenhas et al.), or by random insertion using transposon based systems (Herrero et al. J Bacteriol. 172:6557 (1990); Peredelchuk & Bennett, Gene 187:231 (1997); U.S. Pat. No. 5,595,889 to Richaud et al.; U.S. Pat. No. 5,102,797 to Tucker et al.). In general, the microbial strains containing an insertion are selected on the basis of an acquired antibiotic resistance gene that is supplied by the integrated construct. However, complementation of auxotrophic mutants can also be used. The same methods can be used to introduce any of the transgenes encopding the various metabolic pathways described herein. Some of these methods are described in more detail below with respect to the pha genes.
Expression of the genes of interest for chromosomal integration can be achieved by including a transcription-activating sequence (promoter) in the DNA construct to be integrated. Site-directed, homologous recombination can be combined with amplification of expression of the genes of interest, as described by U.S. Pat. No. 5,000,000 to Ingram et al. Chromosomal integration can also be achieved by the methods of Datsenko and Wanner (Proc. Natl. Acad. Sci. U S A. 97:6640-6645 (2000)), and as used in Example 7, below. A series of expression cassettes have been developed for inserting heterologous genes into bacterial chromosomes. These cassettes are based on the transposon delivery systems described by Herrero et al., J Bacterial. 172:6557-67 (1990); de Lorenzo et al., J Bacterid. 172:6568 (1990). Although these systems specify RP4-mediated conjugal transfer and use only transposon TnIO and Tn5, any combination of transposon ends and delivery system could be adapted for the technology described, resulting in sustained and homogeneous PHA production.
The following general approach is used for generating transgenic E. coli PHB producers: (1) a promoterless antibiotic resistance (abr) gene is cloned in the polylinker of a suitable plasmid such as pUC18NotI or pUClδSfil so that the major part of the polylinker is upstream of abr; (2) pha genes, genes encoding a GABA transaminase, a succinic semi-aldehyde reductase, and a 4-hydroxybutyrate-CoA transferase are subsequently cloned upstream of and in the same orientation as the abr gene; (3) \hepha-abr cassette is excised as a Noil or Avrlϊ fragment (Avrll recognizes the Sfil site in pUClSSfiϊ) and cloned in the corresponding sites of any plasmid like those from the pUT- or pLOF-series; (4) the resulting plasmids are maintained in E. coli λpir strains and electroporated or conjugated into the E. coli strain of choice in which these plasmids do not replicate; and (5) new strains in which the pha-abr cassette has successfully integrated in the chromosome are selected on selective medium for the host (e.g., naladixic acid when the host is naladixic acid resistant) and for the cassette (e.g., chloramphenicol, kanamycin, tetracyclin, mercury chloride, bialaphos). The resulting pha integrants are screened on minimal medium in the presence of glucose for growth and PHB formation.
Several modifications of this procedure can be made. If the promotorless antibiotic resistance marker is not used, the insertion of the PHA genes is selected based on a marker present in the vector and integrated strains producing the desired level of PHA are detected by screening for PHA production. The pha genes may have, but do not need, endogenous transcription sequences, such as upstream activating sequences, RNA polymerase binding site, and/or operator sequences, ϊf the pha genes do not have such sequences, the described approach is limited to the use of vectors like the pUT series in which transcription can proceed through the insertion sequences. This limitation is due to the inability of RNA polymerase to read through the TnIO flanking regions of the pLOF plasmids. The abr gene may carry its own expression sequences if so desired. Instead of an abr gene, the construct may be designed such that an essential gene serves as selective marker when the host strain has a mutation in the corresponding wild-type gene. Examples of genes useful for this purpose are generally known in the art. Different constructs can be integrated into one host, either subsequently or simultaneously, as long as both constructs carry different marker genes. Using multiple integration events, /?/zα genes can be integrated separately, e.g., the PHB polymerase gene is integrated first as aphaC-cat cassette, followed by integration of the thiolase and reductase genes as aphaAB-kan cassette. Alternatively, one cassette may contain all pha genes whereas another cassette contains only some pha genes required to produce a desired PHA polymer.
In some cases a transposon integration vector such as pJMSl 1 (Panke et al. Appϊ. Enviro, Microbiol 64: 748-751) may be used such that the selectable marker can be excised from the chromosome of the integrated strain. This is useful for a number of reasons including providing a mechanism to insert multiple transposon constructs using the same marker gene by excising the marker following each insertion event.
3. Sources of pha and Other Genes Involved in PHA Formation
A general reference for genes involved on PHA formation is Madison and Huisman, 1999, Microbiology and Molecular Biology Reviews 63: 21- 53. Th&pha genes may be derived from different sources and combined in a single organism, or from the same source. i. Reductase Encoding Genes
Reductase-encoding genes have been isolated from A. latus, R, eutropha (Peoples & Sinskey, J Biol. Chem. 264(26): 15298-303 (1989); Acinetobacter sp. (Schembri, et al., J Bacteriol. 177(15):4501-7 (1995)), C. vinosum (Liebergesell & Steinbuchel, Eur. J. Biochem. 209(1): 135-50 (1992)), P. acidophila, P. denitriflcans (Yabutani, et al., FEMS Microbiol. Lett. 133 (l-2):85-90 (1995)), R. meliloti (Tombolini, et al., Microbiology 141 :2553-59 (1995)), and Z ramigera (Peoples, et al., J. Biol. Chem. 262(l):97-102 (1987)). U.S. Patent No. 7,229,804 discloses transgenic organisms that produce P4HB using the 4HbD gene that encodes the 4- hydroxybutyrate dehydrogenase from C kluyveri (Sohling and Gottschalk, J Bacteriol 178, 871 880 (1996)). 4hbD requires NADH. Preferred reductases include, but are not limited to, those that do not require NADH. Exemplary reductases include AKR7A5 from Mus musculus (Genlnfo Identifier :27659727 )(Hinshelwood, A. et al. FEBS Letters 523:213-218 (2002), GHBDH from Arabidopsis thaliana (GI: 145338934) (Breitkreuz, K. et al. J. Biol Chem. 278:41552-41556, ATEGJ)0539 from Aspergillus fwew (GI: 115491994). ii. CoA Transferase and CoA Synthetase Suitable CoA transferases (EC 2.8.3.n) include but are not limited to orfZ from C. kluyveri. The sequence of orfZ is provided in U.S. Patent No. 7,229,804 to Huisman et ai. and is incorporated by reference in its entirety. Another suitable CoA transferase includes abjT from C. aminobutyricum (Gerhardt et al., Arch Microbiol 74:189-199 (2000)). Other enzymes that could produce acyl-CoA include CoA synthetases (EC 6.2.1.3). These enzymes use ATP and free CoA to catalyze the covalent addition of CoA to the carboxylic acid and have been described in van Beilen et al. {Molec Microbiol (1992) 6:3121-3136) and Aquin et al, (WO 02/40690 A2). iii. PHA Polymerase-Encoding Genes
PHA polymerase-encoding genes have been isolated from Aeromonas caviae (Fukui & Doi, J Bacteriol. 179(15):4821-30 (1997)), A. latus, R. eutropha (Peoples & Sinskey, J Biol. Chem. 264(26): 15298-303 (1989); Acinetobacter (Schembri, et al., J Bacteriol. 177(15):4501-7 (1995)), C vinosum (Liebergesell & Steinbuchel, Eur. J. Biochem. 209(1): 135-50 (1992)), Methylobacterium extorquens (Valentin & Steinbuchel, Appl. Microbiol. Biotechnol 39(3):309-17 (1993)), Nocardia corallina (GenBank Ace. No. AF019964), Nocardia salmonicolor, P. acidophila, P. denitrificans (Ueda, et al., J. Bacteriol 178(3):774-79 (1996)), Pseudomonas aeruginosa (Timm & Steinbuchel, Eur. J. Biochem. 209(1): 15-30 (1992)), Pseudomonas oleovorans (Huisman, et al., J Biol. Chem. 266:2191-98 (1991)), Rhizobium etli (Cevallos, et al, J Bacteriol 178(6): 1646-54 (1996)), R. meliloti (Tombolini, et al., Microbiology 141 (Pt 10):2553-59 (1995)), Rhodococcus ruber (Pieper & Steinbuchel, FEMS Microbiol Lett. 96(l):73-80 (1992)), Rhodospirήlum rubrum (Hustede, et al., FEMS Microbiol. Lett. 93:285-90 (1992)), Rhodobacter sphaeroides (Steinbuchel, et al., FEMS Microbiol. Rev. 9(2-4):217-30 (1992); Hustede, et al., Biotechnol Lett. 15:709-14 (1993)), Synechocystis sp. (Kaneko, DNA Res. 3(3): 109-36 (1996)), T. violaceae (Liebergesell & Steinbuchel, Appl. Microbiol. Biotechnol. 38(4: 493-501 (1993)), and Z ramigera (GenBank Ace. No. U66242).
Other genes that have not been implicated in PHA formation but which share significant homology with the pha genes and/or the corresponding gene products may be used as well. Genes with significant homology to the phaB gene encoding acetoacetyl CoA reductase have been isolated from several organisms, including Azospirillum brasiliense (NCBI Accession Nos. X64772, X52913) Rhizobium sp. (NCBI Accession Nos. U53327, Y00604), E. coli (NCBI Accession No. D90745), Vibrio harveyi (NCBI Accession No.U39441), H. influenzae (NCBI Accession No.U32701), B. subtilis (NCBI Accession No. U59433), P. aeruginosa (NCBI Accession No. U91631), Synechocystis sp. (NCBI Accession No. D90907), H. pylori (NCBI Accession No. AE000570), Arabidopsis thaliana (NCBI Accession No. X64464), Cuphea lanceolata (NCBI Accession No. X64566) and Mycobacterium smegmatis (NCBI Accession No. U66800). IH. Methods of Producing PHAs containing 5HV and C5 Chemicals
Methods for producing polyhydroxyalkanoates using renewable carbon sources as feedstock are provided. In a preferred embodiment, bacteria are transformed or transfected with one or more nucleic acid constructs encoding the genes necessary for producing 5-aminopentanoate (5AP), 5-hydroxyvalerate (5HV), glutarate, and 1,5-pentanediol (PDO) and polymers thereof from renewable resources, IV. Methods of Use The disclosed transgenic organisms can be used to produce C5 chemicals such as 5-arninoρentanoate (5AP), 5-hydroxyvalerate (5HV), glutarate, and 1,5-pentanediol (PDO) as well as PHA biopolymers comprising 5HV monomers. In the case of the production of the C5 chemicals the recombinant organism expressing the appropriate transgenes(s), optionally including having genes encoding competing patheways inactiviated or deleted are grown on a renewable fermentation substrate. The substrate is selected from carbohydrates, lysine, proline vegetable oils, fatty acids or combinations thereof. A useful combination for some embodiments would be a mixture of glucose and lysine. Another suitable combination would be sucrose and lysine. Preferably the feedstock comprises predominantly one substrate, for example glucose or sucrose. Suitable carbohydrate substrates comprise one or more sugars selected from glucose, fructose, xylose, arabinose, sucrose, lactose and maltose. For the production of C5 chemicals, the recombinant organism is grown on the renewable substrate until the desired end product accumulates in the growth medium at which time the cells are removed by flocculation, settling, centrifugation or filtration and the product is recoverd from the cell-free medium by standard procedures. Such procedures are known in the art for the recovery of other fermentation produed acids and diols such as lactic acid, succinic acid, 3-hydroxypropionic acid, 1,3 -propanediol and 1,4- butanediol.
The recombinant organisms can be used for the fermentation production of 5HV containing PHA biopolymers including the homopolymer P5HV and 5HV containing PHA copolymers by culturing the organisms in the presence of renewable carbons sources such as glucose, lysine, etc. and other substrates selected to provide a desired polymer or copolymer. The recombinant organsims are grown on the substrates until the PHA polymers have accumulated inside the cells at which point the PHA polymers are extracted from the cells by methods known to those skilled in the art. The
5HV containing PHA polymers obtained from the organisms can be used in a wide range of industrial plastics applications such as for films, fibers, foams, injection molded goods, blow molded bottles, paper coating and the like. They can also be used for biomedical applications including tissue augmentation, to produce heart valves, as sutures, and to produce vascular grafts. Exemplary co-polymers include, but are not limited to, PHB5HV and p(3 -hydroxypropionate-co- 5 -hydroxy valerate) p(4-hydroxy butyrate-co-5 - hydroxy valerate). The recombinant organisms can be genetically engineered to include additional genes such as beta-ketothiolase and acetoacetyl-CoA reductase as needed for the production of copolymers.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. .
