CN105316271A - Method for constructing bacterium strain with high output of rhamnolipid - Google Patents
Method for constructing bacterium strain with high output of rhamnolipid Download PDFInfo
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Abstract
The invention provides a method for constructing a bacterium strain with a high output of rhamnolipid. Specifically, the invention provides a method for enhancing the performance of cells on producing rhamnolipid. The method comprises steps of increasing the expression or activity of AlgC polypeptide in cells, and/or reducing the formation of Psl and/or Pel polysaccharide in the cells. The experiment results show that the provided method can increase the output of rhamnolipid in a culture medium by 120% or more.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to a kind of method utilizing genetic engineering means to build producing rhamnolipid with high yield bacterial strain.
Background technology
The type biological surfactant that rhamnolipid is mainly produced by pseudomonas, in oil production, the field tools such as biological medicine, environment protection and food have been widely used.The acquisition of producing rhamnolipid with high yield bacterial strain, for reduction production cost, improves product competitiveness and has important practical significance.
At present, the acquisition of producing rhamnolipid with high yield bacterial strain is generally that carry out shake flask fermentation screening, and physics and chemistry mutagenesis has very large randomness, therefore screening efficiency is very low by after various physical and chemical factor mutagenesis; Therefore those skilled in the art are devoted to build rhamnolipid high-yield strain by genetic engineering means always, improve the production efficiency of rhamnolipid.
Summary of the invention
The object of the present invention is to provide a kind of method building high yield rhamnosyl bacterial strain.
Another object of the present invention is to provide a kind of producing rhamnolipid with high yield bacterial strain and preparation thereof.
A first aspect of the present invention, provides a kind of method improving cell generation rhamnolipid ability, comprises expression or activity that step improves AlgC polypeptide in described cell; And/or
Reduce the formation of Psl and/or Pel polysaccharide in described cell.
In another preference, in the described cell of described reduction, the formation of Psl and/or Pel polysaccharide comprises: the expression or the activity that raise RhlI and/or RhlR polypeptide.
In another preference, described AlgC polypeptide is selected from lower group:
(ia) there is the polypeptide of aminoacid sequence shown in SEQIDNO.:2;
(iia) by the aminoacid sequence such as shown in SEQIDNO.:2 through the replacement of one or several amino-acid residue, disappearance or interpolation is formed, can improve the polypeptide derivative by (i) that cell produces rhamnolipid ability; Or
(iiia) sequence shown in aminoacid sequence and SEQIDNO.:2 homology >=95% (preferably >=98%), can improve cell produce rhamnolipid ability polypeptide.
In another preference, the encoding gene of described AlgC polypeptide is selected from lower group:
(a1) polynucleotide of coding polypeptide as shown in SEQIDNO.:2;
(b1) polynucleotide of sequence as shown in SEQIDNO.:1;
(c1) polynucleotide of homology >=95% (preferably >=98%) of sequence shown in nucleotide sequence and SEQIDNO.:1;
(d1) 5 ' end of polynucleotide and/or the polynucleotide of 3 ' end brachymemma or interpolation 1-60 (preferably 1-30, more preferably 1-10) Nucleotide as shown in SEQIDNO.:1;
(e1) polynucleotide of arbitrary described polynucleotide complementation with (a1)-(d1).
In another preference, the above-mentioned AlgC polypeptide being selected from (ia), (iia) or (iiia) of described encoding gene encodes.
In another preference, described RhlI polypeptide is selected from lower group:
(ib) there is the polypeptide of aminoacid sequence shown in SEQIDNO.:4;
(iib) by the aminoacid sequence such as shown in SEQIDNO.:4 through the replacement of one or several amino-acid residue, disappearance or interpolation is formed, can improve the polypeptide derivative by (i) that cell produces rhamnolipid ability; Or
(iiib) sequence shown in aminoacid sequence and SEQIDNO.:4 homology >=95% (preferably >=98%), can improve cell produce rhamnolipid ability polypeptide.
In another preference, the encoding gene of described RhlI polypeptide is selected from lower group:
(a2) polynucleotide of coding polypeptide as shown in SEQIDNO.:4;
(b2) polynucleotide of sequence as shown in SEQIDNO.:3;
(c2) polynucleotide of homology >=95% (preferably >=98%) of sequence shown in nucleotide sequence and SEQIDNO.:3;
(d2) 5 ' end of polynucleotide and/or the polynucleotide of 3 ' end brachymemma or interpolation 1-60 (preferably 1-30, more preferably 1-10) Nucleotide as shown in SEQIDNO.:3;
(e2) polynucleotide of arbitrary described polynucleotide complementation with (a2)-(d2).
