Method for constructing genetic engineering strains for producing (R)-acetoin and application of genetic engineering strains

A technology of genetically engineered strains and construction methods, which is applied to the construction of highly optically pure-acetoin genetically engineered strains and the application field of producing highly optically pure-acetoin, which can solve the problem of ineffective regeneration of oxidized coenzyme NAD, Low yield of acetoin, pathogenicity of strains and other problems, to achieve the effect of releasing transcription inhibition, low cost and stable properties

Active Publication Date: 2017-09-05
GUANGXI ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention aims at the high cost of raw materials, low yield of (R)-acetoin, and oxidized coenzyme NAD in the existing synthetic (R)-acetoin technology + Can not be effectively reproduced, or strains have pathogenicity and many other problems, provide a method for the production of (R)-acetoin genetically engineered strains and its application

Method used

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  • Method for constructing genetic engineering strains for producing (R)-acetoin and application of genetic engineering strains
  • Method for constructing genetic engineering strains for producing (R)-acetoin and application of genetic engineering strains

Examples

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Embodiment 1

[0034] The construction of (R)-acetoin genetically engineered strain ECRA1:

[0035] The nucleotide sequences of the α-acetolactate synthase gene budB, the α-acetolactate decarboxylase gene budA derived from Enterobacter cloacae and the NADH oxidase gene noxE derived from Lactobacillus brevis were codon optimized, adding Containing the nucleotide sequence TAAGGAGGATATACA of the ribosome binding site, and then using the method of artificial synthesis to obtain the gene cluster budB-budA-noxE, the nucleotide sequence length is 3849 bases, and the nucleotide sequence is as SEQ ID NO.1 mentioned. The gene cluster budB-budA-noxE was inserted behind the promoter of the plasmid pTrc99A by double enzyme digestion and ligation to obtain the polycistronic recombinant plasmid pTrc99A-budB-budA-noxE, and then the recombinant plasmid pTrc99A-budB-budA- noxE was introduced into the host strain E.coli MG1655 to obtain (R)-acetoin-producing genetically engineered strain ECRA1.

Embodiment 2

[0037] Construction of the production (R)-acetoin genetic engineering strain ECRA2:

[0038] The genome sequence of Paenibacillus polymyxa DSM 365 was analyzed, and the nucleotide sequences of the α-acetolactate synthase gene alsS and α-acetolactate decarboxylase gene alsD of the strain were obtained. The length of the alsS gene nucleotide sequence is 1701 bases, and the nucleotide sequence is as described in SEQ ID NO.2; the length of the alsD gene nucleotide sequence is 747 bases, and the nucleotide sequence is as described in SEQ ID NO.3 stated. Codon optimization was performed on the nucleotide sequences of alsS, alsD and noxE, and the nucleotide sequence TAAGGAGGATATACA containing a ribosome binding site was added in front of each gene, and then the gene cluster alsS-alsD-noxE was obtained by artificial synthesis. The length of its nucleotide sequence is 3837 bases, and the nucleotide sequence is as described in SEQID NO.4. Then the gene cluster alsS-alsD-noxE was inser...

Embodiment 3

[0040] The construction of (R)-acetoin genetically engineered strain ECRA3:

[0041] Through analysis, it was found that the by-products of the fermentation of engineering strains ECRA1 and ECRA2 were (S)-acetoin, 2,3-butanediol, succinic acid, lactic acid, and acetic acid, and the key genes of the synthesis pathway were dar, gldA, frdABCD, ldhA and pta. Using the principle that the Red recombination system derived from Escherichia coli λ phage can efficiently mediate homologous recombination events in bacteria, first replace the above target gene with an antibiotic resistance gene with FRT sites on both sides, and then induce exogenous temperature The sensitive plasmid expresses FLP recombinase to delete the antibiotic resistance gene to achieve the purpose of knocking out the target gene. The specific steps are as follows:

[0042] Transform the pKD46 plasmid into host cells to prepare electroporation-competent cells; use primers to carry out PCR to construct the targeting ...

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Abstract

The invention discloses a method for constructing genetic engineering strains for producing (R)-acetoin and application of the genetic engineering strains. The method includes optimizing codons of nucleotide sequences of alpha-acetolactate synthase genes, alpha-acetolactate decarboxylase genes and NADH (reduced form of nicotinamide adenine dinucleotide) oxidase genes and acquiring each gene cluster with three genes by the aid of artificial synthesis processes; inserting the gene clusters into expression vectors to obtain polycistron recombinant plasmids; introducing the polycistron recombinant plasmids into host bacteria E. coli and knocking out key genes of main byproduct synthesis paths to obtain the genetic engineering strains for producing the (R)-acetoin. The method and the application have the advantages that raw materials for the genetic engineering strains can come from wide sources and are low in cost, the strains are free of pathogenicity, oxidized form coenzymes NAD+ (nicotinamide adenine dinucleotide+) can be effectively regenerated, the strains are high in (R)-acetoin yield and production efficiency, the maximum yield can reach 72.1 g/L, and the optical purity can reach 99% at least; the (R)-acetoin is produced by the aid of non-grain cassava flour and inexpensive nitrogen sources which are used as fermentation raw materials, and accordingly the production cost can be reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the construction of a genetically engineered bacterial strain for producing highly optically pure (R)-acetoin, and its use in the production of highly optically pure (R)- Application of Acetomarin. Background technique [0002] Acetoin, whose chemical name is 3-hydroxy-2-butanone, naturally exists in dairy products, butter, cheese, wine, corn, grapes, apples, strawberries, cocoa, meat and other foods, and has a strong Creamy, fatty, buttery aroma, highly diluted with a pleasant milky aroma. The national standard GB2760-86 stipulates that it is an edible flavor that is allowed to be used. The safety number of the American Food Flavor and Extract Manufacturers Association (FEMA) is 2008. It is often used as a flavor enhancer at home and abroad, and is also used to configure milky and meaty flavors. , Strawberry-flavored flavor, is a widely used edible flavor. At the same time, aceto...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/26C12R1/19
CPCC12N9/0036C12N9/1022C12N9/88C12P7/26C12Y106/99003C12Y202/01006C12Y401/01005
Inventor 谢能中黄日波陈先锐李检秀黄艳燕
Owner GUANGXI ACAD OF SCI
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