CN103571785A - Method for efficiently producing mevalonic acid from fatty acid and constructed gene engineering bacterium - Google Patents
Method for efficiently producing mevalonic acid from fatty acid and constructed gene engineering bacterium Download PDFInfo
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Abstract
The invention discloses a method for efficiently producing mevalonic acid from fatty acid and a constructed gene engineering bacterium. The method comprises the following steps: overexpressing a fatty acid transport protein gene (fadL) and an acyl-CoA synthetase gene (fadD) in an Escherichia coli, thus obtaining a recombinant Escherichia coli; and using the recombinant Escherichia coli to produce the mevalonic acid through fermentation. According to the method disclosed by the invention, long-chain fatty acid is used as a carbon source for synthesis of the mevalonic acid, the mevalonic acid yield can be up to 230mg/L, and the productivity can be up to 56%. The method disclosed by the invention can also be used for producing isoprene taking mevalonic acid as an intermediate product and terpenoid compounds synthesized by taking isoprene as a unit through fermentation.
Description
Technical field
The present invention relates to a kind of method of lipid acid production mevalonic acid and genetic engineering bacterium that structure obtains of efficiently utilizing.
Background technology
Mevalonic acid is the mesostate that mevalonate pathway generates foreign matter alkene tetra-sodium (IPP), is the important intermediate of synthetic sterol, terpene, isoprenoid, meanwhile, in chemical industry, also has important purposes.At present, the methods such as that mevalonic acid is synthetic mainly contains that enzyme process is synthetic, biocatalysis and chemical catalysis combine is synthetic, chemosynthesis, but it is rare all to exist raw material, step is complicated, contaminate environment, the problem such as cost is higher, and the synthetic mevalonic acid of biological process can be good at overcoming the above problems.In biomass cells, mevalonic acid is to be obtained through reaching three step catalyzed reactions by acetyl-CoA, only has the active mevalonic acid of a kind of tool in synthetic product, has higher economic worth.Therefore the synthetic mevalonic acid of biological process has broad prospects.
Biological process synthesizes mevalonic acid, mainly take glucose as carbon source, through glycolysis-, produces pyruvic acid; Pyruvic acid generates acetyl-CoA under the effect of pyruvate dehydrogenase system; Acetyl-CoA enters mevalonic acid pathways metabolism, at acetoacetyl-coenzyme A thiolase, under the catalyzer of HMG-CoA synthase and HMG-CoA reductase enzyme, generates mevalonic acid.Investigator has crossed expression acetoacetyl-coenzyme A thiolase/HMG-CoA reductase gene (mvaE) and HMG-CoA synthase gene (mvaS) in intestinal bacteria, and the glucose of take ferments as carbon source, and final mevalonic acid output can reach 47gL
-1(Tabata et al., Biotechnology Letters26,1487-1491,2004).Yet using glucose as initial substrate, a part glucose is lost two carbon atoms and is generated acetyl-CoA, and acetylcoenzyme enters mevalonate pathway and generates mevalonic acid, and its theoretical yield is only 54.8%.How improving the utilising efficiency of substrate in mevalonic acid route of synthesis, is the problem that prior art need to solve.
Summary of the invention
The invention provides a kind of product mevalonic acid restructuring colibacillus, is in intestinal bacteria, to cross expression fatty acid transport protein gene (fadL) and fatty acyl-CoA synthetase gene (fadD), the restructuring colibacillus obtaining.
Described fatty acid transport protein gene (fadL) refers to coding outer membrane protein FadL, as the acceptor of longer chain fatty acid, is the important albumen that regulation and control longer chain fatty acid is transported to inner membrance across adventitia, preferably Genbank accession number: AAC75404.2.
Described fatty acyl-CoA synthetase is that lipid acid is converted into aliphatic alcohol, preferably Genbank accession number: AAC74875.1.
The present invention also provides a kind of method of utilizing lipid acid production mevalonic acid, is in intestinal bacteria, to import fatty acid transport protein gene (fadL) and fatty acyl-CoA synthetase gene (fadD), utilizes recombination bacillus coli fermentative production mevalonic acid.
The described method of utilizing lipid acid production mevalonic acid, concrete steps are as follows:
1) obtain longer chain fatty acid transporter gene (fadL) and fatty acyl-CoA synthetase gene (fadD);
2) build the recombinant plasmid that contains longer chain fatty acid transporter gene (fadL) and fatty acyl-CoA synthetase gene (fadD);
3) by step 2) plasmid and the plasmid pYJM11 importing intestinal bacteria that build;
4) utilize the restructuring colibacillus that step 3) obtains to utilize lipid acid fermentative production mevalonic acid.