EXAMPLES
EXAMPLE 1: Biosynthesis of P(SHV) horaopolymer from sodium 5- hydroxyvalerate
As a first demonstration of the ability to synthesize 5HV-containing PHAs, sodium 5 -hydroxy valerate (Na5HV) was fed to E. coli strains expressing both a CoA transferase or CoA synthetase and a PHA synthase, in order to determine if the fed 5HV monomer would be accepted and incorporated directly into P(SHV) homopolymer. NaSHV was synthesized via base hydrolysis of δ-valero lactone (DVL). This procedure involved adding 0,1 mol NaOH to 50 mL methanol with stirring until dissolved. To this, 0.1 mol DVL was added with vigorous stirring. The resulting precipitate was dried, dissolved in water, pH-adjusted to 8.5, and filter- sterilized with a 0.2 μM filter. Analysis of the salt solution was performed on a Waters Alliance HPLC in order to confirm that all of the DVL had been saponified,
Different combinations of CoA transferase/synthetase and PHA synthase genes were tested in order to find the best combination suitable for P(SHV) production. The CoA transferases/synthetases tested were C. kluyveri orfZ and P. oleovorans alkK, and the PHA synthases were R. eutrophaphaC and T. pfenigii phaEC (Table IA). All strains used in this experiment were derived from MGl 655 (Jensen, J. Bacteriol, 175(11):3401~ 3407 (1993)). Four expression plasmids (pFS92, pMS96; pMS93, and pMS102), each containing different combinations of CoA transferase/synthetase and PHA synthase, were constructed as described in the following paragraphs. Plasmid construction
Plasmid ρFS30 was constructed by digesting pAET41 (Peoples and Sinskey, J. Biol Chem. 264(26): 15298-15303 (1989)) with Xmal and Stul in order to remove a fragment containing R. eutropha phaC and its native promoter (P/?e). Plasmid pAET41 is a pUC18 vector (Accession No. L08752) that contains a chromosomal fragment from R. eutropha Hl 6 encompassing the phaC gene. Plasmid pFS16 (Skraly and Peoples, U. S. Patent No. 6,323,010), which is a derivative of pTrc99a (Pharmacia, Uppsala, Sweden) containing the orfZ gene from C. kluyveri (Table IA) under the Vm promoter, was digested with BamHI, blunted with T4 polymerase, and digested a second time with Xmnl prior to ligation with the ?Re-phaC Xmal-Stul fragment. The resulting plasmid was designated pFS30, and contained the phaC-orfZ operon fusion under the constitutively- expressed P^, promoter.
Plasmid pFS92 was made in a multi step process. First, T. pfenigii phaE was amplified from pMON25893 (Reiser et al., Appl. Microbiol, Biotechnol 53(2):209-218 (2000)) with primers FS-E5' and FS-E3', which contain engineered EcoRI and Acc65I restriction sites. Thep/zαE PCR product was then digested with restriction enzymes EcoRI and Acc65I and ligated to similarly digested pTrcN (Gerngross et al,, Biochemistry 33:931 1- 9320 (1994)) to form pFS89. The primer sequence for FS-E5' is (5')- GGAATTCAGGAGGTTTTTATGAACGATACGGCCAACAAGACCAGC (SEQ ID NO:1) and the primer sequence for FS-E3' is (5')-
GGGGTACCTCACTGGCCGGTGGTGGGCTTGGTGGTCTTGCGGCG (SEQ ID NO:2). Next, T. pfenigii phaC was amplified from pMON25894 (Reiser et al., Appl, Microbiol. Biotechnol. 53(2):209-218 (2000)) with primers FS-C5' and FS-C3', which contain engineered Acc65I and BamHI restriction sites. The phaC PCR product was then digested with restriction enzymes Acc65I and BamHI and ligated to similarly digested pTrcN to form pFS90. The primer sequence for FS-C5' is (5>
GGGGTACCAGGAGGTTTTTATGTCCCCATTCCCGATCGACATCCG (SEQ ID NO:3) and the primer sequence for FS-C3' is (5> CGGGATCCTCAGCCGCGTTCGTTCAGCCAGCGGCCGATCGCCG (SEQ ID NO:4). After T, pfenigii phaE and phaC were individually cloned into pTrcN to form pFS89 and pFS90, respectively, phaE was cloned upstream of phaC by digesting pFS89 with Mluϊ and Acc65I, isolating the phaE containing fragment, and ligating it to a similarly digested preparation of pFS90. The resulting plasmid, pFS91, contained the phaEC operon fusion under the ?Trc promoter. Finally, pFS92 was created by ligating a MIuI- BamHI fragment from pFS91 containing phaEC to pFS16 that had been digested with MIuI and BamHI. Plasmid pFS92 contains the phaEC-orfZ operon fusion under the Pτ>c promoter.
Plasmid pMS96 was made by cloning alkK (see Table IA) into pFS91 downstream of phaEC, First, alkK was PCR amplified from P. oleovorαns genomic DNA using primers K5-1 and K3-1, which were engineered to incorporate BamHI sites onto the ends of the PCR product. The sequence for primer K5-1 is (5')-
GCTGAGGATCCAGGAGGTTTTTATGTTAGGTCAGATGATGCGTAA TC (SEQ ID NO:5) and the sequence for primer K3-1 is (5')- CTAGAGGATCCTTATTCACAGACAGAAGAACTACTG (SEQ ID NO:6). Following amplification, the αlkK PCR fragment and pFS91 (described in the paragraph above) were digested with BamHI and ligated to form pMS96. The orientation of αlkK in pMS96 was verified to be in the same direction as phαEC by restriction enzyme digestion, thus ensuring the proper construction of a phαEC-αlkK operon fusion under the Pr,r promoter. Plasmids pMS93 and pMS102 were constructed by first digesting pACYC 177 (Accession #X06402) with BspHI in order to remove the kαn marker and ligating it into the unique BspHI site of ρFS30 to form pFS73, which contains phαC-orfZ under the control of tandem promoters VTK and ?Re. The P/?e promoter region was removed by replacing the EcoRI-BspEI fragment of ρFS73 containing both ~PRe and 837 bp from the 5' end of the phαC CDS with an EcoRI-BspEI fragment from ρKAS4 (Peoples et al., U.S. Patent No. 5,480,794) that contained only the 837 bp from the 5' end of phαC. This resulting plasmid, which contains phαC-orfZ under only the ?τrc promoter, was designated pMS93. To create pMS102, the or/Z gene was removed from pFS73 by digesting with Dral and self-ligating the plasmid backbone to form pMS74. The αlkK gene was PCR-amplified from plasmid pTrcN-A.eut-AIkK (described below) using primers K5-2 and K3-2. The sequence for primer K5-2 is (5')-
AATTCAGGAGGTTTTTATGTTAGGTCAGATGATGCGTAATC (SEQ ID NO:7) and the sequence for primer K3-2 is (5 s)- GATCCTTATTCACAGACAGAAGAACTACTG (SEQ ID NO; 8). Plasmid pMS74 was then digested with Spel and Sbfl and then made blunt- ended via Klenow fill-in and the alkK PCR fragment ligated to the blunted pMS74 backbone to form pMS92. Plasmid pMS92 thus contains XhsphaC- alkK operon fusion under the control of tandem promoters ΫTK and ¥&, In order to express the operon exclusively from the IPTG-inducible P^ promoter, the P&, promoter region was removed by replacing the EcoRI- BspEI fragment of pMS92 containing both P&, and 837 bp from the 5' end of the phaC CDS with an EcoRI-BspEI fragment from ρMS93 that contained only the 837 bp from the 5' end of phaC. This resulting plasmid, which contains phaC-alkK under only the Prre promoter, was designated pMS102.
Plasmid pTrcN-A.eut-AlkK was created by first PCR-amplifying alkK from P. oleovorans genomic DNA using primers Posynrbs.c (5!- GGAATTCAGGAGGTTTTTATGTTAGGTCAGATGATGCGTAATCAG) (SEQ ID NO:9) and Posynrbs.r (5'-
CGGGATCCTTATTCACAGACAGAAGAACTACTGCG) (SEQ ID NO: 10). The resulting PCR product was digested with EcoRI and BamHI and ligated to similarly digested pTrcN to create pTrcN- AlkK. The P/& promoter was then PCR-amplified from Ralslonia eutropha genomic DNA using primers A.eutPhaG.c (5'-
GGAATTCGGATCCCAAGTACCTTGCCGACATCTATGCGCTGGC) (SEQ ID NO:11) and A.eut.EcoRI.r (5'-
GGAATTCCCGGCTCCGGGATTGCCCTGGCCGGACT) (SEQ ID NO: 12). The resulting PCR product was digested with EcoRI and ligated to similarly-digested pTrcN-AlkK in order to create pTrcN-A.eut-AllcK.
Plasmids pFS92, pMS96, pMS93, and pMS102 were individually transformed into MGl 655 (Jensen, J Bacterid. 175(11):3401-3407 (1993)) to create four plasmid-bearing strains that contained different combinations of CoA transferase/synthetase (orfZ or alkK) and PHA synthase iphaC or phaEC). These strains were grown in 250 niL shake flasks to characterize P(5HV) homopolymer production, as described in the following section. Media, growth conditions, and testing for production of P(SHV) homopolymer in shake flask cultures
Each plasmid-bearing MG 1655 strain was grown overnight in a test tube containing 3 mL LB (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (2001)) supplemented with appropriate antibiotic at 370C with 250 rpm shaking. Antibiotics appropriate for each strain are as follows: 100 μg/mL ampicillin was added to overnight cultures of pFS92- and pMS96-beaπng MGl 655 strains; and 50 μg/mL Km was added to overnight cultures of pMS93- and ρMS102-bearing MG1655 strains. The next day, 0.5 mL of each overnight culture was used to inoculate a shake flask containing 50 mL of fresh LB supplemented with the appropriate antibiotic and grown at 370C with 250 rpm shaking. At 3.5 hours, 0.1 mM IPTG was added to the liquid cultures, and at 5 hours, the culture was spun down at 4150 rpm (Sorvall Legend RT benchtop centrifuge) and resuspended in 50 mL of production medium containing 0.1 mM IPTG and the same antibiotic. The production medium consists of Ix E2 minimal salts solution containing 10 g/L glucose, 2.5 g/L LB, 10 g/L NaSHV5 2mM MgSO4, and Ix Trace Salts Solution. 50x E2 stock solution consists of 1.275 M NaNH4HPO4-4H2O, 1.643 M K2HPO4, and 1.36 M KH2PO4. 100Ox stock Trace Salts Solution is prepared by adding per 1 L of 1.5 N HCL: 50 g FeSO4-7H20, 11 g ZnSO4-7H2O, 2.5 g MnSO4-4H2O, 5 g CuSO4-5H2O, 0.5 g (NH4)6Mo7024-4H20, 0.1 g Na2B4O7, and 10 g CaCl2-2H2O.
The resuspended cultures were transferred to 250 mL shake flasks and incubated at 3O0C for 24 to 72 hours with shaking. At the end of the experiment, cultures were spun down at 4150 rpm, washed once with distilled water, frozen at -8O0C for at least 30 minutes, and lyophilized overnight. The next day, a measured amount of lyophilized cell pellet was added to a glass tube, followed by 3 mL of butanolysis reagent that consists of an equal volume mixture of 99.9% n-butanol and 4.0 N HCl in dioxane with 2 mg/mL diphenylmethane as internal standard. After capping the tubes, they were vortexed briefly and placed on a heat block set to 930C for six hours with periodic vortexing. Afterwards, the tube was cooled down to room temperature before adding 3 mL distilled water. The tube was vortexed for approximately 10 s before spinning down at 620 rpm (Sorvall Legend RT benchtop centrifuge) for 2 min. 1 mL of the organic phase was pipetted into a GC vial, which was then analyzed by gas chromatography- fiame ionization detection (GC-FID) (Hewlett-Packard 5890 Series II). The quantity of P(SHV) homopolymer in the cell pellet was determined by comparing against standard curves that were made by adding defined amounts of DVL in separate butanolysis reactions. Three DVL standards ranging from 2-6 mg were used to create the standard curves. Experimental results
Table 2 shows that all constructs were able to generate P(5HV). However, MGl 655 [pMS93] generated significantly more P(5HV) than any of the other strains demonstrating that the optimal gene combination to polymerize P(5HV) is phaC and orfZ.
Table 2. P(5HV) homopoiymer production
Relevant genotype P(5HV)
Strain [Plasmid]
(%dcw)
MGl 655 [pMS93] Ptrc-phaC-orfZ 54.6
MGl 655 [pFS92] Ptrc-phaEC-orβ 5.7 ± 1.2
MGl 655 [pMS96] Ptrc-phaEC-alkK 3.82
MG1655 [pMS102] PtrcψhaC-alkK 35.2
EXAMPLE 2: Biosynthesis of P(3HB-co-5HV) copolymer from sodium 5-hy d roxy valerate
The next experiment was to demonstrate the production of P(3HB~co- 5HV) copolymer in an E coli strain capable of synthesizing the 3HB-CoA and 5HV-CoA monomers and incorporating them into PHA. Strain construction
The strain used in this example was MBX2641, which is an MG 1655 derivative that contains an operon consisting of R eutropha H16 bktB-B megaterium phaB-kan randomly integrated into the chromosome To carry out the operon integration, strain Sl7-lλpir (Miller and Mekalanos, J Bacteriol 170(6):2575-2583 (1988)) containing pCJ022 (described below) was mated with MBX1987, which is a nalidixic acid-resistant mutant of MGl 655, using a protocol taken from the literature (De Lorenzo and Timmis, Methods Enzymol 235:386-405 (1994)). Derivatives of MBX1987 carrying the bktB-phaB-kan cassette in the chromosome were selected on LB plates containing 30 μg/mL nalidixic acid (Nl) and 50 μg/mL kanamycin (Km). One such integrant displaying a N1R KmR phenotype was saved as MBX2079. Then, phaEC-cat was randomly integrated into the chromosome of MBX2079 by mating with an S\7 -\λpir strain carrying the integration vector pUT-C16~cat (described below). Integrants of MBX2079 carrying the phaEC-cat cassette in the chromosome were selected on LB plates containing 25 μg/mL chloramphenicol (Cm). Several integrants possessing the N1R KmR CmR phenotype were pooled and subjected to nitrosoguanidine mutagenesis (Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory (1972)), with selection on LB plates containing 100 μg/mL Cm. One mutant was isolated and designated MBX21 14. Finally, MBX2641 was created by raising a Pl lysate on MBX21 14 and transducing into MGl 655 as described by Miller (Experiments in Molecular Genetics, Cold Spring Harbor Laboratory (1972)) using KmR as selection. One such transductant was saved and designated MBX2641, which is MGl 655 with the bktB-phaB-kan gene cassette randomly integrated into the genome.