In another preference, the above-mentioned RhlI polypeptide being selected from (ib), (iib) or (iiib) of described encoding gene encodes.
In another preference, described RhlR polypeptide is selected from lower group:
(ic) there is the polypeptide of aminoacid sequence shown in SEQIDNO.:6;
(iic) by the aminoacid sequence such as shown in SEQIDNO.:6 through the replacement of one or several amino-acid residue, disappearance or interpolation is formed, can improve the polypeptide derivative by (i) that cell produces rhamnolipid ability; Or
(iiic) sequence shown in aminoacid sequence and SEQIDNO.:6 homology >=95% (preferably >=98%), can improve cell produce rhamnolipid ability polypeptide.
In another preference, the encoding gene of described RhlR polypeptide is selected from lower group:
(a3) polynucleotide of coding polypeptide as shown in SEQIDNO.:6;
(b3) polynucleotide of sequence as shown in SEQIDNO.:5;
(c3) polynucleotide of homology >=95% (preferably >=98%) of sequence shown in nucleotide sequence and SEQIDNO.:5;
(d3) 5 ' end of polynucleotide and/or the polynucleotide of 3 ' end brachymemma or interpolation 1-60 (preferably 1-30, more preferably 1-10) Nucleotide as shown in SEQIDNO.:5;
(e3) polynucleotide of arbitrary described polynucleotide complementation with (a3)-(d3).
In another preference, the above-mentioned RhlR polypeptide being selected from (ic), (iic) or (iiic) of described encoding gene encodes.
In another preference, described AlgC, RhlI or RhlR polypeptide or its encoding gene derive from Pseudomonas aeruginosa (Pseudomonasaeruginosa), pseudomonas stanieri or denitrifying pseudomonas (Pseudomonasstutzeri), pseudomonas putida (Pseudomonasputida).
In another preference, described cell can produce rhamnolipid.
In another preference, described cell comprises microorganism cells.
In another preference, described microorganism comprises Pseudomonas aeruginosa (Pseudomonasaeruginosa), pseudomonas stanieri or denitrifying pseudomonas (Pseudomonasstutzeri), pseudomonas putida (Pseudomonasputida).
In another preference, described method comprises step:
A () provides the expression vector of foreign gene-carrying, described foreign gene is selected from lower group: algC gene, RhlI gene, RhlR gene or its combination;
B described expression vector proceeds in host cell by ();
C () cultivates described host cell.
In another preference, described expression vector is identical carrier or different carriers.
In another preference, in step (a), the expression vector carrying described foreign gene is high copy number plasmid.
In another preference, in the genome of the host cell in step (c) or karyomit(e) containing or additionally integrate the gene being selected from lower group: algC gene, RhlI gene, RhlR gene or its combination.
In another preference, in the host cell in described step (c), the copy number c 1-50 respectively of copy number b, RhlR gene of copy number a, RhlI gene of algC gene, preferably 5-50, more preferably 10-30, and a+b+c >=5, preferably >=10, more preferably >=20.
In another preference, in step (a), comprise step: algC gene described in external pcr amplification, in wherein said PCR, the primer of use is:
5'-G
GAATTCATGAGCACTGCAAAAGCAC-3'；
5'-C
GGATCCTCAGAAGGGCACGGGC-3'。
In another preference, described host cell comprises microorganism cells or vegetable cell.
In another preference, described microorganism is Pseudomonas aeruginosa (Pseudomonasaeruginosa), pseudomonas stanieri or denitrifying pseudomonas (Pseudomonasstutzeri), pseudomonas putida (Pseudomonasputida).
In another preference, the encode primer of polynucleotide of described rhlRI polypeptide of pcr amplification is:
5'-CTAG
TCTAGAATGAGGAATGACGGAGGC-3'；
5'-CC
CAAGCTTTCACACCGCCATCGACAG-3'。
A second aspect of the present invention, provide a kind of bacterial strain of modified raising rhamnolipid throughput, its expression of polypeptide or the activity that are selected from lower group in described bacterial strain strengthen to some extent or improve compared with its wild-type, wherein, described polypeptide is selected from lower group: AlgC polypeptide, RhlI polypeptide, RhlR polypeptide or its combination.
