CN103571785B - The genetic engineering bacterium that a kind of method of efficiency utilization lipid acid production mevalonic acid and structure obtain - Google Patents
The genetic engineering bacterium that a kind of method of efficiency utilization lipid acid production mevalonic acid and structure obtain Download PDFInfo
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Abstract
The present invention discloses the genetic engineering bacterium that a kind of method of efficiency utilization lipid acid production mevalonic acid and structure obtain, process LAN fatty acid transport protein gene (fadL) and fatty acyl-CoA synthetase gene (fadD) in intestinal bacteria, the restructuring colibacillus obtained, utilizes recombination bacillus coli fermentative production mevalonic acid.The inventive method is utilized to be that carbon source synthesizes mevalonic acid with longer chain fatty acid, mevalonic acid output can reach 230mg/L, productive rate can reach 56%, and the inventive method also can be used for isoprene that fermentative production take mevalonic acid as intermediate product and take isoprene as the terpenoid of unit synthesis.
Description
Technical field
The present invention relates to the genetic engineering bacterium that a kind of method of efficiency utilization lipid acid production mevalonic acid and structure obtain.
Background technology
Mevalonic acid is the mesostate that mevalonate pathway generates foreign matter alkene tetra-sodium (IPP), is the important intermediate of synthesis sterol, terpene, isoprenoid, meanwhile, also has important purposes in chemical industry.At present, mevalonic acid synthesis mainly contains enzymatic clarification, biocatalysis and chemical catalysis and to combine the methods such as synthesis, chemosynthesis, but it is rare all to there is raw material, step is complicated, contaminate environment, the problems such as cost is higher, and biological process synthesis mevalonic acid can be good at overcoming the above problems.In biomass cells, mevalonic acid is obtained through reaching three step catalyzed reactions by acetyl-CoA, only has the active mevalonic acid of a kind of tool, have higher economic worth in synthetic product.Therefore biological process synthesis mevalonic acid has broad prospects.
Biological process synthesis mevalonic acid is mainly carbon source with glucose, 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-CoA thiolase, generates mevalonic acid under the catalyzer of HMG-CoA synthase and HMG-CoA reductase.Investigator is process LAN acetoacetyl-CoA thiolase/HMG-CoA reductase gene (mvaE) and HMG-CoA synthase gene (mvaS) in intestinal bacteria, be that carbon source is fermented with glucose, final mevalonic acid output can reach 47gL
-1(Tabataetal., BiotechnologyLetters26,1487-1491,2004).But using glucose as starting material, 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 needs to solve.
Summary of the invention
The invention provides a kind of product mevalonic acid restructuring colibacillus, is process LAN fatty acid transport protein gene (fadL) and fatty acyl-CoA synthetase gene (fadD) in intestinal bacteria, the restructuring colibacillus obtained.
Described fatty acid transport protein gene (fadL) refers to encoding outer membrane proteins FadL, as the acceptor of longer chain fatty acid, is regulate and control longer chain fatty acid across the important albumen of adventitia to membrane transport, preferred Genbank accession number: AAC75404.2.
Described fatty acyl-CoA synthetase is aliphatic alcohol by convert fatty acids, preferred Genbank accession number: AAC74875.1.
The present invention also provides a kind of method utilizing lipid acid production mevalonic acid, be in intestinal bacteria, import fatty acid transport protein gene (fadL) and fatty acyl-CoA synthetase gene (fadD), utilize recombination bacillus coli fermentative production mevalonic acid.
The described method utilizing lipid acid production mevalonic acid, concrete steps are as follows:
1) longer chain fatty acid transporter gene (fadL) and fatty acyl-CoA synthetase gene (fadD) is obtained;
2) recombinant plasmid containing longer chain fatty acid transporter gene (fadL) and fatty acyl-CoA synthetase gene (fadD) is built;
3) by step 2) plasmid that builds and plasmid pYJM11 import intestinal bacteria;
4) the restructuring colibacillus utilizing step 3) to obtain utilizes lipid acid fermentative production mevalonic acid.
Described step 2) adopt pET-30a carrier.
Described plasmid pYJM11 (pACY-mvaE-mvaS) builds for this seminar early stage, is published in: JianmingYangetal., BioresourceTechnology104,642-647,2012.