Plasmids pFS92 (see Example 1), and pJB84 (construction described below) were individually transformed into MBX2641 that contains bktB- phaB-kan randomly integrated into the chromosome. Plasmid construction Plasmid pCJ022 was made by creating a mini-TnJ integration vector containing bktB-phaB upstream of a kan marker. To do this, the bktB-phaB operon was assembled on pSE380 (Invitrogen, Carlsbad, CA) by first PCR- amplifying bktB with primers MS069 and MS070 from pMON25765 (Slater et al, J. Bacteriol 180(8): 1979- 1987 (1998)). The sequence for primer MS069 is (5>
GGTGGATCCTTAAGAGGAGGTTTTTATGACGCGTGAAGTGGTAGT GG (SEQ ID NO: 13) and the sequence for primer MS070 is (5> GGTGCTAGCTCAGATACGCTCGAAGATGGCG (SEQ ID NO: 14). The resulting PCR fragment was digested with BamHI and Nhel and ligated to ρSE380 that had been cut with the same enzymes. The resulting plasmid was designated pSE380-bktB. Next,phaB was PCR-amplified from pGMIO (McCool and Cannon, J Bacterial 181(2):585-592 (1999)) with primers MS071 and MS072. The sequence for primer MS071 is (5')-
GGTCCTAGGTTAAGAGGAGGTTTTTATGACAACATTACAAGGTAA AG (SEQ ID NO: 15) T and the sequence for primer MS072 is (5> GGTGCGGCCGCTTACATGTATAAGCCGCCGTTAAT (SEQ ID NO; 16). The resulting PCR fragment was digested with Avrll and Notl and ligated to pSE38O~bktB that had been treated with the same enzymes. The resulting plasmid was designated pSE380-bktBphaB19. After the operon had been assembled, it was moved to pTrcN-fam (a derivative of pTrcN that had the bla gene replaced with the lean gene) by digesting pSE380- bklBphaB19 with EcoRI and Spel and ligating the fragment containing bktB- phaB to pTrcN-Aαw that had been cut with EcoRI and Xbal. The resulting plasmid was designated pMSl 15. The bktB-phaB operon was then transferred from pMSl 15 to the pBSLl 18 integration vector (Alexeyev et al., Can. J. Microbiol. 41: 1053-1055 (1995)) by digesting pMSl 15 and pBSLl 18 with BamHI and ligating together to produce plasmid ρCJ022, which was used to integrate bktB-phaB into the chromosome of MGl 655.
Plasmid pUT-C16cat was made by removing phaEC from pFS91 with EcoRI and Xbal and blunting the sticky ends using Klenow. Integration vector pUT-cat (De Lorenzo and Timmis, Methods Enzymol. 235:386-405 (1994)) was digested with Avrlϊ, blunted using Klenow fill-in, and then ligated to the phaEC fragment. After verifying that phaEC was in the same orientation as the downstream cat marker, the plasmid was designated pUT- Clόcat.
Plasmid pJB84 was constructed by PCR-amplifying the aacC (GmR) marker from pBSL202 (Alexeyev et al., Can. J, Microbiol 41 : 1053-1055 (1995)) using primers JB 123b and JB 124a, which were engineered to include BspHI sites, on the 5' flanking ends. The sequence for primer JB 123b is (5 >TT ATTTCATGAACCCTCGAATTGACGCGCTC (SEQ ID NO: 17) and the sequence for primer JB 124a is (5')- TTATTTCATGAGCTTATCGATACCGTCGACC (SEQ ID NO: 18). The resulting PCR fragment containing the aacC gene was digested with BspHI and ligated to pMS93 that had been digested with the same enzyme to form pJB84. Note that this last step was done to replace the original bla (ApR) marker on pMS93 with aacC (GmR) in the same orientation. Experimental results
MBX2641 strains carrying plasmids pFS92 (described in Example 1) or pJB84 were grown and prepared for GC-FID analysis as described in Example 1. MBX2641 [pJB84] made more copolymer (69.5%dcw) and incorporated more 5HV into copolymer (82.3%PHA) as shown in Table 3.
Table 3. P(3HB-co-5HV) production
Figure imgf000048_0001
EXAMPLE 3: Tunable 5HV monomer composition in P(3HB-co-5HV) copolymer and effect on material properties The copolymer composition was modulated by altering the amounts of NaSHV and glucose added to the production medium. Alternate ways to accomplish this include (1) feeding different amounts of L-Iy sine to the growth medium to a recombinant cell that can produce 5HV from L-lysine as is shown in Example 6, or (2) deregulating L-lysine pathway genes in recombinant cells that can produce 5HV from glucose via L-lysine as shown in Example 9 and 10.
To demonstrate tunable 5HV monomer composition in P(3HB-co- 5HV) copolymer, strain MBX2641 [ρFS30] was used that expresses the enzymes for the 3HB pathway φktB and phaB) in addition to the CoA- transferase (orβ) and PHA synthase iphaC). Parallel cultures of MBX2641 [ρFS30] were grown in either decreasing concentrations of glucose (10, 5, 1, 0.5, 0.1, 0 g/L) or NaSHV (10, 5, 1, 0.5, 0.I5 0 g/L) and analyzed for polymer content as described in Example 1. Table 4 shows that various amounts of 5HV can be incorporated into the P(3HB-co-5HV) copolymer. Table 4. Effect of co-feed on 5HV incorporation into P(3HB-co-5HV)
Figure imgf000049_0001
In another experiment, a total of 10 P(3HB-co-5HV) copolymer samples that had a wide range of 5HV compositions were generated and then extracted for differential scanning calorimetry (DSC) analysis. Table 5 shows that the glass transition temperature (Tg) decreased as the percent composition of 5HV increased in the P(3HB-co-5HV) copolymer. This demonstrates that a wide range of material properties can be obtained by modulating the 5HV comonomer composition.
Table 5. Material properties of extracted polymers
Figure imgf000050_0001
EXAMPLE 4: Synthesis of 3-hydroxypropionate from NaSHV via the fatty acid degradation system
In order to determine if the fatty acid degradation (FAD) system of E coli could break down 5HV to acetyl-CoA and 3-hydroxypropkmyl-CoA (3HP-CoA)5 plasmid pMS93 (expressing phaC-orfZ from a Ptrc promoter; see Example 1) was transformed into E coli Kl 2 strains that were either fadR+, atoC+ (repressed FAD) ovfadR', atoCcomt (derepressed FAD) MGl 655 and LS5218 (Spratt et al, J Bacterial 146(3) 1166-1169 (1981)) were used as the fadR*, atoC* said fadR", atoCconsi strains, respectively. MG1655 [pMS93] and LS5218 [pMS93] were tested by feeding NaSHV in shake flasks as described in Example 1 GC-FID and GC-Mass Spectroscopy (MS) analysis demonstrated that LS5218 [pMS93] produced P(5HV-co-3HP), whereas MG1655 [pMS93] did not (Table 6). This shows that active fatty acid degradation will produce 3HP from 5HV. Table 6. Incorporation of 3HP into P(5HV-co-3HP)
Figure imgf000050_0002
EXAMPLE 5: Biosynthesis of P(SHV) homopoϊymer from L-lysine
The pathway devised to convert L-lysine to P(SHV) is schematically diagrammed in Figure 2B and requires six heterologous genes to be cloned and expressed in E. coli: P. putida davB, P. putida davA, P. putida davT, A, thaliana gsaR/n, C. kluyveri orfZ, and R. eutropha phaC (see Table 1 A). A cloning strategy was designed such that the davBAT genes would be cloned into pACYC184 (Chang and Cohen, J. Bacterid. 134: 1141-1156 (1978)) and gsaRdt, orfZ, and phaC would be cloned into pSE380. These plasmids are designated ρJB91 and pMZ28, respectively, and their assembly is described in the next section. Plasmid construction
The multiple cloning site of plasmid pSE380 was PCR-amplified with primers JB134 (5'- TGAGCGGATAACAATTTCAC) (SEQ ID NO: 19) and JBl 35 (5'- AATAACACGTCAACGCAAAAAGGCCATCCGT) (SEQ ID NO:20). The resulting PCR product was digested with BmgBI and cloned into plasmid pACYC184 that was digested with EcoRV and Nruϊ to create pJB78.
Plasmid pJB91 was constructed in a three step process. First, the davBA genes from P. putida were PCR-amplified from a genomic DNA preparation using primers JBl 36 and JBl 37, which were engineered to incorporate Ndel and BsrGI restriction sites, respectively, on the 5' and 3' ends of the davBA PCR product, The sequence for primer JBl 36 is (5')- TTTTTCATATGAGGAGGTTTTTATGAACAAGAAGAACCGCCA (SEQ ID NO:21) and the sequence for primer JB137 is (5>
TTTTTTGTACATCAGCCTTTACGCAGGTGCA (SEQ ID NO:22). The resulting PCR product was digested with Ndel and BsrGI and ligated to pJB78 that had been treated with the same enzymes, to give plasmid pJB79. Next, the davT gene from P, putida was PCR-amplified from genomic DNA using primers JB 138 and JB 139, which were engineered to incorporate Spel and ApaLI restriction sites, respectively, on the 5' and 3' ends of the davT PCR product. The sequence for primer JB138 is (5')- TATATACTAGTAGGAGGATAATATGAGCAAAACCAACGAATC (SEQ ID NO:23) and the sequence for primer JB 139 is (5*)- TTTTTGTGCACTCAGGCGATTTCAGCGAAGC (SEQ ID NO:24). The resulting PCR product was digested with Spel and ApaLΪ and ligated to pJB79 that had been digested with the same enzymes, thus creating plasmid pJB80. Finally, the ompA promoter was PCR-amplifϊed from E. coli Kl 2 genomic DNA using primers JB 141 and JB 142, which were engineered to incorporate BmgBI and Asel restriction sites, respectively, on the 5' and 3' ends. The resulting PCR product was digested with BmgBI and Asel and ligated to pJB80 that had been digested with SnaBI and Ndel to form plasmid pJB82. Plasmid pJB91 was constructed by digesting a davBA PCR product created with primers JB 136 and JB 137 (as described above) with DraIII and ligating the 507 bp fragment to ρJB82 that had been digested with the same enzyme, thus creating plasmid pJB91. This construction was done in order to correct a nonsense mutation that had been discovered in the davB CDS of pJB82. Plasmid ρJB80 contains the davBAT ap&tm under the constitutive ?,et promoter, while plasmid pJB91 contains the same operon under the strong T?OmpA promoter.
Plasmid pMZ28 was constructed by digesting plasmid pJ31:7950, which is a construct created by DNA 2.0 (Menlo Park, CA) and contained gsaRAt that is codon-optimized for expression in E. coli Kl 2, with BsrGI. The resulting fragment containing gsaR^ was ligated to pFS30 that had also been cut with BsrGI. After verifying that the orientation of gsaRAt was in the same direction as phaC-orfZ by restriction enzyme digestion, the resulting plasmid was designated pMZ28. Experimental results MGl 655 [pMZ28] that expresses an incomplete L-lysine to P(5HV) pathway and MGl 655 [pMZ285 pJB91] that expresses the entire L-lysine to P(5HV) pathway were inoculated in a test tube containing 3 mL LB supplemented with appropriate antibiotic (25 μg/mL chloramphenicol for pJB91; 100 μg/mL ampicillin for pMZ28) and grown overnight at 370C with 250 rpm shaking. The next day, 0.5 mL of each overnight culture was used to inoculate a shake flask containing 50 mL of fresh LB supplemented with the appropriate antibiotic(s) and grown at 370C with 250 rpm shaking. At 3.5 hours, 0.1 mM IPTG was added to the liquid cultures, and at 5 hours, the cultures were spun down at 4150 rpm (Sorvall Legend RT benchtop centrifuge) and resuspended in 50 mL of production medium that consisted of Ix E2 minimal salts solution containing 10 g/L glucose, 2.5 g/L LB, 10 g/L L-lysine, 2mM MgSO4, Ix Trace Salts Solution, and 0.1 mM IPTG. The recipes for E2 salts and Trace Salts Solution are given in Example 1. Shake flask growth conditions and the analysis protocol for PHA content are as described in Example 1. Table 7 shows that eight-fold more P(SHV) was produced after introduction of the davBAT opsmn.