In another preference, described bacterial strain is the false protein fungus bacterial strain of verdigris, pseudomonas stanieri or denitrifying pseudomonas, pseudomonas putida.
In another preference, described AlgC polypeptide, RhlI polypeptide, RhlR polypeptide and encoding gene thereof are described above.
In another preference, the expression of described AlgC polypeptide, RhlI polypeptide and/or RhlR polypeptide or activity strengthen respectively or improve more than 30% compared with wild-type type, and preferably more than 50%, more preferably, more than 100%, most preferably, more than 200%.
A third aspect of the present invention, provides a kind of microbiobacterial agent, the bacterial strain containing the modified raising rhamnolipid throughput described in second aspect present invention in described microbiobacterial agent.
A fourth aspect of the present invention, provides a kind of method of producing rhamnolipid, comprises step:
(1) bacterial strain described in fermentation culture second aspect present invention; With
(2) rhamnolipid is extracted.
A fifth aspect of the present invention, provides the purposes of a kind of isolated polypeptide or its encoding gene, and described polypeptide or its encoding gene produce the ability of rhamnolipid for improving cell; Wherein, described polypeptide is selected from lower group: AlgC polypeptide, RhlI polypeptide, RhlR polypeptide or its combination.
In another preference, described AlgC polypeptide, RhlI polypeptide, RhlR polypeptide and encoding gene thereof are described above.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
Fig. 1 shows the swarming motor capacity contrast of engineering strain PAO1/algC and contrast bacterial strain PAO1/vector.
Fig. 2 shows engineering strain PAO1/algC and the contrast of contrast bacterial strain PAO1/vector rhamnolipid synthesis capability.
Fig. 3 A shows swarming motor capacity when engineering strain PAO1/rhlRI adds the inductor pectinose of 1% and do not add inductor; Fig. 3 B shows the expression of inducing rhlRI in PAO1, significantly can reduce the generation of Psl exocellular polysaccharide.
Embodiment
The present inventor is by extensive and deep research, be surprised to find that, algC gene can improve the ability that microorganism produces rhamnolipid significantly, and experimental result shows, be that algC gene proceeds in Pseudomonas aeruginosa by carrier with high copy number plasmid, the output of rhamnolipid increases 130%.In experimentation further, applicant reduces applicant reduces Psl and/or Pel polysaccharide in cell formation by genetic engineering means, also can significantly improve the output of rhamnolipid, complete the present invention on this basis.
Term
Rhamnolipid
Rhamnolipid is a kind of bio-surfactant with biological metabolism character produced by pseudomonas or Burkholderia class.It belongs to a kind of glycolipid analog anion surfactants.Rhamnolipid comprises two parts: fatty acid chain portion and rhamanopyranosyl.The normally single rhamnolipid of rhamnolipid of Pseudomonas aeruginosa synthesis and the mixture of two rhamnolipid, wherein modal single rhamnolipid is Rha-C
10-C
10, modal pair of rhamnolipid is Rha-Rha-C
10-C
10, the structure of these two kinds of rhamnolipids is as follows:
Psl and Pel polysaccharide
Psl polysaccharide is that one is secreted into extracellular polysaccharide--exocellular polysaccharide, it is repetition six sugar of seminose, rhamnosyl, glucose composition, it plays a crucial role in the biofilm load of non-mucus type bacterium is formed, and is also the crucial polysaccharide that mucoid Pseudomonas aeruginosa forms biofilm load simultaneously.
AlgC gene
As used herein, a kind of canonical nucleotide sequence of algC gene of the present invention is as shown in SEQIDNO.:1.
The nucleotide sequence of algC gene is as follows:
The typical aminoacid sequence of one of AlgC polypeptide of the present invention is as shown in SEQIDNO.:2.
The aminoacid sequence of AlgC is as follows:
RhlI gene
The nucleotide sequence of rhlI gene is as follows:
The aminoacid sequence of rhlI is as follows:
RhlR gene
The nucleotide sequence of rhlR gene is as follows:
The aminoacid sequence of rhlR genes encoding is as follows:
The invention provides a kind of gene improving cell generation rhamnolipid ability, described gene is for deriving from algC gene and the variant thereof of microorganism (preferably from Pseudomonas aeruginosa or similar microorganism).AlgC gene coded protein AlgC, AlgC albumen is a bifunctional protein enzyme, there are phosphoglucomutase and mannose-phosphate mutase two kinds of functions, 6-phosphate-dextrose and 6-phosphoric acid-seminose can be separately converted to 1-phosphate-dextrose and 1-phosphoric acid-seminose, 1-phosphate-dextrose can be further converted to TDP-L-rhamnosyl, thus is may be used for improving the gene that cell produces rhamnolipid ability for the synthesis gene of the present invention of rhamnolipid.
