Method for efficiently producing mevalonic acid from fatty acid and constructed gene engineering bacterium

A technology for producing mevalonate and mevalonate, applied in genetic engineering, microbe-based methods, biochemical equipment and methods, etc., can solve problems such as complex steps, high cost, and environmental pollution

Active Publication Date: 2014-02-12
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the synthesis of mevalonate mainly includes enzymatic synthesis, combined synthesis of biocatalysis and chemical catalysis, chemical synthesis, etc., but

Method used

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  • Method for efficiently producing mevalonic acid from fatty acid and constructed gene engineering bacterium
  • Method for efficiently producing mevalonic acid from fatty acid and constructed gene engineering bacterium
  • Method for efficiently producing mevalonic acid from fatty acid and constructed gene engineering bacterium

Examples

Experimental program
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Effect test

Embodiment 1

[0035] The construction of the co-expression vector of long-chain fatty acid transporter gene (fadL) and fatty acyl-CoA synthetase gene (fadD) is as follows:

[0036] Use oligonucleotides 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' as primers, and E. coli genomic DNA as template , using the polymerase chain reaction (PCR) method to amplify the long-chain fatty acid transferase (fadL), and introduce EcoI and HindIII restriction sites at the 5' end and 3' end respectively, and then use the above restriction sites to decompose This gene is cloned on the pET-30a vector to obtain the recombinant plasmid pET-fadL( figure 1 ); with oligonucleotides 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' as primers, Escherichia coli genomic DNA As a template, acyl-CoA synthetase (fadD) was amplified by polymerase chain reaction (PCR), and KpnI and BamHI restriction sites were int...

Embodiment 2

[0038] The preparation of engineering Escherichia coli strain BL21(DE3) / pET-fadL-fadD / pYJM11 for synthesizing mevalonate, and using the strain to ferment and transform fatty acid into mevalonate, the specific process is as follows:

[0039] The recombinant plasmid pET-fadL-fadD constructed in Example 1 and the recombinant plasmid pYJM11 (Jianming Yang et al., Bioresource Technology 104, 642-647, 2012) constructed in our laboratory were extracted by alkaline lysis, and transformed by heat shock 5μl recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, and then 50μl of the transformed bacteria liquid was applied to the cells containing 34μg·mL -1 Chloramphenicol and 50 μg·mL -1 Positive clones were screened on the ammonia LB plate, and the grown colonies were recombinant Escherichia coli strains co-expressing long-chain fatty acid transferase and fatty acyl-CoA synthetase. Pick a single colony of recombinant Escherichia coli that has been constru...

Embodiment 3

[0041] Preparation of engineering Escherichia coli strain W3110 / pET-fadL-fadD / pYJM11 for synthesizing mevalonate, and using this strain to ferment and convert fatty acids into mevalonate, the specific process is as follows:

[0042] The recombinant plasmid pET-fadL-fadD constructed in Example 1 and the recombinant plasmid pYJM11 (Jianming Yang et al., Bioresource Technology 104, 642-647, 2012) constructed in our laboratory were extracted by alkaline lysis, and transformed by heat shock Transform 5 μl of the recombinant plasmid into Escherichia coli W3110 competent cells, and then take 50 μl of the transformed bacterial solution and spread it on a medium containing 34 μg·mL -1 Chloramphenicol and 50 μg·mL -1 Positive clones were screened on the ammonia LB plate, and the grown colonies were recombinant Escherichia coli strains co-expressing long-chain fatty acid transferase and fatty acyl-CoA synthetase. Pick a single colony of recombinant Escherichia coli that has been constru...

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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 [0001] The invention relates to a method for efficiently utilizing fatty acids to produce mevalonate and the constructed genetically engineered bacteria. Background technique [0002] Mevalonate is an intermediate metabolite of isoenzyme pyrophosphate (IPP) produced by the mevalonate pathway, and is an important intermediate in the synthesis of sterols, terpenoids, and isoprenoids. At the same time, it also has important uses in the chemical industry. At present, the synthesis of mevalonate mainly includes enzymatic synthesis, combined synthesis of biocatalysis and chemical catalysis, chemical synthesis, etc., but there are problems such as rare raw materials, complicated steps, pollution of the environment, and high cost. Valeric acid can well solve the above problems. In biological cells, mevalonate is obtained from acetyl coenzyme A through a three-step catalytic reaction. There is only one optically active mevalonate in the synthetic product, which has h...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/54C12N15/52C12N15/70C12P7/42C12P7/04C12R1/19
Inventor 咸漠程涛曹玉锦邹慧斌
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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