Table 7. P(SHV) production from L-Iysine
Total PHA
, Strain [Plasraiill . , Relevant geftotype (%dcw) ,
MGl 655 [pMZ28] pMZ28: VTrc-gsaRAr^Re-phaC-orβ 0.01
MGl 655 [pMZ28, pMZ28: YTrc-gsaRAr?Re-phaC-orβ
0 08 pJB91] pJB91: ?ompA-davBAT
EXAMPLE 6: Biosynthesis of P(3HB-co-5HV) copolymer from L-ϊysine
The pathway devised to convert L-Iy sine to P(SHV) was also introduced into strain MBX2641 expressing bktB and phaB in order to produce P(3 HB-co- 5HV) copolymer from L- lysine and eventually glucose. Plαsmid construction
The genes comprising the pathway in this example include: P putidα dαvB, P putidα dαvA, P putidα dαvT, C kluyveri orfZ, R. eutrophαphαC, and either A thαliαnα gsαRΛt or A terreus gsaR^ (see Table IA).
Plasmid pJB90, which contains an alternate pathway consisting of the gsaRAt2, phaC, and orfZ genes, was created in the following manner. The A terreus gsaRAt2 gene, codon-optimized for expression in E coli K.12, was PCR-amplified from ρSG40 (a construct created by DNA 2.0 (Menlo Park, CA)) using primers JB 145 and JB 146. Both primers contained BgIII sites at the 5' ends. The sequence for primer JB 145 is (5')- TTTTTAGATCTAGGAGGTTTTTATGCTGCGTGCTGCTTCTCG (SEQ ID NO;25) and the sequence of primer JB146 is (5')- TTTTTAGATCTTTAGCGGAAATAGTTTGGAC (SEQ ID NO:26). The resulting PCR fragment was digested with BgIII and ligated into the corresponding site of pJB84 to create pJB90. Experimental results
Strains MBX2641 [ρJB78, pJB84], MBX2641 [pJB91, pMZ28], and MBX2641 [pJB91, pJB90] were grown in shake flasks and analyzed for PHA content and composition as described in Example 1 and 2, in order to characterize the production of P(3HB-co-5HV) from L-lysine and glucose. MBX2641 [pJB78, pPB84], MBX2641 [pJB91, pMZ28], and MBX2641 [ρJB91, pJB90) were inoculated in test tubes containing 3 mL LB supplemented with 25 μg/mL chloramphenicol and 100 μg/mL ampicillin and grown overnight at 370C with 250 rpm shaking. The next day, 0.5 mL of each overnight culture was used to inoculate a shake flask containing 50 mL of fresh LB supplemented with the same antibiotic and grown at 370C with 250 rpm shaking. At 3.5 hours, 0.1 raM IPTG was added to the liquid cultures, and at 5 hours, the cultures were spun down at 4150 rpm (Sorvall Legend RT benchtop centrifuge) and resuspended in 50 mL of production medium that consisted of Ix E2 minimal salts solution containing 10 g/L glucose, 2.5 g/L LB, 10 g/L L-lysine, 2mM MgSO4, Ix Trace Salts Solution, and 0.1 mM IPTG. The recipes for E2 salts and Trace Salts Solution are given in Example 1.
As shown in Table 8, strain MBX2641 [pJB785 pJB84], which does not have the genes that convert L-lysine to 5HV, was unable to produce 5HV from L-lysine and produced only P(3HB) homopolymer. Strains MBX2641 [pJB91, ρMZ28] and MBX2641 [pJB91, pJB90], both of which contain the entire pathway from L-lysine to 5HV-CoA, incorporated 5HV to 2.5%wt and 5%wt, respectively, of the total copolymer. This demonstrates that the davBAT&nd gsaR genes need to be expressed in order to produce 5HV- containing copolymers from L-lysine. Table 8. P(3HB-co-5HV) production from L-lysine
Figure imgf000055_0001
EXAMPLE 7: Improved biosynthesis of 5HV-eontaining PHA polymers from L-lysine Due to the fact that 5HV was incorporated at an unexpectedly low level, the existence of a competing pathway was considered. In order to see if glutarate could be produced from L-lysine, MGl 655 [pJB91] expressing the davBAT genes from the plasmid was grown in LB medium containing 25 mg/L chloramphenicol at 30 0C with shaking for 6h. Aliquots of 25 μL mid- log phase cultures were inoculated into 475 μL EO minimum medium and incubated at 300C with shaking at 250 rpm for 48 h. The EO minimum medium consisted of 10 g/L glucose, 4 g/L lysine, 58 mM K2HPO4, 27 mM KH2PO4, 2 mM MgSO4, 25 μg/mL chloramphenicol, 0.1 mM IPTG, and trace elements. Glutarate, present in the supernatant, was measured by GC- MS as outlined below: supernatants from fermentation broth were obtained by centrifugation and 1 μL of the sample was pipetted to a fresh Eppendorf tube containing 1 μL of 1 g/L 4-aminobutyrate (GABA) as internal standard. Samples were dried in a Labconco centrivap and resuspended in 100 μL acetonitrile (ACN): N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) 1 :1 solution by sonication for 3 h Samples were then derivatized at 65°C for 30 min, centrifuged to remove insoluble material and supernatants injected into an Agilent 5975 GC-MS equipped with a HP-5ms column using the following acquisition parameters: carrier gas Helium flow rate 2 ml/min, scan mode m/z 65-700, solvent delay for 3.5 min, oven program: 15O0C for 2 min, then ramp up to 2800C at 3°C/min, ion source temperature 230°C, the quadrupole mass filter temperature 150°C.
Interestingly, 0.6 g/L glutarate was produced from L-lysine when the davBA T operon was overexpressed in MG 1655. The davBΛ T operon expresses genes encoding enzymes that convert L-lysine to GSA. Glutarate may be produced by an endogenous E. coli gene whose encoded enzyme can oxidize GSA to glutarate.
Examination of probable enzymatic reactions from GSA to glutarate led to the identification of two likely endogenous E. coli GSA dehydrogenase genes, gabD and/or ynel (see Table IA). These two genes have been identified earlier to oxidize succinic semialdehyde to succinic acid (Dennis and Valentin, U.S. Patent No. 6,117,658) but have not been shown to oxidize GSA to glutarate. To test if a gabD- and jwe/-negative strain still produces glutarate from L-lysine, the following strains were constructed.
Single null gabD and ynel mutants were constructed by the Red/ET Recombineering method described by Datsenko and Wanner (Proc. Natl, Acad. Sci. USA. 97:6640-6645 (2000)). The process of deleting gabD from the chromosome of MG 1655 involved the replacement of gabD with an FRT-flanked kan marker via PCR-mediated homologous recombination. The FRT-flanked kan marker was PCR-arnplified from plasmid pKD4 (Datsenko and Warmer, Proc. Natl. Acad. ScL USA. 97:6640-6645 (2000)) by using primers RF314 5'- GCAAGCCAGAGTAACCCCGGACGCACGCTGCGAGCGGCACGTAG TGTGGATGCCTTACACGCCGCATTTAATCAATAACCTTGAGCGAT TGTGTAGGCTGGAGCTGCTTC (SEQ ID NO:27) and RF315 5'- GAATTTGCCCAACGCCACGGGGAATCGCCTGACTGCGGCGCTGCA TTAACTCTTTATTGCTGTTCATTCGCATTCTCCAGATGGGAATTAG CCATGGTCCATATGAATAT (SEQ ID NO:28). The ynel gene was deleted from the chromosome of MGl 655 by replacement with an FRT-flanked kan marker. This marker was PCR- amplified from plasmid pKD4 using primers MS220 5'- GCAAGAGTAAATCTGCGTATCTTCATACCATGACTCATAAAGGAG ATACCCCGGTGTAGGCTGGAGCTGCTTC (SEQ ID NO:29) and MS217 5'-
ACCGCAGGTCTGAAAAGACCTGCGAGTATATCAGAGCTGAATATG TCGCGCATATGAATATCCTCCTTAGT (SEQ ID NO:30) that introduced 50 bp flanking regions of homology to the gene to be deleted. Replacement of ynel with this DNA fragment did not work, and hence, another PCR fragement was created that had increased regions of homology for gene replacement. To accomplish this, an additional round of PCR was performed with the PCR fragment generated above as template and primers MS223 5'- TCGATTCGTGAATAAGTGGCTTAATATTATTCATTTTAAAGCAAG AGTAAATCTGCGTATC (SEQ ID NO:31) and MS224 5'-
GCCACTTTCTACTCCTGGACCGCAGGTCTGAAAAGACCTGCGAGT ATATCAGAGCTG (SEQ ID NO:32). Successful replacement of ywlwith FRT-Arαrc-FRT was achieved. The kctn marker was then removed as described in Datsenko and Wanner (Proc. Natl. Acad. Sci. U S A. 97:6640- 6645 (2000)).
The MG1655 AgabD: :FRT-λαw-FRT, Δj/ne/::FRT was constructed by Pl-mediated transduction from MG\655AgabD::FKT-kcm-FRT to MG1655Δyrøe/::FRT, and the remaining kan marker was further removed using the same method as described above. The resulting strain MGl 655 AgabD: :FRT, Ayneϊ. :FRT was transformed with pJB91 and analyzed for glutarate production from L-lysine in an experiment analogous to the one described above with MG1655 [pJB91] expressing the davBAT genes from the plasmid.
In constrast to MG1655 [pJB91] that produced 0.6 g/L glutarate from L-lysine, strain MGl 655 AgabDv.FRΥ, AyneI::FRT [pJB91] did not produce any glutarate from L-lysine demonstrating that either the E, coli endogenous gabD and/or ynel were responsible for converting GSA to glutarate. Improved production of PβHB-co-5HV) copolymers from L-lysine
Improved 5HV flux from L-lysine was accomplished by deleting the endogenous GSA dehydrogenase encoding genes gabD and ynel in a strain that produced the 3HB-co-5HV copolymer.
MBX2855 was constructed by transforming plasmids pJB91 and pJB90 into strain MBX2641. This strain has all the genes to produce P(3HB- co-SHV) from glucose and L-lysine. MG 1655 AgabDv.FKT, Δywe/::FRT was Pl -transduced with donor strain MBX21 14 that conferred PHB producing capabilities as described in Example 2. This strain was further transformed with pJB90 and pJB91, which expressed the L-lysine to P(5HV) pathway genes as described in 5 Examples 6 and 5, respectively. The resulting strain was designated as MBX3378 and has all the genes to produce P(3HB-co-5HV) from glucose and L-lysine but unlike MBX2855 has both the gabD and ynel genes removed from the genome.
A shake-plate fermentation was conducted using strain MBX2855 0 and its GSA dehydrogenase-deficient counterpart MBX3378. Cells were incubated and analyzed under the same conditions as described above (shaking at 300 rpm at 300C). The EO minimum medium consisted of 10 g/L glucose, 2 g/L L-lysine, 58 niM K2HPO4, 27 mM KH2PO4, 2 mM MgSO4, 25 μg/mL chloramphenicol, 5 μg/mL gentamicin, 0.1 mM IPTG, and trace 5 elements as described in a previous example. The carbon flux from L-lysine to 5HV was dramatically improved in the GSA dehydrogenase-deficient strain MBX3378 as compared to MBX2855 that contained wild-type GSA dehydrogenase activity as shown in Table 9. In order to significantly improve production of 5HV containing PHA, GSA dehydrogenase genes 0 such as gabD and ynel need to be removed from the genome of production hosts.