Pseudomonas aeruginosa AlgC albumen has mannose-phosphate mutase simultaneously and phosphoglucomutase is active.This enzyme high conservative, except microorganism, all has isozyme (ProteinScience200413:2130-2138) in higher organism (as rabbit and human cell).In rhamnolipid route of synthesis, its synthesis of glucose-1-phosphoric acid (Glc-1-P), this sugar precursor is directly used in the TDP-L-Rhamonos (rhamnosyl) of rhamnolipid synthesis in Pseudomonas aeruginosa by other enzymic synthesis.AlgC is uniquely can the albumen of synthesis of glucose-1-phosphoric acid in Pseudomonas aeruginosa, and thus its copy number and expression are one of key rate-limiting step in rhamnolipid synthesis.
Rhamnolipid synthesis is also by the regulation and control of RhlR (transcriptional regulator) and RhlI (synthesized micromolecule signal), and their are synthesized by transcribing thus affecting rhamnolipid of control rhlABC.Three albumen coded by rhlABC to be responsible for rhamnosyl to transfer to respectively from TDP-L-Rhamonos on fatty acid chain thus to be synthesized single rhamnolipid and two rhamnolipid respectively.RhlR and rhlI transcribes in the mode of an operon.
Gene order of the present invention also comprises and to have 50% or more with SEQIDNO.:1,3,5 (preferably more than 60%, more than 70%, more than 80%, more preferably more than 90%, more preferably more than 95%, most preferably more than 98%, as 99%) nucleic acid of homology, described nucleotide sequence also effectively can improve cell and produce rhamnolipid ability." homology " refers to according to the identical per-cent in position, the similar level (i.e. sequence similarity or identity) between two or more pieces nucleic acid.
In the present invention, the variant of described gene by inserting or delete regulation and control region, can carry out random or rite-directed mutagenesis etc. and obtains.
In the present invention, the nucleotide sequence in SEQIDNO.:1,3,5 is through replacing, lacking or add one or more Nucleotide, the derived sequence of generation.Due to the simple well of codon, though with SEQIDNO.:1,3, the homology of 5 is lower, also basic coding can go out SEQIDNO.:1, aminoacid sequence coded by 3,5.In addition, " nucleotide sequence in SEQIDNO.:1,3,5 is through replacing, lacking or add at least one Nucleotide; generate SEQIDNO.:1,3, the derived sequence of 5 " implication also comprise can under moderate stringency conditions, better under high stringent condition with SEQIDNO.:1,3, the nucleotide sequence of the nucleotide sequence hybridization of 5.These variant forms comprise (but and little be limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance of Nucleotide, insertion and/or replacement, and to add at 5' and/or 3 ' end and severally (be generally within 60, within being preferably 30, within being more preferably 10, within being 5 best) Nucleotide.
Should understand, although what provide in example of the present invention derives from Pseudomonas aeruginosa, but derive from other similar microorganism, with gene order of the present invention (preferably, sequence as SEQIDNO.:1,3, shown in 5) there is the gene order of certain homology, the information that those skilled in the art provide according to the application after having read the application is also included within scope of the present invention, as long as can be separated easily from other microorganism obtain this sequence.
Present invention also offers a kind of polypeptide and the variant thereof that improve cell generation rhamnolipid ability, in a preference of the present invention, the aminoacid sequence of described polypeptide is as shown in SEQIDNO.:2,4,6.Polypeptide of the present invention can improve the ability that cell produces rhamnolipid effectively.
The present invention also comprise with SEQIDNO.:2 of the present invention, 4,6 sequences have 50% or more (preferably more than 60%, more than 70%, more than 80%, more preferably more than 90%, more preferably more than 95%, most preferably more than 98%, as 99%) polypeptide with same or similar function of homology.
Described " same or similar function " refers to: " improving the ability that cell produces rhamnolipid ".
In the present invention, described polypeptide variants is the aminoacid sequence as shown in SEQIDNO.:2,4,6, (1-50 is generally through several, preferably 1-30, more preferably 1-20,1-10 best) replace, lack or add the derived sequence of at least one amino acid gained, and add one or several at C-terminal and/or N-terminal and (be generally within 20, within being preferably 10, within being more preferably 5) amino acid.Such as, in described albumen, when replacing with similar nature or similar amino acid, usually can not change the function of protein, C-terminal and/or end add the function that or several amino acid also can not change polypeptide usually.These conservative variation preferably carry out replacing according to table 1 and produce.