Described step 2) adopt pET-30a carrier.
Described plasmid pYJM11 (pACY-mvaE-mvaS) Wei Ben seminar builds in earlier stage, is published in: Jianming Yang et al., Bioresource Technology104,642-647,2012.
The described method of utilizing lipid acid production mevalonic acid, preferred steps is as follows:
1) clone respectively the longer chain fatty acid transporter gene (fadL) of Genbank accession number: AAC75404.2 and the fatty acyl-CoA synthetase gene (fadD) of Genbank accession number: AAC74875.1;
2) at gene 5 ' end described in step 1) and 3 ' end, introduce respectively restriction enzyme site, described gene clone, to pET-30a carrier, is built to recombinant vectors pET-fadL-fadD;
3) by step 2) the recombinant vectors pET-fadL-fadD that obtains and pYJM11 (pACY-mvaE-mvaS) import e. coli bl21 (DE3) and obtain the colibacillus of recombinating;
4) utilize the intestinal bacteria that step 3) obtains to utilize lipid acid fermentative production mevalonic acid.
Said gene source is chemosynthesis, increases from microorganism or increase from recombinant plasmid.
Said gene can be not specifically limited, as long as the synthetic protein of any gene has described function and can be described as fatty acid transport protein gene or fatty acyl-CoA synthetase gene.
The engineering colon bacillus that the structure of usining obtains is as biological catalyst, the longer chain fatty acid (C12 acid (lauric acid), C14 acid (tetradecanoic acid), C16 (palmitinic acid) or C18 (stearic acid)) of take is carbon source, and adopting fermentation method to transform lipid acid is mevalonic acid.Fermentation condition is: temperature cultivate for 30-37 ℃, stirring velocity be to be cultured to OD under 400-800rpm, pH6-8 and more than 20% condition of dissolved oxygen
600be about 12-20, add inductor IPTG to final concentration be 0.1-1.0mmolL
-1, add lipid acid fermentation 24-72 hour, the output of mevalonic acid can reach 230mg/L, and productive rate can reach 56%.
Further, the present invention also provides a kind of and take mevalonic acid as intermediate product, the method for production object product.Be on the basis of high yield mevalonic acid, further utilize the synthetic terpenoid of mevalonic acid fermentative production object product isoprene and prenyl unit.
It should be noted that, the molecular biology method that the present invention adopts is routine techniques means, and the method for the target product that fermentative production mevalonic acid or the mevalonic acid of take are intermediate product is also common fermentation process optimization.
The present invention has following beneficial effect:
1. it is mevalonic acid that the intestinal bacteria that the present invention builds can transform longer chain fatty acid efficiently.
2. utilizing the inventive method to take longer chain fatty acid (C12 acid (lauric acid), C14 acid (tetradecanoic acid), C16 (palmitinic acid) or C18 (stearic acid)) is carbon source, and the output of mevalonic acid can reach 230mg/L, and productive rate can reach 56%.
3. the present invention not only can production mevalonic acid, and the inventive method also can be used for fermentative production and take the leavened prod that mevalonic acid is intermediate product.
Accompanying drawing explanation:
Fig. 1 pET-fadL restriction enzyme mapping
Fig. 2 pET-fadL-fadD restriction enzyme mapping
Fig. 3 C12-C18 is carbon source mevalonic acid output
Fig. 4 C12-C18 is carbon source mevalonic acid productive rate
Specific embodiments
Embodiment 1:
The structure of longer chain fatty acid transporter gene (fadL) and fatty acyl-CoA synthetase gene (fadD) co-expression carrier, detailed process is as follows:
Take oligonucleotide 5 '-CAT GCC ATG GGC ATG TCC TCA GCC ATC TAT CCC AG-3 ' and 5 '-CGCGGA TCC TCA ACG CCA GCC GGC GTC GAT C-3 ' is primer, take genome of E.coli DNA as masterplate, adopt polymerase chain reaction (PCR) method to amplify longer chain fatty acid transferring enzyme (fadL), and introduce respectively EcoI and HindIII restriction enzyme site at 5 ' end and 3 ' end, then use above-mentioned restriction enzyme site by this gene clone to pET-30a carrier, obtain recombinant plasmid pET-fadL (Fig. 1); Take oligonucleotide 5 '-GGG AAT TCC ATA TGA CCG CTC AGG TTA CAT GCG-3 ' and 5 '-CCG CTC GAG TTA AAC CAG ACG AAC TTC GTG C-3 ' is primer, take genome of E.coli DNA as masterplate, adopt polymerase chain reaction (PCR) method to amplify acyl CoA synthetic enzyme (fadD), and introduce respectively KpnI and BamHI restriction enzyme site at 5 ' end and 3 ' end, then use above-mentioned restriction enzyme site by this gene clone to pET-fadL carrier, obtain recombinant plasmid pET-fadL-fadD(Fig. 2).
