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Naringenin in-vitro enzymatic synthesis method based on malonyl coenzyme A regeneration

The technology of a malonyl coenzyme and a synthesis method is applied in the field of in vitro enzymatic synthesis of naringenin to achieve the effects of simple product separation, simple composition and easy reaction process

Active Publication Date: 2021-11-09
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no similar strategy to reduce the high production cost caused by malonyl-CoA in the in vitro synthesis system

Method used

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  • Naringenin in-vitro enzymatic synthesis method based on malonyl coenzyme A regeneration
  • Naringenin in-vitro enzymatic synthesis method based on malonyl coenzyme A regeneration

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Embodiment 1

[0042] 1. Construction of recombinant plasmids

[0043] Escherichia coli acetyl-CoA synthetase gene provided by GenBank database ACS and yeast acetyl-CoA carboxylase gene ACC1 nucleotide sequence information, design two pairs of primer sequences for the ACS The coding region of the gene was cloned into the Escherichia coli expression vector pET-32a(+), and the ACC1 The coding region of the gene was cloned into the Pichia pastoris expression vector pGAP-Neo:

[0044] PCR amplification ACS The forward primer of the gene is 5'-GCTGATATCGGATCC GAATTC atgagccaaattcacaaacac-3' (as shown in SEQ ID No.3), the base in italics indicates the enzyme cutting site EcoRI; the reverse primer is 5'-GTGGTGGTGGTGGTG CTCGAG ttacgatggcatcgcgatag-3' (as shown in SEQ ID No.4), the base in italics indicates the restriction site XhoI.

[0045] PCR amplification ACC1 The forward primer of the gene is

[0046] 5'-CTATTTCGAAACGAG GAATTC ATGAGCGAAGAAAGCTTATTC-3' (as shown in SEQ ID No.5), t...

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Abstract

The invention designs a naringenin in-vitro enzymatic synthesis method based on malonyl coenzyme A regeneration. On the basis of constructing recombinant expression plasmids of an acetyl coenzyme A synthetase ACS gene and an acetyl coenzyme A carboxylase ACC1 gene, the recombinant plasmids are respectively transformed into escherichia coli and yeast cells to express a target protein, and the purified ACS and ACC1 recombinant proteins are added into a naringenin in-vitro enzymatic synthesis system to realize the regeneration of malonyl coenzyme A and the in-vitro low-cost enzymatic synthesis of naringenin by using 4-coumaric acid as a substrate. The invention designs a new reaction system, which can be regenerated and utilized without adding expensive malonyl coenzyme A and finally generates naringenin.

Description

technical field [0001] The invention relates to an in vitro enzymatic synthesis method of naringenin based on malonyl-CoA regeneration, the classification number is C12N9, and belongs to the technical field of biomedicine. Background technique [0002] Malonyl-coenzyme A (malonyl-coenzyme A) is a coenzyme A derivative. As a key intermediate metabolite, malonyl-CoA plays an important role in a variety of metabolic pathways and is an important precursor molecule for the synthesis of fatty acids and triglycerides in cells (Kastaniotis A J, et al. Biochim Biophys Acta Mol Cell Biol Lipids,2017, 1862(1): 39-48.), is also a synthetic polyketide compound (Shimizu Y, et al. Chembiochem,2017, 18(1): 50-65.) and many platform compounds (Kildegaard K R , et al. Microb Cell Fact,2016, 15: 53.) raw materials. [0003] Generally speaking, intracellular coenzyme A is catalyzed successively by acetyl-CoA synthase (ACS) and acetyl-CoA carboxylase (acetyl-CoA carboxylase, ACC) to form malon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N15/61C12N15/54C12N15/70C12N15/81C12P17/06
CPCC12N15/52C12N9/93C12N9/90C12N9/1037C12N15/70C12N15/815C12P17/06C12Y604/01002C12Y602/01003C12Y602/01012C12Y505/01006C12Y203/01074
Inventor 张新跃聂也森何妍之张智萍丁笠陈磊廖凯赵晨宏
Owner YANGZHOU UNIV
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