Table 1
The present invention also comprises polypeptide analog required for protection.These analogues and natural polypeptides (or protein) (as SEQIDNO.:2,4,6) difference can be the difference on aminoacid sequence, also can be the difference do not affected on the modified forms of sequence, or have both at the same time.The analogue of these albumen comprises genetic variant that is natural or induction.Induce variation body can be obtained by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, has also knownly divided biological technology by site-directed mutagenesis or other.Analogue also comprises the analogue with the residue (as D-amino acid) being different from natural L-amino acids, and has the analogue of amino acid (as β, gamma-amino acid) that is that non-natural exists or synthesis.Should be understood that albumen of the present invention is not limited to the above-mentioned representational albumen exemplified.
(usually the not changing primary structure) form of modification comprises: in body or the chemically derived form of external albumen as acetoxylation or carboxylated.Modify and also comprise glycosylation, as those carry out glycosylation modified in protein synthesis and processing.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) by being exposed to by albumen and completing.Modified forms also comprises the sequence with phosphorylated amino acid residue (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).
Present invention also offers a kind of recombinant vectors comprising gene of the present invention.As the preferred mode of one, the promotor downstream of recombinant vectors comprises multiple clone site or at least one restriction enzyme site.When goal gene of the present invention expressed by needs, goal gene is connected in applicable multiple clone site or restriction enzyme site, thus goal gene is operably connected with promotor.
As another kind of optimal way, described recombinant vectors comprises in (from 5 ' to 3 ' direction): promotor, goal gene, and terminator.If needed, described recombinant vectors can also comprise the element being selected from lower group: 3 ' polymerized nucleoside acidifying signal; Untranslated nucleotide sequence; Transhipment and target nucleotide sequence; Resistance selective marker (Tetrahydrofolate dehydrogenase, neomycin resistance, hygromycin resistance and green fluorescent protein etc.); Enhanser; Or operator.
Method for the preparation of recombinant vectors is well known to those of ordinary skill in the art.Expression vector can be bacterial plasmid, phage, yeast plasmid, vegetable cell is viral, mammalian cell is viral or other carriers.In a word, as long as it can copy and stablize in host, any plasmid and carrier are all can be adopted.
Those of ordinary skill in the art can use the method known to build the expression vector containing gene of the present invention.These methods comprise recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc.When using gene constructed recombinant expression vector of the present invention, any one enhancement type, composing type, organizing specific type or inducible promoter can be added before its transcription initiation Nucleotide, as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin (Ubiquitin) gene promoter (pUbi) etc., they can be used alone or are combined with other promotor.
Comprise gene of the present invention, expression cassette or carrier may be used for transforming suitable host cell, to make host expresses protein.Host cell can be prokaryotic cell prokaryocyte, as intestinal bacteria, and streptomyces, Agrobacterium: or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as vegetable cell.Persons skilled in the art all know how to select suitable carrier and host cell.Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host is prokaryotic organism (as intestinal bacteria), CaCl can be used
2method process, also can carry out with electroporation.When host is eukaryote, following DNA transfection method can be selected: calcium phosphate precipitation, conventional mechanical methods (as microinjection, electroporation, liposome packaging etc.).Conversion of plant also can use the method such as Agrobacterium-mediated Transformation or via Particle Bombardment Transformation, such as leaf disk method, rataria conversion method, bud infusion method etc.Can ordinary method regeneration plant be used for the vegetable cell transformed, tissue or organ, thus obtain genetically modified plant.
For the ease of identifying transgenic cell and screening, can process expression carrier used thereof, as being added in the gene (gus gene, GFP gene, luciferase genes etc.) of enzyme or the luminophor that can produce colour-change, there is the antibiotic marker thing (gentamicin marker, kantlex marker etc.) etc. of resistance.
Major advantage of the present invention is:
(1) by algC channel genes microorganism, the ability that microorganism produces rhamnolipid can be improved significantly;
(2) use method of the present invention, with the plasmid pUCP18 of height copy for carrier, by algC channel genes pseudomonas aeruginosa, successfully improve the output of rhamnolipid.