The described method utilizing lipid acid production mevalonic acid, preferred steps is as follows:
1) Genbank accession number is cloned respectively: the longer chain fatty acid transporter gene (fadL) of AAC75404.2 and the fatty acyl-CoA synthetase gene (fadD) of Genbank accession number: AAC74875.1;
2) described in step 1), gene 5 ' end and 3 ' end introduce restriction enzyme site respectively, by described gene clone on pET-30a carrier, build recombinant vectors pET-fadL-fadD;
3) by step 2) recombinant vectors pET-fadL-fadD and the pYJM11 (pACY-mvaE-mvaS) that obtains imports e. coli bl21 (DE3) and obtains colibacillus of recombinating;
4) intestinal bacteria utilizing step 3) to obtain 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 protein of any gene chemical synthesis has described function namely can be described as fatty acid transport protein gene or fatty acyl-CoA synthetase gene.
To build the engineering colon bacillus that obtains as biological catalyst, with longer chain fatty acid (C12 acid (lauric acid), C14 acid (tetradecanoic acid), C16 (palmitinic acid) or C18 (stearic acid)) for carbon source, the acid of fermentation method conversions of fats is adopted to be mevalonic acid.Fermentation condition is: it is 30-37 DEG C that temperature is cultivated, stirring velocity is be cultured to OD under the condition of 400-800rpm, pH6-8 and dissolved oxygen more than 20%
600be about 12-20, adding inductor IPTG to final concentration is 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, present invention also offers a kind of is intermediate product with mevalonic acid, the method for production object product.Be on the basis of high yield mevalonic acid, utilize the terpenoid of mevalonic acid fermentative production object product isoprene and the synthesis of prenyl unit further.
It should be noted that, the molecular biology method that the present invention adopts is routine techniques means, and fermentative production mevalonic acid or be that the method for target product of intermediate product is also common fermentation process optimization with mevalonic acid.
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. utilize the inventive method with longer chain fatty acid (C12 acid (lauric acid), C14 acid (tetradecanoic acid), C16 (palmitinic acid) or C18 (stearic acid)) for carbon source, 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 the leavened prod that fermentative production take mevalonic acid as intermediate product.
Accompanying drawing illustrates:
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:
With oligonucleotide 5 '-CATGCCATGGGCATGTCCTCAGCCATCTATCCCAG-3 ' and 5 '-CGCGGATCCTCAACGCCAGCCGGCGTCGATC-3 ' for primer, with genome of E.coli DNA for masterplate, polymerase chain reaction (PCR) method is adopted to amplify longer chain fatty acid transferring enzyme (fadL), and introduce EcoI and HindIII restriction enzyme site respectively at 5 ' end and 3 ' end, then with above-mentioned restriction enzyme site by this gene clone on pET-30a carrier, obtain recombinant plasmid pET-fadL (Fig. 1); With oligonucleotide 5 '-GGGAATTCCATATGACCGCTCAGGTTACATGCG-3 ' and 5 '-CCGCTCGAGTTAAACCAGACGAACTTCGTGC-3 ' for primer, with genome of E.coli DNA for masterplate, polymerase chain reaction (PCR) method is adopted to amplify acyl CoA synthetic enzyme (fadD), and introduce KpnI and BamHI restriction enzyme site respectively at 5 ' end and 3 ' end, then with above-mentioned restriction enzyme site by this gene clone on 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 synthesis mevalonic acid, and be mevalonic acid with this strain fermentation conversions of fats acid, its detailed process is as follows:
Adopt in alkaline lysis method of extracting embodiment 1 the recombinant plasmid pYJM11 (JianmingYangetal. that the recombinant plasmid pET-fadL-fadD that builds and obtain and this laboratory have built, BioresourceTechnology104,642-647,2012), adopt thermal shock conversion method by 5 μ l recombinant plasmids to transformation of E. coli BL21 (DE3) competent cell, the bacterium liquid then got after 50 μ l conversions is coated containing 34 μ gmL
-1paraxin and 50 μ gmL
-1screening positive clone on the LB flat board of ammonia Bian, the bacterium colony grown 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, 37 DEG C, 180rpm shaking culture spends the night, then by bacterium liquid by volume 1% inoculum size be inoculated into interpolation 34 μ gmL