Table 9. Improved P(3HB-co-5HV) production from L-lysine
Figure imgf000058_0001
EXAMPLE 8: Biosynthesis of L-lysine from glucose 5 Allosteric feedback regulation occurs in the L-lysine pathway through the genes lysC and dap A. Therefore, this control needs to be eliminated in order to enable increased L-lysine production from glucose. The procedure to do this is well-established and has been described for both genes (Kojima et al., U.S. Patent No. 6,040,160). E. coli mutants possessing deregulated lysC and dap A can be obtained first by deleting metL and thrA from E. coli. LysC, MetL and ThrA are isozymes that all catalyze the same aspartate kinase reaction, so it will be necessary to eliminate the latter two before mutations in lysC can be positively selected. Once a AmetL AthrA strain has been made, it can be mutated with N-methyl-N'-nitro-N- nitrosoguanidine (NTG). The resulting mutant pool would then be grown in a minimal medium containing S-2-aminoethylcysteine (AEC), a non- metabolizable analog of L-lysine, in order to put pressure on lysC and dap A. Since metL and thrA are missing, only mutations that desensitize lysC and dap A to L-lysine (or its AEC analog) will allow the cell to synthesize L- lysine, threonine, and methionine, and thus survive. Further manipulations can be carried out by overexpressing deregulated lysC, deregulated dap A, and other pathway genes from a recombinant promoter in order to increase flux capacity and to eliminate transcriptional regulation. EXAMPLE 9: Biosynthesis of P(SHV) homopolymer from glucose
P(SHV) was produced from glucose as the sole carbon source in an E. coli strain capable of synthesizing the 5HV-CoA monomers from glucose and incorporating them into PHA. For that, a strain was constructed that expressed not only the genes required to produce P(5HV) homopolymer from L-lysine, but which also expressed a mutated dapA gene called daρA^r that encodes a L-lysine feedback-resistant dihydrodipicolinate synthase. The first part of this example will describe the construction of the plasmids required to demonstrate this ability. Plasmids construction
In E. coli, allosteric regulation occurs in the L-lysine pathway through aspartate kinase III and dihydrodipicolinate synthase encoded by the lysC and dapA genes, respectively (Figure 6). In order to increase production of L-lysine and eventually P(SHV) homopolymer from glucose, the allosteric regulation needs to be reduced or entirely eliminated. The procedure to do this is well-established and has been described for both genes (Kojima et al, U.S. Patent No. 6,040,160). An L-lysine feed-back resistant dapA^r gene was constructed that had the 352lK nucleotide residue changed from cytosine to thymine {dap A ). This was obtained by PCR amplification of the chromosomal E. coli dapA gene generating two DNA fragments using primers that introduced the desired base change, followed by splicing by overlap extension PCR (SOE-PCR) to fuse the two DNA fragments. The SOE-PCR method has been described earlier (Ho et al., Gene 77(l):51-9 (1989). In detail, one DNA fragment contained the 1st to 366th nucleotide pairs of the dap A gene that was amplified with primers DE081 (5>-
AAAAGAATTCTTAATTAATTCTAGAAGGAGGTTTCATATGTTCAC GGGAAGTATT GTC) (SEQ ID NO:33) and DE082 (5'- AGCGATGGCTTTGAAATACTGATACAAACCTTC) (SEQ ID NO:34), and the other DNA fragment contained the 337th to 879th nucleotide pairs of dapA amplified with primers DE083 (5 ' -
GAAGGTTTGTATCAGTATTTCAAAGCCATCGCT) (SEQ ID NO:35) and DE084 (5'-
CCCGAGCTCGTTTAAACTTAATTAAGACTAGTTTTACAGCAAACC GGCATGCTT) (SEQ ID NO:36). The primers DE082 and DE083 are reverse complementary and were designed to introduce the cytosine to thymine base change at the 352th nucleotide residue. The two DNA fragments from the first round of PCRs were fused by SOE-PCR using primers DE081 and DE084. The resulting PCR product was digested with Xbal and Sad and ligated to pDE031 that had been digested with Spel and Sad, thus creating plasmid pDE035. Plasmid pDE031, containing a synthetic constitutive promoter (FSynd was constructed by digesting a synthesized 63 bp double-stranded DNA fragment (5'-
TTTTTCTAGATTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCA CTAGTGTTTAAACCCCC ) (SEQ ID NO:37) with Xbal and Pmel and ligated into the same restriction enzyme sites of a pBluescript II SK(+) plasmid (Stratagene, La Jolla, CA) that was previously engineered to contain these sites. The ΫsyflrdapACi52τ gene construct in pDE035 was digested with Xhol and Pmel, which was followed by ligation with the plasmid pJB90 (described in Example 6) that had been digested with BsrGI, blunted with Mung Bean nuclease, and digested a second time with Xhol to generate plasmid pJG22 that expressed phaC~g$aRΛrorfl operon from the Phc promoter and the dapA03521 gene from the Psy,,i promoter. Experimental results Plasmid ρJG22 was transformed along with plasmids pJB91
(described in Example 5) into strain MBX3342 (MGl 655 ΔgabDy.FRT AyneI::ΕKT) to form strain MBX3342 [pJB91 , pJG22]. Plasmids pSE380 and pACYC184, the empty vectors used to construct pJG22 and ρJB91, respectively, were also transformed into strain MBX3342 to create the negative control strain MBX3342 [pSE380, pACYC 184] . These strains were incubated for 48 h shaking at 300 rpm at 30σC in 2x E2 medium and analyzed as described in the earlier examples. The medium consisted of 15 g/L glucose, 52 mM NaNH4HPO4, 66 niM K2HPO4, 54 mM KH2PO4, 2 mM MgSO4, 0.1 mM IPTG, and trace elements as described above. For the culture media for MBX3342 [p JB91 , p JG22] , 25 μg/mL chloramphenicol and 5 μg/mL gentamicin were supplemented, while 25 μg/mL chloramphenicol and 100 μg/mL ampicillin were added for MBX3342 [pSE380, pACYC184], The data in Table 10 shows that MBX3342 [pJB9, pJG22] produced 2.60% dry cell weight (DCW) P(5HV) homopolymer while strain MBX3342 [pSE380, pACYC184] did not produce any PHA. These results demonstrate that a strain expressing the feedback resistant dapA gene in addition to the L-lysine to P(5HV) pathway genes can produce P(5HV) from glucose as the sole carbon source.
Table 10. P(SHV) production from glucose
< Strain , , Total PHA
Relevant Plasmid Genotype • [jPlasroW] (%dcw)
MBX3342 pSE380: empty vector control
0.0 [pSE380, pACYC184] pACYC184: empty vector control pJG22: Ϋfrc -phaC-gsaRArorfZ
MBX3342 ?ml- dapAC352τ 2.60 [pJG225 pJB91] pJB91: P onψA-davBAT EXAMPLE 10: Biosynthesis of P(3HB-co-5HV) copolymer from glucose
The next experiment was to demonstrate the production of P (3 HB -co- 5HV) copolymer from glucose in an E. coli strain capable of synthesizing the 3HB-CoA and 5HV-CoA monomers and incorporating them into PHA. Experimental results
The btkB-phaB-kan genes from strain MBX2114 were Pl -transduced into MBX3342 generating strain MBX3344. MBX3344 was transformed with plasmids pJB91 and pJG22 to create strain MBX3344 [pJB91, pJG22], The plasmids pSE380 and pACYC1845 the empty vectors used to construct pJG22 and pJB91, respectively, were also transformed into MBX3344 to create a negative control strain, MBX3344 [pSE38O, pACYC184]. These strains were incubated for 48 h shaking at 300 rpm at 30°C in 2x E2 medium and analyzed as described in the earlier examples. The medium consisted of 15 g/L glucose, 52 mM NaNH4HPO4, 66 mM K2HPO4, 54 mM KH2PO4, 2 mM MgSO4, 0.1 mM IPTG, and trace elements as described above. For the culture media for MBX3344 [pJB91, pJG22], 25 μg/mL chloramphenicol and 5 μg/mL gentamicin were supplemented while 25 μg/mL chloramphenicol and 100 μg/mL ampicillin were added for MBX3344 [pSE380, ρACYC184]. The culture broth was supplemented with 10 g/L glucose after 24 hours incubation. Table 11 shows that 5HV could be incorporated into the strain containing all the P(3HB-co-5HV) metabolic pathway genes from glucose as the sole carbon source.
Table 11. P(3HB-co-5HV) production from glucose
Figure imgf000062_0001
Next, strain MBX3824 (W31 10 AgabD::FRT AyneliiFRT AcadA::FRT AldcCr.FRΥ AargOv.FRT bktB-phaB-kan) was tested as the host strain to produce P(3HB-co-5HV) copolymer from glucose. In this strain, the competing pathways that may divert L-lysine away from the 5HV-CoA co-monomer were removed from the E. coli genome.
The first competing pathway may convert L-lysine to cadaverine and consists of two L-lysine decarboxylase enzymes (EC number 4.1.1.18) encoded by cadA (Meng and Bennett, J. Bacterid. 174(8):2659- 2669 (1992); EcoCyc accession number: EG10131) and ldcC (see Table IA; Yamamoto et al., Genes Genet. Syst. 72(3):167-72 (1997); EcoCyc accession number: G6094).
A second competing pathway may export L-lysine out of the microbial cell. In Corynebacterium glutamicum, the L-lysine export protein has been identified as LysE (see Table IA; Vrljic et al., MoI. Microbiol. 22 (5): 815-826 (1996)). In order to identify putative L-lysine exporter genes in E. coli, several literature and patent searches as well as BLAST and Psi-BLAST searches using C. glutamicum LysE as the query were conducted. Six proteins were found to be targets for removal from the E. coli genome in order to prevent L-lysine export outside of the cell. They include: (1) ArgO (a.k.a. YggA, Nandineni and Gowrishankar, J.
Bacteriol 186:3539-3546 (2004)), (2) YfiK (a.k.a. EamB; Franke et al., J. Bacterial 185:1161-1166 (2003)), (3) RlitB (formerly called YigK; Zakataeva et al,, FEBS Lett. 452(3):228-32 (1999)), (4) YahN (Kutukova et al., MoL Biol (Most) 39(3);374-378 (2005)), (5) RhtC (formerly called YigJ; Zakataeva et al., FEBS Lett, 452(3):228-32 (1999)), and (6) YeaS
(a.k.a. LeuE; Kutukova et al., FEBS Lett. 579(21):4629-34 (2005)). ArgO appeared to be the most likely candidate to export L-lysine out of E. coli cells based on its lowest e-value of 2e-22 in BLASTP searches using LysE from C glutamicum as the query, the closest clustered with LysE in a Neighbor Join Tree after ClustalX (Thompson et al,, Nucleic Acids Res. 25: 4876-4882 (1997)) with C glutamicum LysE and the six E. coli homologues, and the reported 3 -fold increased resistance to L-lysine as well as a 38% higher L-lysine accumulation when argO was overexpressed as compared to vector-only control strains (Livshits et al., U.S. Patent No. 6,979,560). However, the other identified proteins may export L-lysine too and therefore are also targets for gene deletion.
Another competing pathway may convert L-lysine to (R)-β-lysine which is catalyzed by lysine 2,3-aminomutase (EC number 5.4.3.-) encoded by yjeK of E. coli (EcoCyc accession number: G7836; Behshad et al, Biochemistry 45(42): 12639-46 (2006)).
Single null cadA and ldcC mutants were constructed by the Red/ET Recombineering method from Gene Bridges as described previously using the following primers: DEI l 8 (5'- TGTCCCATGTGTTGGGAGGGGCCTTTTTTACCTGGAGATATGACTG TGTAGGCTGGAGCTGCTTC) (SEQ ID NO:38) and DEl 19 (5'- GAGCAAAAAAGGGAAGTGGCAAGCCACTTCCCTTGTACGAGCTA AATGGGAATTAGCCATGGTCC) (SEQ ID NO:39) for a ΔcαdL4::FRT- kan-FRT mutation and DE 122 (5'- GTTTGAGCAGGCTATGATTAAGGAAGGATTTTCCAGGAGGAACAC GTGTAGGCTGGAGCTGCTTC) (SEQ ID NO:40) and DEl 23 (5 s- TATTTGTTAACAGCACGTTACTCGCCCGGAAGCCGCTCTGGCAAG ATGGGAATTAGCCATGGTCC) (SEQ ID NO:41) for AldcC::FRT~cat- FRT mutation. A single null argO mutation was constructed by the Red/ET Recombineering method using primers DEl 06 (5'-
GTGTTTTCTTATTACTTTCAAGGTCTTGCACTTGGGGCGGCTATGG TGTAGGCTGGAGCTGCTTC) (SEQ ID NO:42) and DEl 07 (5'- CTAACTGAACAAGGCTTGTGCATGAGCAATACCGTCTCTCGCCAG ATGGGAATTAGCCATGGTCC) (SEQ ID NO:43). The W31 10 AgabD: :FRT Ayneh:FRT AcadA::FRT AldcC.-.FRT AargOv.FRT was constructed by iterative Pl -mediated transductions of AgabDvJFRΥ-kan-FRT, AyneI::FRT- kan-FRT, AcadA::FR7-kan-FRΥ, AldcC::FRT-cat-FRT, AcadAv.FRT-kan- FRT cassettes into strain W3110 (Bachmann, Bacterial Rev., 36 (4):525-557 (1972)), which was followed by removal of kan or cat markers after each Pl -mediated transduction as described in earlier examples. The resulting strain MBX3818 was Pl -transduced with donor strain MBX2114 to finish the construction of MBX3824. The plasmids pJG22 and ρJB91 were transformed into MBX3824 and the resulting strain MBX3824 [pJG22, ρJB91] was tested for the production of P(3HB-co-5HV) copolymer along with MBX3344 [ρJG22, ρJB91]. These strains were incubated for 48 h shaking at 300 rpm at 300C in 1.5x E2 medium and analyzed as described in the earlier examples. The medium consisted of 15 g/L glucose, 39 mM NaNH4HPO4, 49.5 mM K2HPO4, 40.5 mM KH2PO4, 2 mM MgSO4, 0.1 mM IPTG, and trace elements as described above. The culture media were supplemented with 25 μg/mL chloramphenicol and 5 μg/mL gentamicin. Table 12 shows that various strains with different genetic backgrounds have the ability to produce P(3HB-co-5HV) from glucose with different compositions of 5HV in the polymer. In particular, removing competing 0 pathways that divert carbon away from 5HV-CoA, such as removing the L- lysine export protein argO or the two lysine decarboxylase genes cadA and idcC, increase 5HV incorporation into PHA.