(3) method of the present invention is used, with the plasmid pHerd20T of height copy for carrier, by rhlR and rhlI channel genes pseudomonas aeruginosa, by abduction delivering RhlR and RhlI, while reducing exocellular polysaccharide Psl, significantly improve the output of rhamnolipid.
Below in conjunction with specific embodiment, state the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted detailed conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
Experiment material used in the embodiment of the present invention all can obtain from commercially available channel if no special instructions, wherein, Pseudomonas aeruginosa PAO1 is typical type strain, can obtain from China General Microbiological preservation administrative center (CGMCC) that (see reference document CompletegenomesequenceofPseudomonasaeruginosaPAO1, anopportunisticpathogen.Nature2000,406:959-964.); Plasmid pUCP18 sees reference document Developmentofbroad-host-rangevectorsandgenebanks:self-cl oningofthePseudomonasaeruginosaPAOchromosome.J.Bacteriol.1982,150(1),60-69。PHERD20T plasmid sees reference document AppliedEnviromentalMicrobiology2008,74:7422-7426.
The structure of embodiment 1 engineering strain PAO1/algC and contrast bacterial strain PAO1/vector
According to algC gene order design primer on Pseudomonas aeruginosa PAO1 genome, upstream and downstream primer sequence is respectively:
5'-G
GAATTCATGAGCACTGCAAAAGCAC-3'(SEQIDNO.:7)；
5'-C
GGATCCTCAGAAGGGCACGGGC-3'(SEQIDNO.:8)；
(underlined sequences is respectively EcoRI and BamHI restriction enzyme site), through order-checking after pcr amplification, nucleotide sequence is consistent with expection.By pcr amplification and enzyme be connected with the carrier pUCP18 of identical restriction enzyme ferment treatment after cutting, obtain carrier pUCP18/algC, transform finally by electricity, the plasmid pUCP18/algC built and carrier pUCP18 is imported in Pseudomonas aeruginosa PAO1, obtains engineering strain PAO1/algC and contrast bacterial strain PAO1/vector.Extract engineering strain PAO1/algC plasmid and digestion verification, determine that correct plasmid has proceeded in Pseudomonas aeruginosa.
The contrast of embodiment 2 engineering strain PAO1/algC and contrast bacterial strain PAO1/vectorswarming motor capacity
Swarming motion is relevant to the output of Pseudomonas aeruginosa rhamnolipid, is moved, indirectly can compare the size of rhamnolipid synthesis capability between bacterial strain by swarming.Specific practice is for meeting engineering strain PAO1/algC and contrasting (yeast powder 5g/L in the dull and stereotyped single bacterium colony of bacterial strain PAO1/vector and LBNS liquid nutrient medium, peptone 10g/L, pH7.2, 121 DEG C of sterilizing 30min) in 37 DEG C, after 200rpm shaking culture 12h, select and connect 1 μ L culture in nutrient broth semi-solid dull and stereotyped upper (agar concentration is 0.5%), 37 DEG C of incubated overnight, the motor capacity of engineering strain PAO1/algC and contrast bacterial strain PAO1/vector is compared by swarming circle, the swarming motion circle of two strain bacterium is respectively 2.4 ± 0.1cm and 1.3 ± 0.1cm, engineering strain PAO1/algC is apparently higher than contrast bacterial strain PAO1/vector, imply that engineering strain synthesizes more rhamnolipid.Fig. 1 shows the contrast of the swarming motor capacity of engineering strain PAO1/algC and contrast bacterial strain PAO/vector.
The comparison of embodiment 3 engineering strain PAO1/algC and contrast bacterial strain PAO1/vector rhamnolipid synthesis capability.
Respectively by single colony inoculation in nutrient broth medium, in 37 DEG C, 200rpm shaking culture 2 days.Then, the centrifugal 10min of 5000rpm, gets supernatant, adds concentrated hydrochloric acid and adjusts pH to 2.Then isopyknic chloroform/methanol (v:v=2:1) solution is added, high speed whirlpool concussion 1min, extracting twice, then the organic phase of collection is merged, vacuum volatilization, finally obtains rhamnolipid product, then detects total rhamnolipid concentration by sulfuric acid anthrone method.