-1enlarged culturing is carried out in the fermention medium of paraxin, under the condition of culture temperature 37 DEG C, 180rpm shaking culture, be cultured to OD600 be about 0.8, collected by centrifugation thalline, resuspended, be inoculated in lipid acid be sole carbon source substratum in, 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 in fermented liquid supernatant and mevalonic acid content, and with the lipid acid of purchase and mevalonic acid standard substance in contrast.Calculated by external standard method, the output of mevalonic acid can reach 230mg/L, and productive rate can reach 56%.The bacterial strain mevalonic acid output of non-process LAN two enzyme 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 synthesis mevalonic acid, and be mevalonic acid with this strain fermentation conversions of fats acid, its detailed process is as follows:
Adopt in alkaline lysis method of extracting embodiment 1 the recombinant plasmid pYJM11 (JianmingYangetal. that the recombinant plasmid pET-fadL-fadD that builds and obtain and this laboratory have built, BioresourceTechnology104,642-647,2012), adopt thermal shock conversion method by 5 μ l recombinant plasmids to transformation of E. coli W3110 competent cell, the bacterium liquid then got after 50 μ l conversions is coated containing 34 μ gmL
-1paraxin and 50 μ gmL
-1screening positive clone on the LB flat board of ammonia Bian, the bacterium colony grown 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, 37 DEG C, 180rpm shaking culture spends the night, then by bacterium liquid by volume 1% inoculum size be inoculated into interpolation 34 μ gmL
-1carry out enlarged culturing in the fermention medium of paraxin, under the condition of culture temperature 37 DEG C, 180rpm shaking culture, be cultured to OD
600be about 0.8, collected by centrifugation thalline, resuspended, be inoculated in lipid acid be sole carbon source substratum in, 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 in fermented liquid supernatant and mevalonic acid content, and with the lipid acid of purchase and mevalonic acid standard substance in contrast, result as shown in Figure 3.Calculated by external standard method, the output of mevalonic acid can reach 180mg/L, and productive rate can reach 56%.The bacterial strain mevalonic acid output of non-process LAN two enzyme is 57mg/L, and productive rate is only 17.3%.
Claims (6)
1. utilize a method for lipid acid production mevalonic acid, it is characterized in that, concrete steps are as follows:
1) longer chain fatty acid transporter gene fadL and fatty acyl-CoA synthetase gene fadD is obtained;
2) recombinant plasmid containing longer chain fatty acid transporter gene fadL and fatty acyl-CoA synthetase gene fadD is built;
3) by step 2) plasmid that builds and plasmid pYJM11 import intestinal bacteria;
4) step 3 is utilized) the restructuring colibacillus that obtains utilizes lipid acid fermentative production mevalonic acid.
2. method according to claim 1, is characterized in that, described fatty acid transport protein gene Genbank accession number: AAC75404.2.
3. method according to claim 1, is characterized in that, described fatty acyl-CoA synthetase gene Genbank accession number: AAC74875.1.
4. method according to claim 1, it is characterized in that, concrete steps are as follows:
1) Genbank accession number is cloned respectively: the fatty acyl-CoA synthetase gene fadD of the longer chain fatty acid transporter gene fadL of AAC75404.2 and Genbank accession number: AAC74875.1;
2) in step 1) described gene 5 ' end and 3 ' end introduce restriction enzyme site respectively, by described gene clone on pET-30a carrier, builds recombinant vectors pET-fadL-fadD;
3) by step 2) recombinant vectors pET-fadL-fadD and the pYJM11 (pACY-mvaE-mvaS) that obtains imports e. coli bl21 (DE3) and obtains colibacillus of recombinating;
4) step 3 is utilized) intestinal bacteria that obtain utilize lipid acid fermentative production mevalonic acid.
5. method described in claim 1 in fermentative production is the application in the product of mesostate with mevalonic acid.
6. apply according to claim 5, it is characterized in that, recombination bacillus coli described in claim 1 is used for fermentative production isoprene or is the terpenoid that unit synthesizes with isoprene.
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| 脂肪酸转运蛋白家族(FATPs)的研究进展;卢向阳;《生命科学研究》;20130831;348-353 * |
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