Table 12. P(3HB-co-5HV) production from glucose
Figure imgf000065_0001
EXAMPLE 11: Biosynthesis of P(4HB-co-5HV) copolymer from sodium 4-hydroxybutyrate and sodium 5-hydroxyvalerate
The next experiment was to demonstrate the production of P(4HB-co- 5HV) copolymer in an E. coli strain capable of synthesizing the 4HB-CoA 0 and 5HV-CoA monomers and incorporating them into PHA. Methods for engineering 4HB co-monomers in recombinant organisms have been described in detail in U.S. Pat. No. 6,117,658, U.S. Pat. No. 6,316,262, U.S. Pat. No. 6,689,589, U.S. Pat No. 7,081,357, U.S. Pat. No. 7,229,804, U.S. Pat. No. 6,759,219, and U.S. Pat. App. Pub. 2004/0253693, which are hereby incorporated by reference in their entirety. In an experiment similar to the one described in Example 1, sodium 5 -hydroxy valerate (Na5HV) was fed to strain MG 1655 [pMS93] along with sodium 4-hydroxybutyrate (Na4HB). Strain MG 1655 [pMS93] contains the genes orfZ and phaC, both of which are required to generate 4HB-CoA and 5HV-CoA from Na4HB and NaSHV, respectively, as well as to polymerize the precursors to the P(4HB-co-5HV) copolymer. Na4HB for use as substrate was prepared analogous to the method described for preparation of NaSHV in Example 1 with use of γ- butyrolactone (GBL) in place of DVL. The culture conditions used for copolymer production was also the same as that described in Example 1 with the difference that 4 g/L of Na4HB was added to the production medium. Following the PHA production period, analysis of polymer content of the MG 1655 [pMS93] culture proceeded as described in Example 1 except that a standard curve for determining 4HB content was made using GBL standards in addition to the standard curve made for determining the 5HV content. This analysis showed that the MGl 655 [pMS93] culture cofed Na4HB and NaSHV generated P(4HB-co-5HV) copolymer that comprised 67% dew and had a composition that was 67% 4HB and 33% 5HV. An extracted sample of this polymer was analyzed using DSC and was determined to have a Tg of -54.90C and no detectable Tm. EXAMPLE 12: Biosynthesis of gϊutarate from glucose
In order to differentiate which of the two gene products were the primary GSA dehydrogenase as identified in Example 7, wild-type strain MG1655 [pJB91], MG1655Δy«e/::FRT [pJB91], and MG1655ΔgαδD::FRT [pJB91]were compared for their ability to produce glutarate from 5- aminopentanoate, an intermediate metabolite between L-lysine and GSA (see Figure 2). All three strains contain plasmid pJB91 which express davBAT from the PompA promoter as described in Example 5. The three strains were grown in EO minimum medium containing 2 g/L 5-aminopentanoate as described earlier and glutarate was measured by a GC-MS method from culture supernatants. The incubation method and conditions were the same as described in Example 7.
Unlike the other two strains, MG1655ΔgabD [pJB91] did not accumulate any detectable glutarate. Thus, the dehydrogenase encoded by gab D has the major activity towards GSA. Therefore, if high amounts of glutarate is to be produced, production hosts need to express gabD or homologues (see Table IP) thereof. However, since MGl 655AyneI [pJB91] accumulated slightly lower amounts of glutarate from 5-aminopentanoate as compared to MG1655 [pJB91], 0.75 g/L versus 1.0 g/L glutarate, respectively, the dehydrogenase encoded by ynel also has moderate activity towards GSA. Thus, overexpression of yneloτ homologues (see Table IQ) thereof, may also yield high amounts of glutarate from GSA, L-lysine or glucose.
The two best GabD homologues present in Corynebacterium glutamicum include (1 ) the NAD-dependent aldehyde dehydrogenase (Accession No. NP_599302) and (2) the hypothetical protein cgR_0068 (Accession No. YP_001136931). Unexpectedly, these two C. glutamicum proteins were also identified as the two closest homologues to E. coli Ynel. Next, glutarate was produced from glucose. To provide an L-lysine- overproducing strain, plasmid pDE033 which contains the L-lysine feedback resistant dapACi52τ gene was constructed as follows: the product of SOE-PCR for the fabrication of dapAC352T gene described in Example 9 was digested with EcoRI and Sad, followed by ligation with ρSE380 that had been digested with the same enzymes, thus creating plasmid pDE033. The dapAQ5τϊ gene in pDE033 is under the IPTG-inducible promoter P,,c. Plasmids pDEO33 and pJB91 (described above) were transformed into MG 1655 strain to create strain MG 1655 [pDE033, pJB91]. The strains MG1655 and MG1655 [pDEO33, pJB91] were incubated for 48 h with shaking at 300 rpm at 3O0C in a medium that contained 25 g/L glucose, 16 g/L (NH4) 2SO3, 1 g/L KH2PO4, 1 g/L MgSO4, 2 g/L yeast extract, 30 g/L CaCO3, 0.1 mM IPTG, and trace elements as described above. The culture media for MG1655 [pDE033, pJB91] was supplemented with 100 μg/mL ampicillin and 25 μg/mL chloramphenicol. Glutarate was measured as described in Example 7. The data shown in Table 13 demonstrates that MG 1655 [pDE033, pJB91] secreted 0.7 g/L glutarate into the medium while the negative control strain MGl 655 did not produce any glutarate. This result clearly shows that using a feedback-resistant dapA gene to accumulate L- lysine, together with the davBAT opeτon to convert L-lysine to GSA in a host cell that encodes a GSA dehydrogenase, is sufficient to produce glutarate from glucose as the sole carbon source.
Table 13. Glutarate production from glucose
1 Strain > * Glutaraie
Relevant Plasmid Genotype t (g/L)
MG1655 0
MG1655 pDE33: P frc - dapA∞21'
0.7 [pDE033, pJB91] pJB91 : VompA'davBAT
EXAMPLE 13: Biosynthesis of 1,5-pentanediol from sodium 5- hydroxyvalerate Strain construction
Strain MBX3017 (LS5218 AadhE::¥RT, A!dhA::FRT, AackA- ptav.f KT) and K- 12 strain MGl 655 were used as a host strain to assess if 1 ,5-pentanediol (PDO) could be accumulated and secreted into the medium. Each single deletion strain was constructed by the Red/ET method from Gene Bridges. Primers for constructing knock-out cassettes for the three pathways are listed in Table 14. Briefly, the following primers were used for the construction of the various chromosomal deletions: MS286 and MS287 for the AadhE cassette; MS289 and MS290 for the AackA-pta cassette; and MS292 and MS293 for the MdhA cassette. The LS5218 AadhE\:FRT, AldhAr.FRT, ΔackA-ptavSKl was obtained by iteratively Pl transducing each single null mutation into LS5218 and removing the marker as described in an earlier example. Table 14. Primer used for PDO studies
Figure imgf000069_0001
The CoA-dependent propionaldehyde dehydrogenase encoding pduP from S. typhimurium (see Table IA; Leal, Arch. Microbiol. 180:353-361 (2003)) was amplified by primers JRG47 and JRG48. The 1,3 -propanediol dehydrogenase encoding dhaT from Klebsiella pneumoniae (see Table IA; Tong et al, Appl. Environ. Microbiol. 57(12):3541-3546 (1991)) was amplified with primers JRG49 and JRG50. The two genes were fused together by SOE-PCR using primers JRG47 and JRG50. The resulting DNA
10 fragment was cloned into pJB78 via BamHI and BspHI sites, and the resulting plasmid was designated as pJGlO.
Strain MBX3017 or MG1655 harboring pFS16 or pSE380 and pJGlO or pJB78 were used for PDO studies. Strains were grown under oxygen limited conditions in 5HV- containing E2 medium for 40 h. The medium consisted of: 10g/L glucose, 2g/L 5HV, 26 niM NaNH4HPO4, 33 mM K2HPO4, 27 mM KH2PO4, 2 niM MgSO4, 25 μg/mL chloramphenicol, 100 μg/mL ampicillin, 0.1 mM IPTG, and trace elements as described above. Overnight cultures were inoculated into a sealed culture tube containing fresh medium to a final OD6oo of approximately 0.2, the headspace for the culture tube was small to ensure oxygen limitation of the culture. The cultures were incubated at 3O0C. After 48 h of incubation, 100 μL sample was removed, centrϊfuged, and the resulting supernatant was spiked with 1,4-butanediol (0.1 g/L) as internal standard, dried in a Labconco centrivap and resuspended in 100 μL acetonitrile (ACN) by sonication for 3 h. The acetonitrile solution was centrifuged to remove insoluble material, and the supernatant was injected into an Agilent 5975 GC-MS equipped with a DB-225ms column using the following acquisition parameters: carrier gas Helium flow rate 1 ml/min, scan mode m/z 30-400, oven program: 4O0C for 2 min, then ramp up to 2200C at 10°C/min, ion source temperature 2300C, the quadrupole mass filter temperature 1500C.
The measured PDO results are shown in Table 14. The combination of host strain MBX3017 and overexpression of orfZ-dhaT-pduP gave the highest PDO yield of 0,32 g/L. Strain MGl 655 harboring these three genes gave a lower yield of 0.22 g/L, possibly due to its active ethanol, acetate and lactate pathway, which are known electron acceptors (Clark, FEMS Microbiol. Rev. 5:223-34 (1989)) and could compete with the 5HV pathway for NAD(P)H. Interestingly, MGl 655 harboring just orfZ also produced small amounts of PDO, while the MBX3017 host did not yield any detectable PDO (Table 15). This indicates that a endogenous alcohol dehydrogenase, e.g. adhEi has weak activity towards 5HV-CoA. The measured PDO was confirmed by GC-MS against the PDO standard and NIST library PDO reference spectrum as shown in Figure 8. These results demonstrate that PDO can be produced from NaSHV when the orfZ gene is expressed to generate 5HV-CoA and the pduP-dhaT genes are expressed converting 5HV-CoA to 5-hydroxypentenal and to PDO. Table 15. PDO titer of strains grown in 5HV containing mediuma
Figure imgf000071_0001
Results are the average of three independent replicated experiments
Gene ID 001 Nucleotide Sequence: Aspergillus terreus NIH2624 glutarate semialdehyde reductase gene gsaR-Aα