Specific practice is, the rhamnolipid 100 μ L solution good with methanol dilution is joined in the anthrone solution (solvent is the sulfuric acid of 70%) of 1mL0.1%, 80 DEG C of process 30min, then cool to room temperature, detect 625nm light absorption value, do typical curve with the rhamnosyl of different concns simultaneously, draw the concentration of surveyed liquid rhamnosyl finally by rhamnolipid typical curve, then be multiplied by relation conefficient 3.0 thus calculate to obtain the concentration of rhamnolipid.The engineering strain PAO1/algC that sulfuric acid anthrone method records and the rhamnolipid concentration contrasting bacterial strain PAO1/vector total are respectively 339 ± 8mg/L and 262 ± 5mg/L, and engineering strain PAO1/algC increases 130% relative to the output of contrast bacterial strain PAO1/vector rhamnolipid.Fig. 2 shows engineering strain PAO1/algC and the contrast of contrast bacterial strain PAO/vector rhamnolipid synthesis capability.
Embodiment 4 induces RhlR and RhlI to express the synthesis of reduction Psl to improve the output of rhamnolipid
In an experiment, the present inventor is surprised to find that, significantly can be reduced the synthesis of Pcl and Pel, and in culture, the output of rhamnolipid significantly improves by induction intervention school-based rhlRI high expression level.
By quorum sensing gene rhlR and rhlI of regulation and control rhamnolipid, (rhlR and rhlI shares promotor, hereinafter abbreviated as rhlRI) after the promotor that is cloned into arabinose-inducible, induced the great expression of intervention school-based by pectinose in a large number, thus start a large amount of synthesis of rhamnolipid.
Respectively with following primer amplification gene:
rhlRI：Primer1，CTAG
TCTAGAATGAGGAATGACGGAGGC(SEQIDNO.:9)；Primer2，CC
CAAGCTTTCACACCGCCATCGACAG(SEQIDNO.:10)。
Italic is respectively the restriction enzyme site of restriction enzyme XbaI and HindIII.After the PCR primer of amplification is cut by restriction enzyme XbaI and HindIII enzyme, be connected with the carrier pHERD20T being cut process by same enzyme, obtain plasmid pHERD20T/rhlRI, then turned by electricity, plasmid pHERD20T/rhlRI is imported in Pseudomonas aeruginosa PAO1, obtains engineering strain PAO/rhlRI.
Then detect engineering strain PAO1/rhlRI by above-mentioned same method and add the inductor pectinose of 1% and swarming motor capacity when not adding inductor.Result as shown in Figure 3, Fig. 3 A shows, when not adding with interpolation 1% pectinose, the swarming motion circle of engineering strain PAO1/rhlRI is respectively 2.5 ± 0.1cm and 4.5 ± 0.2cm, proves that inducible strain may synthesize more rhamnolipid in induction situation.Fig. 3 B shows, and induces the expression of rhlRI in PAO1, significantly can reduce the generation of Psl exocellular polysaccharide.Further detection engineering strain PAO1/rhlRI adds inductor pectinose and the synthesis capability of rhamnolipid when not adding inductor of 1%, found that, the amount of the rhamnolipid synthesized when adding the inductor of 1% pectinose is 124% when not adding pectinose inductor.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. improve the method that cell produces rhamnolipid ability, it is characterized in that, comprise expression or activity that step improves AlgC polypeptide in described cell; And/or
Reduce the formation of Psl and/or Pel polysaccharide in described cell.
2. in the method for claim 1, wherein described described cell of reduction, the formation of Psl and/or Pel polysaccharide comprises: the expression or the activity that raise RhlI and/or RhlR polypeptide.
3. the method for claim 1, wherein described AlgC polypeptide is selected from lower group:
(ia) there is the polypeptide of aminoacid sequence shown in SEQIDNO.:2;
(iia) by the aminoacid sequence such as shown in SEQIDNO.:2 through the replacement of one or several amino-acid residue, disappearance or interpolation is formed, can improve the polypeptide derivative by (i) that cell produces rhamnolipid ability; Or
(iiia) sequence shown in aminoacid sequence and SEQIDNO.:2 homology >=95% (preferably >=98%), can improve cell produce rhamnolipid ability polypeptide; And/or
Described RhlI polypeptide is selected from lower group:
(ib) there is the polypeptide of aminoacid sequence shown in SEQIDNO.:4;
(iib) by the aminoacid sequence such as shown in SEQIDNO.:4 through the replacement of one or several amino-acid residue, disappearance or interpolation is formed, can improve the polypeptide derivative by (i) that cell produces rhamnolipid ability; Or
(iiib) sequence shown in aminoacid sequence and SEQIDNO.:4 homology >=95% (preferably >=98%), can improve cell produce rhamnolipid ability polypeptide; And/or
Described RhlR polypeptide is selected from lower group:
(ic) there is the polypeptide of aminoacid sequence shown in SEQIDNO.:6;
(iic) by the aminoacid sequence such as shown in SEQIDNO.:6 through the replacement of one or several amino-acid residue, disappearance or interpolation is formed, can improve the polypeptide derivative by (i) that cell produces rhamnolipid ability; Or
(iiic) sequence shown in aminoacid sequence and SEQIDNO.:6 homology >=95% (preferably >=98%), can improve cell produce rhamnolipid ability polypeptide.