Embodiment 2:
The preparation of engineering colon bacillus bacterial strain BL21 (the DE3)/pET-fadL-fadD/pYJM11 of synthetic mevalonic acid, and be mevalonic acid with this strain fermentation conversion lipid acid, its detailed process is as follows:
Recombinant plasmid pYJM11 (the Jianming Yang et al. that the recombinant plasmid pET-fadL-fadD that in employing alkaline lysis method of extracting embodiment 1, structure obtains and this laboratory have built, Bioresource Technology104,642-647,2012), adopt thermal shock conversion method by 5 μ l recombinant plasmids to transforming e. coli bl21 (DE3) competent cell, then get bacterium liquid after 50 μ l transform and coat and contain 34 μ gmL
-1paraxin and 50 μ gmL
-1screening positive clone on the LB flat board of ammonia Bian, the bacterium colony growing is the recombinant escherichia coli strain of coexpression longer chain fatty acid transferring enzyme and acyl CoA synthetic enzyme.The recombination bacillus coli list bacterium colony that picking builds, is seeded to LB liquid nutrient medium, and 37 ℃, 180rpm shaking culture spend the night, then by bacterium liquid by volume 1% inoculum size be inoculated into and add 34 μ gmL
-1in the fermention medium of paraxin, carry out enlarged culturing, under the condition of 37 ℃ of culture temperature, 180rpm shaking culture, be cultured to OD600 and be about 0.8, centrifugal collection thalline, resuspended, be inoculated in and take in the substratum that lipid acid is sole carbon source, fatty acid concentration is 4g/L, adds inductor IPTG to final concentration 0.5mmolL
-1, ferment 72 hours; Nutrient solution is carried out centrifugal, isolated cell and supernatant, adopt complexion spectrometry to detect lipid acid and the mevalonic acid content in fermented liquid supernatant, and with the lipid acid of purchase and mevalonic acid standard substance in contrast.By external standard method, calculate, the output of mevalonic acid can reach 230mg/L, and productive rate can reach 56%.Not crossing the bacterial strain mevalonic acid output of expressing two enzymes is 78mg/L, and productive rate is only 17.3%, and result is as shown in accompanying drawing 3,4.
Embodiment 3:
The preparation of the engineering colon bacillus bacterial strain W3110/pET-fadL-fadD/pYJM11 of synthetic mevalonic acid, and be mevalonic acid with this strain fermentation conversion lipid acid, its detailed process is as follows:
Recombinant plasmid pYJM11 (the Jianming Yang et al. that the recombinant plasmid pET-fadL-fadD that in employing alkaline lysis method of extracting embodiment 1, structure obtains and this laboratory have built, Bioresource Technology104,642-647,2012), adopt thermal shock conversion method by 5 μ l recombinant plasmids to transforming intestinal bacteria W3110 competent cell, then get bacterium liquid after 50 μ l transform and coat and contain 34 μ gmL
-1paraxin and 50 μ gmL
-1screening positive clone on the LB flat board of ammonia Bian, the bacterium colony growing is the recombinant escherichia coli strain of coexpression longer chain fatty acid transferring enzyme and acyl CoA synthetic enzyme.The recombination bacillus coli list bacterium colony that picking builds, is seeded to LB liquid nutrient medium, and 37 ℃, 180rpm shaking culture spend the night, then by bacterium liquid by volume 1% inoculum size be inoculated into and add 34 μ gmL
-1in the fermention medium of paraxin, carry out enlarged culturing, under the condition of 37 ℃ of culture temperature, 180rpm shaking culture, be cultured to OD
600be about 0.8, centrifugal collection thalline, resuspended, be inoculated in and take in the substratum that lipid acid is sole carbon source, fatty acid concentration is 4g/L, adds inductor IPTG to final concentration 0.5mmolL
-1, ferment 72 hours; Nutrient solution is carried out centrifugal, isolated cell and supernatant, adopt complexion spectrometry to detect lipid acid and the mevalonic acid content in fermented liquid supernatant, and with the lipid acid of purchase and mevalonic acid standard substance in contrast, result as shown in Figure 3.By external standard method, calculate, the output of mevalonic acid can reach 180mg/L, and productive rate can reach 56%.Not crossing the bacterial strain mevalonic acid output of expressing two enzymes is 57mg/L, and productive rate is only 17.3%.