ATGCCACTGGTTGCTCAAAATCCACTGCCACGTGCTATTCTGGGTCTGATG ACTTTCGGTCCGAGCGAAAGCAAAGGTGCGCGTATCACTTCCCTGGATGAG TTTAACAAGTGCCTGGATTACTTCCAGCAGCAGGGCTTCCAGGAAATCGAT ACCGCGCGCATCTACGTCGGCGGTGAACAGGAGGCATTCACGGCGCAGGCA AAGTGGAAAGAACGCGGCCTGACGCTGGCGACTAAGTGGTATCCGCAGTAC CCGGGTGCGCACAAACCGGATGTCCTGCGTCAGAACCTGGAGCTGTCCCTG AAAGAACTGGGCACGAACCAGGTCGATATCTTCTATCTGCACGCCGCGGAT CGTTCTGTGCCGTTCGCGGAAACTCTGGAAACTGTTAACGAACTGCACAAA GAAGGCAAATTTGTTCAGCTGGGTCTGTCTAACTACACCGCTTTCGAAGTA GCTGAAATCGTGACCCTGTGTAACGAGCGTGGTTGGGTTCGTCCGACTATC TACCAGGCGATGTATAACGCTATCACCCGTAACATCGAAACTGAACTGATC CCGGCGTGCAAGCGTTACGGTATTGACATTGTTATCTACAACCCACTGGCG GGTGGCCTGTTCAGCGGCAAATACAAAGCACAGGACATCCCGGCTGAAGGT CGTTACAGCGACCAATCTTCCATGGGCCAGATGTACCGCAACCGTTACTTT AAGGACGCAACCTTTGACGCTCTGCσCCTGATCGAACCGGTTGTTGCGAAG CACGGCCTGACGATGCCGGAAACCGCGTTCCGCTGGGTCCACCACCACTCC GCACTGAACATGGAAGATGGCGGCCGTGACGGCATCATTCTGGGTGTAAGC AGCCTGGCTCAGCTGGAAAACAACCTGAAAGACATTCAGAAAGGTCCGCTG CCGCAGGAGGTTGTAGACGTCCTGGATCAGGCTTGGCTGGTGGCTAAGCCG ACGGCTCCAAACTACTGGCATCTGGACCTGAAATACACGTACGACACCCAG GAAGCTCTGTTCAAACCGAAATCTAAGGCGTAA (SSQ ID NO: 56)
Gene ID 001 Amino Acid Sequence: Aspergillus terreus NIH2624 glutarate semialdehyde reductase gene gsaR.At2
MPLVAQNPLPRAILGLMTFGPSESKGARITSLDEFNKCLDYFQQQGFQEID TARIYVGGEQEAFTAQAKWKERGLTLATKWYPQYPGAHKPDVLRQNLELSL KELGTNQVDIFYLHAADRSVPFAETLETVNELHKEGKFVQLGLSNYTAFEV AEIVTLCNERGWVRPTIYQAMYNAITRNIETELIPACKRYGIDIVIYNPLA GGLFSGKYKAQDIPAEGRYSDQSSMGQMYRNRYFKDATFDALRLIEPWAK HGLTMPETAFRWVHHHSALNMEDGGRDGIILGVSSLAQLENNLKDIQKGPL PQEWDVLDQAWLVAKPTAPNYWHLDLKYTYDTQEALFKPKSKA { SEQ
ID NO : 57 )
Gene ID 002 Nucleotide Sequence: Arabidopsis thaliana glutarate semialdehyde reductase gene gsaRAt
ATGGAAGTAGGTTTTCTGGGTCTGGGCATTATGGGTAAAGCTATGTCCATG AACCTGCTGAAAAACGGTTTCAAAGTTACCGTGTGGAACCGCACTCTGTCT AAATGTGATGAACTGGTTGAACACGGTGCAAGCGTGTGCGAGTCTCCGGCT GAGGTGATCAAGAAATGCAAATACACGATCGCGATGCTGAGCGATCCGTGT GCAGCTCTGTCTGTTGTTTTCGATAAAGGCGGTGTTCTGGAACAGATCTGC GAGGGTAAGGGCTACATCGACATGTCTACCGTCGACGCGGAAACTAGCCTG AAAATTAACGAAGCGATCACGGGCAAAGGTGGCCGTTTTGTAGAAGGTCCT GTTAGCGGTTCCAAAAAGCCGGCAGAAGACGGCCAGCTGATCATCCTGGCA GCAGGCGACAAAGCACTGTTCGAGGAATCCATCCCGGCCTTTGATGTACTG GGCAAACGTTCCTTTTATCTGGGTCAGGTGGGTAACGGTGCGAAAATGAAA CTGATTGTTAACATGATCATGGGTTCTATGATGAACGCGTTTAGCGAAGGT CTGGTACTGGCAGATAAAAGCGGTCTGTCTAGCGACACGCTGCTGGATATT CTGGATCTGGGTGCTATGACGAATCCGATGTTCAAAGGCAAAGGTCCGTCC ATGACTAAATCCAGCTACCCACCGGCTTTCCCGCTGAAACACCAGCAGAAA GACATGCGTCTGGCTCTGGCTCTGGGCGACGAAAACGCTGTTAGCATGCCG GTCGCTGCGGCTGCGAACGAAGCCTTCAAGAAAGCCCGTAGCCTGGGCCTG GGCGATCTGGACTTTTCTGCTGTTATCGAAGCGGTAAAATTCTCTCGTGAA TAA (SEQ ID NO: 58)
Gene ID 002 Amino Acid Sequence: Arabidopsis thaliana glutarate semialdehyde reductase gene gsaR.At
MEVGFLGLGIMGKAMSMNLLKNGFKVTVWNRTLSKCDELVEHGASVCESPA EVIKKCKYTIAMLSDPCAALSWFDKGGVLEQICEGKGYIDMSTVDAETSL KINEAITGKGGRFVEGPVSGSKKPAEDGQLIILAAGDKALFEESIPAFDVL GKRSFYLGQVGNGAKMKLIVNMIMGSMMNAFSEGLVLADKSGLSSDTLLDI LDLGAMTNPMFKGKGPSMTKSSYPPAFPLKHQQKDMRLALALGDENAVSMP VAAAANEAFKKARSLGLGDLDFSAVIEAVKFSRE (SSQ ID NO: 59)
Gene ID 003 Nucleotide Sequence: Pseudomonas putida/Zoogloea ramigera polyhydroxyalkanoate synthase fusion gene phaC3/C5
ATGAGTAACAAGAACAACGATGAGCTGCAGTGGCAATCCTGGTTCAGCAAG GCGCCCACCACCGAGGCGAACCCGATGGCCACCATGTTGCAGGATATCGGC GTTGCGCTCAAACCGGAAGCGATGGAGCAGCTGAAAAACGATTATCTGCGT GACTTCACCGCGTTGTGGCAGGATTTTTTGGCTGGCAAGGCGCCAGCCGTC AGCGACCGCCGCTTCAGCTCGGCAGCCTGGCAGGGCAATCCGATGTCGGCC TTCAATGCCGCATCTTACCTGCTCAACGCCAAATTCCTCAGTGCCATGGTG GAGGCGGTGGACACCGCACCCCAGCAAAAGCAGAΆAATACGCTTTGCCGTG CAGCAGGTGATTGATGCCATGTCGCCCGCGAACTTCCTCGCCACCAACCCG GAAGCGCAGCAAAAACTGATTGAAACCAAGGGCGAGAGCCTGACGCGTGGC CTGGTCAATATGCTGGGCGATATCAACAAGGGCCATATCTCGCTGTCGGAC GAATCGGCCTTTGAAGTGGGCCGCAACCTGGCCATTACCCCGGGCACCGTG ATTTACGAAAATCCGCTGTTCCAGCTGATCCAGTACACGCCGACCACGCCG ACGGTCAGCCAGCGCCCGCTGTTGATGGTGCCGCCGTGCATCAACAAGTTC TACATCCTCGACCTGCAACCGGAAAATTCGCTGGTGCGCTACGCGGTGGAG CAGGGCAACACCGTGTTCCTGATCTCGTGGAGCAATCCGGACAAGTCGCTG GCCGGCACCACCTGGGACGACTACGTGGAGCAGGGCGTGATCGAAGCGATC CGCATCGTCCAGGACGTCAGCGGCCAGGACAAGCTGAACATGTTCGGCTTC TGCGTGGGCGGCACCATCGTTGCCACCGCACTGGCGGTACTGGCGGCGCGT GGCCAGCACCCGGCGGCCAGCCTGACCCTGCTGACCACCTTCCTCGACTTC AGCGACACCGGCGTGCTCGACGTCTTCGTCGATGAAACCCAGGTCGCGCTG CGTGAACAGCAATTGCGCGATGGCGGCCTGATGCCGGGCCGTGACCTGGCC TCGACCTTCTCGAGCCTGCGTCCGAACGACCTGGTATGGAACTATGTGCAG TCGAACTACCTCAAAGGCAATGAGCCGGCGGCGTTTGACCTGCTGTTCTGG AATTCGGACAGCACCAATTTGCCGGGCCCGATGTTCTGCTGGTACCTGCGC AACACCTACCTGGAAAACAGCCTGAAAGTGCCGGGCAAGCTGACGGTGGCC GGCGAAAAGATCGACCTCGGCCTGATCGACGCCCCGGCCTTCATCTACGGT TCGCGCGAAGACCACATCGTGCCGTGGATGTCGGCGTACGGTTCGCTCGAC ATCCTCAACCAGGGCAAGCCGGGCGCCAACCGCTTCGTGCTGGGCGCGTCC GGCCATATCGCCGGCGTGATCAACTCGGTGGCCAAGAACAAGCGCAGCTAC TGGATCAACGACGGTGGCGCCGCCGATGCCCAGGCCTGGTTCGATGGCGCG CAGGAAGTGCCGGGCAGCTGGTGGCCGCAATGGGCCGGGTTCCTGACCCAG CATGGCGGCAAGAAGGTCAAGCCCAAGGCCAAGCCCGGCAACGCCCGCTAC ACCGCGATCGAGGCGGCGCCCGGCCGTTACGTCAAAGCCAAGGGCTGA (SEQ ID NO: 60)
Gene ID 003 Amino Acid Sequence: Pseudomonas putida/Zoogloea ramigera polyhydroxyalkanoate synthase fusion gene PhaC3/C5
MSNKMNDELQWQSWFSKAPTTEANPMATMLQDIGVALKPEAMEQLKNDYLR DFTALWQDFLAGKAPAVSDRRFSSAAWQGNPMSAFNAASYLLNAKFLSAMV
EAVDTAPQQKQKIRFAVQQVIDAMSPANFLATNPEAQQKLIETKGESLTRG
LVNMLGDINKGHISLSDESAFΞVGRNLAITPGTVIYENPLFQLIQYTPTTP
TVSQRPLLMVPPCINKFYILDLQPENSLVRYAVEQGNTVFLISWSNPDKSL AGTTWDDYVEQGVISAIRIVQDVSGQDKLNMFGFCVGGTIVATALAVLAAR GQHPAASLTLLTTFLDFSDTGVLDVFVDETQVALRSQQLRDGGLMPGRDLA STFSSLRPNDLVWNYVQSNYLKGNEPAAFDLLFWNSDSTNLPGPMFCWYLR NTYLENSLKVPGKLTVAGEKIDLGLIDAPAFIYGSREDHIVPWMSAYGSLD ILNQGKPGANRFVLGASGHIAGVINSVAKNKRSYWINDGGAADAQAWFDGA QEVPGSWWPQWAGFLTQHGGKKVKPKAKPGNARYTAIEAAPGRYVKAKG ( SEQ ID KO : 61}
Gene ID 004 Nucleotide Sequence: Thiocapsa phenigii polyhydroxyalkanoate synthase subunit phaE ATGGCTGGTGACCACGTCGTGGAATGCCTTCGAATTCAGGAGGTTTTTATG AACGATACGGCCAACAAGACCAGCGACTGGCTGGACATCCAACGCAAGTAC TGGGAGACCTGGTCGGAGCTCGGCCGCAAGACCTTGGGTCTGGAGAAGACC CCGGCCAATCCTTGGGCCGGCGCCCTCGATCATTGGTGGCAGACGGTCTCG CCCGCCGCCCCCAACGACCTGGTTCGCGACTTCATGGAGAAGCTCGCCGAG CAGGGCAAGGCCTTCTTCGGCCTCACCGACTACTTCACGAAGGGCCTCGGC GGCAGTAGCGGTACGCAGGGCTGGGACACCCTCTCGAAGACCATCGACGAC ATGCAAAAGGCCTTCGCCAGCGGCCGGATCGAAGGCGACGAGACCTTCCGC CGCCTGATGGCCTTCTGGGAGATGCCGCTCGACAACTGGCAGCGCACCATG TCCTCGCTGTCCCCGGTGCCCGGCGACCTGCTGCGCAACATGCCGCACGAC CAAGTCAGGGACAGCGTCGACCGCATCCTCTCGGCACCCGGGCTCGGCTAC ACGCGCGAGGAGCAGGCCCGCTACCAGGATCTGATCCGCCGCTCGCTGGAG TACCAGTCGGCCCTGAACGAATACAACGGCTTCTTCGGCCAGCTCGGTGTC AAGTCCCTCGAGCGGATGCGCGCCTTCCTGCAGGGACAGGCCGAGAAGGGC GTCGCCATCGAGTCGGCGCGCACCCTCTACGACGCCTGGGTCGGCTGCTGC GAAGAGGTCTATGCCGAGGAGGTCAGCTCCGCCGACTACGCGCACATCCAC GGCCGCCTCGTCAACGCCCAGATGGCCCTCAAGCAGCGCATGTCGACCATG GTCGACGAGGTCCTCGGCGCGATGCCGCTGCCGACCCGCAGCGAGCTGCGC ACGCTCCAGGATCGGCTCCAGGAGTCGCGCGGCGAGGGCAAGCGCCAGCGC CAAGAGATCGAGACGCTGAAGCGGCAGGTCGCGGCCTTGGCCGGCGGCGCC CAGCCCGCGCCCCAGGCCTCCGCCCAGCCCAGCACCCGGCCCGCGCCGGCG ACGGCCCCGGCGGCGAGCGCGGCGCCCAAGCGCAGCACCACGACCCGCCGC AAGACCACCAAGCCCACCACCGGCCAGTGA (SEQ ID NO: 62) Gene ID 004 Amino Acid Sequence: Thiocapsa phenigii polyhydroxyalkanoate synthase subunit PhaE
MNDTANKTSDWLDIQRKYWETWSΞLGRKTLGLEKTPANPWAGALDHWWQTV SPAAPNDLVRDFMEKLAEQGKAFFGLTDYFTKGLGGSSGTQGWDTLSKTID
DMQKAFASGRIEGDETFRRLMAFWEMPLDNWQRTMSSLSPVPGDLLRNMPH
DQVRDSVDRILSAPGLGYTREEQARYQDLIRRSLEYQSALNEYNGFFGQLG
VKSLERMRAFLQGQAEKGVAIESARTLYDAWVGCCEEVYAEEVSSADYAHI
HGRLVNAQMALKQRMSTMVDEVLGAMPLPTRSELRTLQDRLQESRGEGKRQ RQΞIETLKRQVAALAGGAQPAPQASAQPSTRPAPATAPAASAAPKRSTTTR
RKTTKPTTGQ {SEC ID NO: 63)
Gene ID 005 Nucleotide Sequence: Thiocapsa phenigii polyhydroxyalkanoate synthase subunit phaC
ATGTCCCCATTCCCGATCGACATCCGGCCCGACAAGCTGACCGAGGAGATG CTGGAGTACAGCCGCAAGCTCGGCGAGGGTATGCAGAACCTGCTCAAGGCC GACCAGATCGACACAGGCGTCACCCCCAAGGACGTCGTCCACCGCGAGGAC AAGCTGGTCCTCTACCGCTACCGGCGCCCGGCGCAGGTGGCGACCCAGACG ATCCCGCTGCTGATCGTCTACGCCCTCGTCAATCGGCCCTACATGACCGAC ATCCAGGAGGATCGCTCGACGATCAAGGGCCTGCTCGCCACCGGTCAGGAC GTCTATCTGATCGACTGGGGCTACCCGGATCAGGCCGACCGGGCGCTGACC CTCGATGACTACATCAACGGCTACATCGACCGCTGCGTCGACTACCTGCGC GAGACCCACGGCGTCGACCAGGTCAACCTGCTCGGGATCTGCCAGGGCGGG GCCTTCAGCCTCTGCTACACGGCCCTGCACTCCGAGAAGGTCAAAAACCTC GTCACCATGGTCACGCCGGTCGACTTCCAGACCCCGGGCAACCTGCTCTCG GCCTGGGTCCAGAACGTCGACGTCGACCTGGCCGTCGACACCATGGGCAAC ATCCCGGGCGAACTGCTCAACTGGACCTTCCTGTCGCTCAAGCCCTTCAGC CTGACCGGCCAGAAGTACGTCAACATGGTCGACCTGCTCGACGACGAGGAC AAGGTCAAGAACTTCCTGCGGATGGAGAAGTGGATCTTCGACAGCCCGGAC CAGGCCGGCGAGACCTTCCGCCAGTTCATCAAGGACTTCTACCAGCGCAAC GGCTTCATCAACGGCGGCGTCCTGATCGGCGATCAGGAGGTCGACCTGCGC AACATCCGCTGCCCGGTCCTGAACATCTACCCGATGCAGGACCACCTGGTG CCGCCGGATGCCTCCAAGGCCCTCGCGGGACTGACCTCCAGCGAGGACTAC ACGGAGCTCGCCTTCCCCGGCGGGCACATCGGCATCTACGTCAGCGGCAAG GCGCAGGAAGGAGTCACCCCGGCGATCGGCCGCTGGCTGAACGAACGCGGC TGA (SEQ ID NO: 64)
Gene ID 005 Amino Acid Sequence: Thiocapsa phenigii polyhydroxyalkanoate synthase subunit PhaC
MSPFPIDIRPDKLTEEMLEYSRKLGEGMQMLLKADQIDTGVTPKDWHRED KLVLYRYRRPAQVATQTIPLLIVYALVNRPYMTDIQEDRSTIKGLLATGQD VYLIDWGYPDQADRALTLDDYINGYIDRCVDYLRETHGVDQVNLLGICQGG AFSLCYTALHSEKVKNLVTMVTPVDFQTPGNLLSAWVQNVDVDLAVDTMGN IPGELLNWTFLSLKPFSLTGQKYVNMVDLLDDEDKVKMFLRMEKWIFDSPD QAGETFRQFIKDFYQRNGFINGGVLIGDQEVDLRNIRCPVLNIYPMQDHLV PPDASKALAGLTSSEDYTELAFPGGHIGIYVSGKAQEGVTPAIGRWLNERG
(SEQ ID NO: 65)

Claims

We claim;
1. A recombinant organism genetically engineered to convert glutarate semialdehyde or 5-aminopentanoate into a 5 carbon monomer, polymer or copolymer thereof, wherein the recombinant organism expresses at least one or more gene(s) encoding one or more enzymes selected from the group consisting of: lysine 2-monooxygenase, EC 1.13.12.2; 5- aminopentanamidase (δ-aminovaleramidase), EC 3.5.1.30; 5- aminopentanoate transaminase (δ-aminovalerate transaminase), EC 2.6.1.48; lysine decarboxylase, EC 4,1.1.18; succinate semialdehyde reductase (a.k.a. glutarate semialdehyde reductase), EC 1.1.1.61; CoA-transferase, EC 2.8.3.14 and EC 2.8.3.n; Acyl-CoA synthetase, EC 6.2.1.3; PHA synthase, EC 2.3. l .n; β-ketoacyl-CoA thiolase, EC 2.3.1.9; acetoacetyl-CoA reductase, EC 1.1.1.36; glutarate-semialdehyde dehydrogenase, EC 1.2.L20; propionaldehyde dehydrogenase, EC 1.2.1.3; alcohol dehydrogenase, EC 1.1.1 , 1; 1 ,3-propanediol dehydrogenase EC 1.1.1.202 and wherein the recombinant organism produces more glutarate semialdehyde or 5- aminopentanoate than an unmodified organism and wherein the glutarate semialdehyde or 5-aminopentanoate is converted into the 5 carbon monomer, polymer or copolymer thereof by the recombinant organism.