4. method as claimed in claim 3, wherein, the encoding gene of described AlgC polypeptide is selected from lower group:
(a1) polynucleotide of coding polypeptide as shown in SEQIDNO.:2;
(b1) polynucleotide of sequence as shown in SEQIDNO.:1;
(c1) polynucleotide of homology >=95% (preferably >=98%) of sequence shown in nucleotide sequence and SEQIDNO.:1;
(d1) 5 ' end of polynucleotide and/or the polynucleotide of 3 ' end brachymemma or interpolation 1-60 (preferably 1-30, more preferably 1-10) Nucleotide as shown in SEQIDNO.:1;
(e1) polynucleotide of arbitrary described polynucleotide complementation with (a1)-(d1); And/or
The encoding gene of described RhlI polypeptide is selected from lower group:
(a2) polynucleotide of coding polypeptide as shown in SEQIDNO.:4;
(b2) polynucleotide of sequence as shown in SEQIDNO.:3;
(c2) polynucleotide of homology >=95% (preferably >=98%) of sequence shown in nucleotide sequence and SEQIDNO.:3;
(d2) 5 ' end of polynucleotide and/or the polynucleotide of 3 ' end brachymemma or interpolation 1-60 (preferably 1-30, more preferably 1-10) Nucleotide as shown in SEQIDNO.:3;
(e2) polynucleotide of arbitrary described polynucleotide complementation with (a2)-(d2); And/or
The encoding gene of described RhlR polypeptide is selected from lower group:
(a3) polynucleotide of coding polypeptide as shown in SEQIDNO.:6;
(b3) polynucleotide of sequence as shown in SEQIDNO.:5;
(c3) polynucleotide of homology >=95% (preferably >=98%) of sequence shown in nucleotide sequence and SEQIDNO.:5;
(d3) 5 ' end of polynucleotide and/or the polynucleotide of 3 ' end brachymemma or interpolation 1-60 (preferably 1-30, more preferably 1-10) Nucleotide as shown in SEQIDNO.:5;
(e3) polynucleotide of arbitrary described polynucleotide complementation with (a3)-(d3).
5. method as claimed in claim 2, wherein, described AlgC, RhlI or RhlR polypeptide or its encoding gene derive from Pseudomonas aeruginosa (Pseudomonasaeruginosa), pseudomonas stanieri or denitrifying pseudomonas (Pseudomonasstutzeri) or pseudomonas putida (Pseudomonasputida).
6. method as claimed in claim 2, wherein, described method comprises step:
A () provides the expression vector of foreign gene-carrying, described foreign gene is selected from lower group: algC gene, RhlI gene, RhlR gene or its combination;
B described expression vector proceeds in host cell by ();
C () cultivates described host cell.
7. the bacterial strain of a modified raising rhamnolipid throughput, it is characterized in that, the expression or the activity that are selected from the polypeptide of lower group in described bacterial strain strengthen to some extent or improve compared with its wild-type, and wherein, described polypeptide is selected from lower group: AlgC polypeptide, RhlI polypeptide, RhlR polypeptide or its combination.
8. a microbiobacterial agent, is characterized in that, the bacterial strain containing the modified raising rhamnolipid throughput described in claim 7 in described microbiobacterial agent.
9. produce a method for rhamnolipid, it is characterized in that, comprise step:
(1) fermentation culture bacterial strain according to claim 7; With
(2) rhamnolipid is extracted.
10. a purposes for isolated polypeptide or its encoding gene, is characterized in that, described polypeptide or its encoding gene produce the ability of rhamnolipid for improving cell; Wherein, described polypeptide is selected from lower group: AlgC polypeptide, RhlI polypeptide, RhlR polypeptide or its combination.
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| CN113249284B (en) * | 2021-04-28 | 2024-01-12 | 南京工业大学 | Pseudomonas putida genetically engineered bacterium, construction method and application thereof |
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