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Engineering bacterium for biosynthesizing resveratrol by taking L-tyrosine as substrate, construction and application

A technology for resveratrol and biosynthesis, applied in the biological field, can solve problems such as difficulties, chemical synthesis pollution safety, questioned promotion, etc., and achieve the effect of increasing the content

Pending Publication Date: 2022-01-14
河北维达康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the method of simply relying on natural plants to extract resveratrol can no longer meet people's needs, and the chemical synthesis method is difficult to popularize because of serious pollution and questioned safety. With the progress of microbial metabolic engineering and synthetic biology technology, in the large intestine The design and construction of efficient heterologous synthesis pathways in hosts such as Bacillus and Saccharomyces cerevisiae, combined with the large-scale fermentation of engineered strains is to realize the industrial production of rare plant-derived natural products such as resveratrol, which can provide a large-scale industrial production of resveratrol. important support

Method used

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  • Engineering bacterium for biosynthesizing resveratrol by taking L-tyrosine as substrate, construction and application
  • Engineering bacterium for biosynthesizing resveratrol by taking L-tyrosine as substrate, construction and application
  • Engineering bacterium for biosynthesizing resveratrol by taking L-tyrosine as substrate, construction and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1p

[0042] Construction method of embodiment 1pINA1269-TAL-4CL-STS plasmid

[0043] 1. Construction of pINA1269-TAL, pINA1269-4CL and pINA1269-STS plasmids

[0044] (1) Using the artificially synthesized RgTAL gene (as a template, carry out PCR amplification with primers RgTAL-F and RgTAL-R, and recover the target fragment RgTAL.

[0045] (2) Using the artificially synthesized Gm4CL gene as a template, PCR amplification was performed with primers Gm4CL-F and Gm4CL-R, and the target fragment Gm4CL was recovered.

[0046] (3) Using the artificially synthesized AhSTS gene as a template, PCR amplification was carried out with primers AhSTS-F and AhSTS-R, and the target fragment AhSTS was recovered.

[0047] (4) Using the artificially synthesized CmTAL gene as a template, PCR amplification was performed with primers CmTAL-F and CmTAL-R, and the target fragment CmTAL was recovered.

[0048] (5) Using the artificially synthesized At4CL gene as a template, PCR amplification was performe...

Embodiment 2

[0073] Embodiment 2 Construction of Yarrowia lipolytica engineering bacteria and shake flask fermentation

[0074] (1) The plasmids pINA1269-RgTAL-Gm4CL-AhSTS, pINA1269-CmTAL-At4CL-VvSTS, and pINA1269-SaTAL-Nt4CL-PcSTS obtained in Example 1 were digested with restriction endonuclease NotI, linearized and then transformed with LiAC Transform into Yarrowia lipolytica Po1f, spread it on SC-Leu yeast-deficient medium, culture at 30°C until transformants grow, pick positive transformants, and obtain the initial project capable of producing resveratrol Strain 1, engineering strain 2 and engineering strain 3.

[0075] (2) Streak the engineering strain 1, engineering strain 2 and engineering strain 3 on the YPD medium and cultivate until a single colony grows, pick a single colony, inoculate it into the seed medium, and cultivate it for 15 hours to obtain the seed liquid. Said seed medium is YPD medium: 20g / L glucose is the carbon source, contains yeast extract 10g / L, peptone 20g / L, ...

Embodiment 3

[0084] Example 3 Expression of acetyl-CoA carboxylase (ACC) increases resveratrol production

[0085] 1. Construction of pINA1311-ACC plasmid

[0086] (1) artificially synthesized CgACC (sequence as described in SEQ ID No.10, length 1776bp), AsACC (sequence as described in SEQ ID No.11, length 6699bp), CaACC gene (sequence as described in SEQ ID No.12 described above, with a length of 6693bp) as a template, respectively utilize CgACC-F and CgACC-R, AsACC-F and AsACC-R, CaACC-F and CaACC-R to carry out PCR amplification, and the amplified products are recovered to obtain CgACC, AsACC , the target fragment of CaACC, the fragment sizes are 1776bp, 6699bp, 6693bp respectively, the primer CgACC-F sequence 5'-CCCCACGTGATGTCTGTTGAAACAAGAAAAAATAACCAAAGTGC-3', as described in SEQ ID No.47, the primer CgACC-R sequence 5' -CGGGGTACCTTATTTGATTTCCAATAAAACCACTCCTTTGTTAACAG-3', as described in SEQ ID No.48; said primer AsACC-F sequence 5'-CCCCACGTGATGAGCGCGTATGATCAT-3', as described in SEQ ...

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Abstract

The invention discloses an engineering bacterium for biosynthesizing resveratrol by taking L-tyrosine as a substrate, construction and application, and belongs to the technical field of biology. According to the invention, a simple and effective implementation method is provided for safe biotransformation of the resveratrol; biosynthesis from the L-tyrosine to the resveratrol is implemented in cells by adopting co-expression on TAL, 4CL and STS modules; the content of the product resveratrol is remarkably increased by expressing an acetyl coenzyme A carboxylase gene and an acetyl coenzyme A synthetase gene; and the yield of the resveratrol biotransformed by the engineering bacterium is as high as 5.5-6 g / L.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the construction and application of an engineering bacterium that uses L-tyrosine as a substrate to biosynthesize resveratrol. Background technique [0002] Resveratrol, also known as stilbenol, is a non-flavonoid polyphenolic organic compound. It is an antitoxin produced by many plants when stimulated. It can be synthesized in grape leaves and grape skins. It is in wine and grape juice. biologically active ingredients. It is easily absorbed orally, and excreted through urine and feces after metabolism. There are four main forms of resveratrol in plants: trans-resveratrol, cis-resveratrol, trans-resveratrol glycosides and cis-resveratrol glycosides, of which the trans form is the main form. , and the biological activity of trans-resveratrol is higher than that of cis-resveratrol. At present, it is known that there are at least 34 families, 69 genera, and 100 different ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/60C12N15/54C12N15/52C12N15/66C12P7/22C12R1/645
CPCC12N9/88C12N9/93C12N9/1029C12N15/815C12N15/66C12P7/22C12Y403/01023C12Y602/01012C12Y604/01002C12Y203/01095C12Y602/01001C12N2800/102C12N2800/22
Inventor 赵云现姜黎崔金旺杨志彬胡江林田昊博赵凯
Owner 河北维达康生物科技有限公司
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