Claims (10)
1. producing a mevalonic acid restructuring colibacillus, is in intestinal bacteria, to cross expression fatty acid transport protein gene (fadL) and fatty acyl-CoA synthetase gene (fadD), the restructuring colibacillus obtaining.
2. intestinal bacteria according to claim 1, is characterized in that fatty acid transport protein gene Genbank accession number: AAC75404.2.
3. intestinal bacteria according to claim 1, is characterized in that fatty acyl-CoA synthetase gene Genbank accession number: AAC74875.1.
4. utilizing a method for lipid acid production mevalonic acid, is in intestinal bacteria, to import fatty acid transport protein gene (fadL) and fatty acyl-CoA synthetase gene (fadD), utilizes recombination bacillus coli fermentative production mevalonic acid.
5. method according to claim 4, is characterized in that, concrete steps are as follows:
1) obtain longer chain fatty acid transporter gene (fadL) and fatty acyl-CoA synthetase gene (fadD);
2) build the recombinant plasmid that contains longer chain fatty acid transporter gene (fadL) and fatty acyl-CoA synthetase gene (fadD);
3) by step 2) plasmid and the plasmid pYJM11 importing intestinal bacteria that build;
4) utilize the restructuring colibacillus that step 3) obtains to utilize lipid acid fermentative production mevalonic acid.
6. according to method described in claim 4 or 5, it is characterized in that described fatty acid transport protein gene Genbank accession number: AAC75404.2.
7. according to method described in claim 4 or 5, it is characterized in that described fatty acyl-CoA synthetase gene Genbank accession number: AAC74875.1.
8. method according to claim 5, is characterized in that, concrete steps are as follows:
1) clone respectively the longer chain fatty acid transporter gene (fadL) of Genbank accession number: AAC75404.2 and the fatty acyl-CoA synthetase gene (fadD) of Genbank accession number: AAC74875.1;
2) at gene 5 ' end described in step 1) and 3 ' end, introduce respectively restriction enzyme site, described gene clone, to pET-30a carrier, is built to recombinant vectors pET-fadL-fadD;
3) by step 2) the recombinant vectors pET-fadL-fadD that obtains and pYJM11 (pACY-mvaE-mvaS) import e. coli bl21 (DE3) and obtain the colibacillus of recombinating;
4) utilize the intestinal bacteria that step 3) obtains to utilize lipid acid fermentative production mevalonic acid.
9. described in claim 4, method be take the application of mevalonic acid in mesostate product in fermentative production.
10. method according to claim 9, is characterized in that, intestinal bacteria are for fermentative production isoprene or the isoprene terpenoid synthetic as unit of take described in claim 1.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104372017A (en) * | 2014-11-05 | 2015-02-25 | 中国科学院青岛生物能源与过程研究所 | Method for enhancing yields of gene engineering bacterium isoprene and derivatives thereof and application thereof |
| CN116925991A (en) * | 2023-07-28 | 2023-10-24 | 天津大学 | Recombinant halomonas strain for high yield of mevalonate and construction method and application thereof |
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| CN102741420A (en) * | 2008-05-22 | 2012-10-17 | 味之素株式会社 | Method for production of L-amino acid |
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| CN102741420A (en) * | 2008-05-22 | 2012-10-17 | 味之素株式会社 | Method for production of L-amino acid |
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| 卢向阳: "脂肪酸转运蛋白家族(FATPs)的研究进展", 《生命科学研究》, 31 August 2013 (2013-08-31), pages 348 - 353 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104372017A (en) * | 2014-11-05 | 2015-02-25 | 中国科学院青岛生物能源与过程研究所 | Method for enhancing yields of gene engineering bacterium isoprene and derivatives thereof and application thereof |
| CN104372017B (en) * | 2014-11-05 | 2017-06-30 | 中国科学院青岛生物能源与过程研究所 | A kind of method and application for improving genetic engineering bacterium isoprene and its derivative yield |
| CN116925991A (en) * | 2023-07-28 | 2023-10-24 | 天津大学 | Recombinant halomonas strain for high yield of mevalonate and construction method and application thereof |
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