2. The recombinant organism of claim 1 wherein the 5 carbon monomer is selected from the group consisting of glutarate, 1,5 pentanediol, 5-aminovalerate and 5-hydroxyvalerate.
3. The recombinant organism of any one of claims 1 -2 wherein the polymer or copolymer comprises polyhydroxyalkanoate.
4. The recombinant organism of any one of claims 1-3 wherein the monomer comprises 5-hydroxyvalerate.
5. The recombinant organism of any one of claims 1 -4 wherein the recombinant organism converts 5-aminopentanoate into glutarate semialdehyde.
6. The recombinant organism of any one of claims 1 -5 wherein the recombinant organism converts lysine into 5-aminopentanoate.
7. The recombinant organism of claim 6 wherein the lysine is fed to the organism.
8. The recombinant organism of any one of claims 1 -7 wherein the organism used to construct the recombinant organism has been modified to overproduce lysine relative to an unmodified organism.
9. The recombinant organism of any one of claims 1-8 wherein the recombinant organism is resistant to the toxic lysine analog S-(2- aminoethyl) cysteine.
10. The recombinant organism of any one of claims 1 -8 wherein the recombinant organism expresses a lysine feedback-resistant dihydrodipicolinate synthase.
11. The recombinant organism of any one of claims 1 -9 wherein the recombinant organism expresses a lysine feedback-resistant aspartate kinase III.
12. The recombinant organism of claims 8- 1 wherein the organism is fed a renewable carbon substrate.
13. The recombinant organism of any one of claims 1-12 wherein the recombinant organism is further engineered to inhibit or block lysine export.
14. The recombinant organism of any one of claims 1-13 wherein the recombinant organism expresses or overexpresses glutarate semialdehyde dehydrogenase and the 5 carbon monomer is glutarate.
15. The recombinant organism of claim 14 wherein the glutarate semialdehyde dehydrogenase is encoded by a gene selected from the group consisting of davD, ynel, and gabD.
16. The recombinant organism of any one of claims 1-13 wherein the recombinant organism releases glutarate into the extracellular environment.
17. The recombinant organism of Claims 1- 13 wherein the organism has been modified to reduce or eliminate glutarate semialdehyde dehydrogenase activity and the 5 carbon monomer is not glutarate.
18. The recombinant organism of claim 17 wherein the glutarate semialdehyde dehydrogenase is reduced or eliminated by deleting or disrupting one or more genes selected from the group consisting of davD, ynel, and gab D or their homologs.
19. The recombinant organism of any one of claims 1-13 and 17- 18 wherein the recombinant organism releases 5-hydroxyvalerate into the extracellular environment.
20. The recombinant organism of claim 17-18 wherein the recombinant organism releases 5-hydroxyvalerate in the extracellular environment and the 5-hydroxyvalerate is in equilibrium with delta- valerolactone.
21. The recombinant organism of any one of claims 1-13 and 17- 18 wherein the recombinant organism converts 5-hydroxyvalerate into 5- hydroxyvalerate CoA.
22. The recombinant organism of any one of claims 1-13 and 21 wherein the recombinant organism converts 5-hydroxyvalerate-CoA into a polyhydroxyalkanoate.
23. The recombinant organism of any one of claims 1-13 and 21 wherein the recombinant organism converts 5-hydroxvalerate into 1,5 pentanediol.
24. The recombinant organism of any one of claims 1-13 and 21 wherein the recombinant organism converts 5-hydroxyvalerate into poly(5- hydroxyvalerate) or a copolymer thereof.
25. The recombinant organism of claim 24 wherein the copolymer is selected from the group consisting of poly(3- hydroxypropionate-co-5HV), poly(3-hydroxybutyrate-co-5HV) and poly(4- hydroxybutyrate-co-5HV)
26. The recombinant organism of any one of claims 1-25 wherein the recombinant organism is prokaryotic.
27. The recombinant organism of any one of claims 1 -26 wherein the recombinant organism is E. coli.
28. The recombinant organism of any one of claims 1-25 wherein the recombinant organism is a eukaryotic microorganism.
29. A recombinant organism for producing polymers from lysine wherein the recombinant organism is genetically engineered to express at least one enzyme selected from the group consisting of lysine 2- monooxygenase, EC 1.13.12.2; 5-aminopentanamidase (δ- aminovaleramidase), EC 3.5.1.30; 5-aminopentanoate transaminase (δ- aminovalerate transaminase), EC 2.6.1.48; lysine decarboxylase, EC 4.1.1.18; succinate semialdehyde reductase (a.k.a. glutarate semialdehyde reductase), EC 1.1.1.61 ; CoA-transferase, EC 2.8.3.14 and EC 2.8.3.n; Acyl- CoA synthetase, EC 6.2.1.3; PHA synthase, EC 2.3.1.n to produce a polyhydroxyalkanoate comprising 5-hydroxyvalerate monomers.
30. The recombinant organism of claim 29 wherein the recombinant organism does not produce lysine.
31. The recombinant organism of claim 29 wherein the recombinant organism does not express a functional glutarate semialdehyde dehydrogenase enzyme activity.
32. A recombinant organism for producing 1 ,5-pentanediol from lysine wherein the recombinant organism is genetically engineered to express at least one enzyme selected from the group consisting of lysine 2- monooxygenase, EC 1.13.12.2; 5-aminopentanamidase (δ- aminovaieramidase), EC 3.5.1.30; 5-aminopentanoate transaminase (δ- aminovalerate transaminase), EC 2.6.1.48; lysine decarboxylase, EC 4.1.1.18; succinate semialdehyde reductase (a.k.a. glutarate semialdehyde reductase), EC 1.1.1.61; CoA-transferase, EC 2.8.3.14 and EC 2.8.3.n; Acyl- CoA synthetase, EC 6.2.1.3, and propionaldehyde dehydrogenase, EC 1 ,2.1.3; alcohol dehydrogenase, EC 1.1.1.1; 1,3 -propanediol dehydrogenase EC 1.1.1.202
33. The recombinant organism of claim 32 wherein the recombinant organism does not produce lysine.
34. The recombinant organism of claim 32 wherein the recombinant organism does not express a functional glutarate semialdehyde dehydrogenase enzyme activity.
35. A method for producing polymers from lysine comprising feeding the recombinant organism of claim 29 with lysine and other renewable carbon feedstock such that the polymer is produced.
?7
36. A method for producing 5-carbon based monomers, polymers or co-polymers thereof comprising providing lysine or other renewable carbon feedstock to genetically engineered cells according to Claim 1 , wherein the genetically engineered cells are engineered to produce more glutarate semialdehyde or 5-aminopentanoate than unmodified cells, and wherein the glutarate semialdehyde or 5-aminopentanoate is converted by the genetically engineered cells into a 5 carbon monomer, polymer or copolymer thereof.
37. The method of claims 36 wherein the renewable carbon feedstock is selected from starch, sucrose, glucose, lactose, fructose, xylose, maltose and arabinose, or combinations thereof.
38. The method of claim 36 wherein the monomer is selected from the group consisting of glutarate, 1,5-pentanediol, and 5- hydroxyvalerate.
39. The method of any one of claims 36 wherein the polymer comprises a polyhydroxyalkanoate.
40. The method of claim 39 wherein the polyhydroxyalkanoate comprises 5 -hydroxy valerate.
41. The method of claim 40 wherein the polyhydroxyalkanote is selected from poly(5-hydroxyvalerate), poly(3-hydroxypropionate-co-5HV), poly(3-hydroxybutyrate-co~5HV) and poly(4-hydroxybutyrate-co-5HV)J
42. The method of claim 39 further comprising recovering the polyhydroxyalkanote, polymer or copolymer thereof.
43. The method of claim 42 wherein polyhydroxyalkanote polymer or copolymers are recovered by solvent extraction or aqueous processing.
44. A method for overproducing glutarate from renewable carbon substrates is provided where a recombinant organism of Claims 1-12 is fed a renewable carbon substrate selected from lysine, starch, sucrose, glucose, lactose, fructose, xylose, maltose and arabinose, or combinations thereof and glutarate is overproduced, secreted to the medium and recovered therefrom.
45. 1,5 Pentanediol produced from renewable carbon substrate.
PCT/US2009/067943 2008-12-12 2009-12-14 Green process and compositions for producing poly(5hv) and 5 carbon chemicals WO2010068953A2 (en)

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EP09793403.8A EP2376622B1 (en) 2008-12-12 2009-12-14 Green process and compositions for producing poly(5hv) and 5 carbon chemicals
KR1020177033765A KR101886012B1 (en) 2008-12-12 2009-12-14 Green process and compositions for producing poly(5hv)and 5 carbon chemicals
BRPI0922410-6A BRPI0922410B1 (en) 2008-12-12 2009-12-14 RECOMBINANT MICROORGANISM GENETICALLY MODIFIED TO CONVERT 5AMINOPENTANOATE INTO A POLYMER OR COPOLYMER OF POLYHYDROXYALKANOATE (PHA) THEREOF COMPRISING 5-HYDROXY-VALERATE, AND METHOD FOR PRODUCING POLYMERS OR COPOLYMERS OF POLYHYDROXYALKANOATE (PHA) WITH BASIS ON CARBON THESE
ES09793403.8T ES2639413T3 (en) 2008-12-12 2009-12-14 Ecological process and compositions for the production of poly (5HV) and 5 carbon chemicals
KR1020167004466A KR20160025043A (en) 2008-12-12 2009-12-14 Green process and compositions for producing poly(5hv)and 5 carbon chemicals
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CN2009801568724A CN102317437B (en) 2008-12-12 2009-12-14 Green process and compositions for producing poly(5hv) and 5 carbon chemicals
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