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Use of branched chain alpha-keto acid dehydrogenase complex in preparing malonyl coenzyme A

A technology of malonyl coenzyme and ketoacid dehydrogenase, which is applied in the biological field and can solve problems such as low yield and biosynthesis limitation

Pending Publication Date: 2021-02-26
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, polyketides such as erythromycin have been successfully synthesized in Escherichia coli, but their yields are still low. The main reason is that the synthesis of polyketides is limited by the content of malonyl-CoA in the cell. Acyl-CoA is also an important precursor for the synthesis of flavonoids, and the biosynthesis of flavonoids is also limited by the content of malonyl-CoA

Method used

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  • Use of branched chain alpha-keto acid dehydrogenase complex in preparing malonyl coenzyme A
  • Use of branched chain alpha-keto acid dehydrogenase complex in preparing malonyl coenzyme A
  • Use of branched chain alpha-keto acid dehydrogenase complex in preparing malonyl coenzyme A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0196] Example 1, the branched-chain α-ketoacid dehydrogenase complex can catalyze the synthesis of malonyl-CoA

[0197] The present invention finds that the branched-chain α-ketoacid dehydrogenase complex can catalyze the synthesis of malonyl-CoA, and this example prepares the coding genes (bkdA, bkdB, bkdC, lpdA1, bkdF, bkdG, bkdH genes), and further knocked out two genes (threonine deaminase ilvA and branched chain amino acid transaminase ilvE genes) in the branched-chain α-keto acid synthesis pathway. The synthesis of malonyl-CoA catalyzed by the α-ketoacid dehydrogenase complex was detected, and the primers used are shown in Table 1.

[0198] (1) Construction of a plasmid expressing the branched-chain α-ketoacid dehydrogenase complex of Streptomyces avermitilis

[0199] (1-a) PCR amplification of bkdA, bkdB, bkdC, lpdA1, bkdF, bkdG, bkdH genes

[0200] Genomic DNA of Streptomyces avermitilis was extracted using a bacterial genome extraction kit (Tiangen Biochemical Tech...

Embodiment 2

[0234] Embodiment 2, phosphoenolpyruvate carboxylase ppc gene can improve the synthesis amount of engineering strain M-FGH malonyl-CoA

[0235] In this example, on the basis of the engineering strain M-FGH in Example 1, the phosphoenolpyruvate carboxylase ppc gene was introduced and the ompT gene was knocked out (the sequence of the ompT gene is sequence 27 in the sequence listing, shown in the coding sequence 28 ompT protein), further increased the amount of synthesis of malonyl-CoA, the primers used are shown in Table 2.

[0236] (4) Construction of expressing phosphoenolpyruvate carboxylase ppc genetic engineering strain

[0237] (4-a) Genome extraction from Corynebacterium glutamicum and Escherichia coli, and PCR amplification of ppc gene, chloramphenicol resistance fragment, and upstream and downstream homology arms of ompT gene

[0238] Using ppc-F and ppc-R as PCR amplification primers, using Corynebacterium glutamicum genomic DNA as a template, amplified fragment tac-...

Embodiment 3

[0250] Example 3. The expression of the branched-chain α-ketoacid dehydrogenase complex gene bkdFGH-lpdA1 of Streptomyces avermitilis can increase the production of 3-hydroxypropionic acid (3-HP).

[0251] 3-hydroxypropionic acid is an important platform compound and a raw material for the synthesis of various chemicals. Malonyl-CoA can be used as a precursor to obtain 3-hydroxypropionic acid through a two-step reduction reaction. The present embodiment is by introducing thermophilic light whole Chlorothycus (Chloroflexusaurantiacus ) malonyl-CoA reductase gene mcr gene to prepare 3-hydroxypropionic acid, and use the BW of Example 1 as a control. The primers used are listed in Table 3.

[0252] (6) Construction of a plasmid expressing the malonyl-CoA reductase gene mcr of Chloroflexus aurantiacus (Chloroflexus aurantiacus)

[0253] (6-a) The nucleotide sequence of the transformed Chloroflexus aurantiacus malonyl-CoA reductase gene mcr gene is sequence 21 in the sequence list...

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Abstract

The invention discloses use of a branched chain alpha-keto acid dehydrogenase complex in preparing a malonyl coenzyme A. The invention discloses a method for preparing a malonyl coenzyme A by using abranched chain alpha-keto acid dehydrogenase complex. The method comprises the following steps: introducing a coding gene of the branched chain alpha-keto acid dehydrogenase complex into a biologicalcell and expressing the coding gene of the branched chain alpha-keto acid dehydrogenase complex to obtain a recombinant cell; and culturing the recombinant cell to obtain the malonyl coenzyme A. The branched chain alpha-keto acid dehydrogenase complex is a complete set of proteins composed of M1) or M2): M1): bkdF, bkdG, bkdH and lpdA1; and M2: bkdA, bkdB, bkdC and lpdA1. Experiments prove that the branched chain alpha-keto acid dehydrogenase complex can be used for preparing the malonyl coenzyme A and also can be used for preparing a target product taking the malonyl coenzyme A as an intermediate product.

Description

technical field [0001] The invention relates to the application of a branched-chain α-ketoacid dehydrogenase complex in the preparation of malonyl-CoA in the field of biotechnology. Background technique [0002] Flavonoids are ubiquitous in plants in nature. They are a class of compounds containing 2-phenylchromone structure and belong to plant secondary metabolites. Flavonoids have anti-free radical and anti-oxidation effects, and these compounds also have antibacterial, anti-tumor and enhanced immune functions. Polyketides are also an important class of secondary metabolites, which are formed by continuous decarboxylation and condensation of lower carboxylic acids such as acetic acid, malonic acid, butyric acid, etc. by bacteria, fungi, actinomycetes or plants. Because of its important biological activity, it is widely used clinically as an antibiotic, such as erythromycin, anticancer drug doxorubicin, antifungal agent amphotericin, antiparasitic agent avermectin, insecti...

Claims

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Application Information

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IPC IPC(8): C12P19/32C12P7/42C12P13/00C12P7/22C12P7/26C12P7/64C12N1/21C12N9/02C12N9/10C12N9/88C12N15/70C12R1/19
CPCC12P19/32C12P7/42C12P13/008C12P7/22C12P7/26C12P7/6409C12N9/0008C12Y102/04004C12N9/88C12Y403/01019C12N9/1096C12Y206/01042C12Y401/01031C12N15/70C12Y102/01075C12N9/1029C12Y203/01156C12N9/10C12R2001/19C12N1/205
Inventor 刘伟丰刘波崔倩倩陶勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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