A kind of method utilizing cheap raw material to produce tetramethylpyrazine

A technology of tetramethylpyrazine and raw materials, which is applied in the direction of introducing foreign genetic material, fermentation, and recombinant DNA technology using a carrier, can solve the problem that the tetramethylpyrazine technology is immature, the production cost is greatly affected, and the economic feasibility is reduced. and other problems, to achieve the effect of improving comprehensive utilization value, shortening production cycle and high production efficiency

Active Publication Date: 2020-12-18
南宁中诺生物工程有限责任公司 +1
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Problems solved by technology

However, due to the relatively late start of research on the use of microbial fermentation to produce acetidine and its conversion into tetramethylpyrazine, the technology for producing tetramethylpyrazine through biotechnology is far from mature
[0004] Chinese patent 201010238685.5 discloses a method for producing tetramethylpyrazine and its production strains. Screened Bacillus subtilis XZ1124, Bacillus licheniformis BL-L1, MT-6 and MT-15 use glucose as carbon source and yeast extract as nitrogen When the source is used, the accumulation of acetoin in the fermenter is 30-44g / L, but the cycle is as long as 72h, and the conversion rate of glucose is too low due to the accumulation of by-products
In addition, this invention needs to use expensive yeast extract as a nitrogen source, and the carbon source glucose is high-purity sugar, which also has the problem of high cost, which further reduces the economic feasibility of this method
The composition of the medium for microbial fermentation has a great influence on the production cost, and the use of high-purity sugar (glucose) will increase the cost of raw materials
In addition, yeast powder / yeast extract is often used as a nitrogen source to ferment acetoin and tetramethylpyrazine because it is rich in nutrients such as amino acids, vitamins, and trace elements, but its use usually greatly increases production costs, such as In some lactic acid fermentations, the cost of yeast powder can account for 30% of the total fermentation cost

Method used

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  • A kind of method utilizing cheap raw material to produce tetramethylpyrazine

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Construction of the genetic engineering strain ECTMP1 for the production of precursor substance acetoin:

[0038] 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 acetoin-producing genetically enginee...

Embodiment 2

[0040] Construction of the genetic engineering strain ECTMP2 for the production of precursor substance acetoin:

[0041] Through the analysis, it was found that the main by-products of the fermentation of the engineering strain ECTMP1 were 2,3-butanediol, succinic acid, lactic acid, and acetic acid, and the key genes of the synthesis pathway were gldA, frdABCD, ldhA and pta. Utilizing the principle that the Red recombination system derived from E. coli 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 thermosensitive The 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 tar...

Embodiment 3

[0046] Construction of the genetic engineering strain ECTMP3 for the production of precursor substance acetoin:

[0047] The difference between this example and Example 1 is that the source strain of the α-acetolactate synthase gene and the α-acetolactate decarboxylase gene is Klebsiella pneumoniae, and the source strain of the NADH oxidase gene is Lactococcuslactis.

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Abstract

The invention discloses a method for producing tetramethylpyrazine from cheap raw materials. The method comprises steps as follows: nucleotide sequences of an alpha-acetolactate synthase gene, an alpha-acetolacetate decearboxylase gene and an NADH oxidase gene are subjected to codon optimization; a gene cluster containing the three genes is obtained through splicing; the gene cluster is inserted into an expression vector, and polycistron recombinant plasmid is obtained; the polycistron recombinant plasmid is introduced into a host strain E.coli, and a gene engineering strain producing acetylmethylcarbinol is obtained; a fermentation medium containing the cheap raw materials is inoculated with the activated strain for fermentation culture, and fermentation broth containing acetylmethylcarbinol is obtained; the fermentation broth is subjected to centrifugal treatment, a supernatant is taken, diammonium hydrogen phosphate is added, acetylmethylcarbinol produced through fermentation reacts with NH4<+>, and tetramethylpyrazine is synthesized. The widely sourced and low-cost raw materials can be effectively utilized by the strain for producing a high-concentration precursor substance, namely, acetylmethylcarbinol, oxidative coenzyme NAD<+> can be regenerated effectively, yield and production efficiency of acetylmethylcarbinol can be increased, so that the production cost of acetylmethylcarbinol is effectively reduced, and production period is shortened.

Description

technical field [0001] The invention belongs to the field of biochemical industry, in particular to a method for producing tetramethylpyrazine by using cheap raw materials. Background technique [0002] Tetramethylpyrazine (2,3,5,6-tetramethylpyrazine), also known as ligustrazine, is the main active alkaloid component of the rhizome of the traditional Chinese medicine Ligusticum chuanxiong. It has pharmacological effects and has certain curative effects in the treatment of cardiovascular and cerebrovascular diseases, respiratory diseases and glomerular diseases, and has been widely used in clinical practice as a standing drug. In addition, tetramethylpyrazine naturally occurs in dairy products, soybean products, cocoa, coffee, peanuts, hazelnuts, and liquor, and has a special aroma of roasting and nuts. Tetramethylpyrazine is also an important aroma compound in Chinese liquor. A certain amount of tetramethylpyrazine has been detected in traditional Chinese liquor, which not...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12P17/12
CPCC12N15/70C12P17/12
Inventor 谢能中黄日波韦航李晓明
Owner 南宁中诺生物工程有限责任公司
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