CN107177620B - A kind of method utilizing cheap raw material to produce tetramethylpyrazine - Google Patents

Abstract

The invention discloses a method for producing tetramethylpyrazine by using cheap raw materials. The nucleotide sequences of α-acetolactate synthase gene, α-acetolactate decarboxylase gene and NADH oxidase gene are codon-optimized; A gene cluster comprising the three genes was obtained; the gene cluster was inserted into an expression vector to obtain a polycistronic recombinant plasmid; then the host strain E.coli was introduced to obtain a genetically engineered strain producing acetoin; the activated strain was inoculated into The fermentation medium containing cheap raw materials is fermented and cultured to obtain a fermentation broth containing acetoin; the fermentation broth is centrifuged, the supernatant is taken, and diammonium hydrogen phosphate is added, and the acetoin produced by fermentation reacts with NH ₄ ⁺ Synthesis of tetramethylpyrazine. The strain provided by the invention can effectively use raw materials with wide sources and low cost to produce high-concentration precursor substance acetoin, and the oxidized coenzyme NAD ⁺ can be effectively regenerated, and the yield and production efficiency of acetoin can be improved, thereby effectively reducing the tetramethyl value of acetoin. The production cost of pyrazine shortens the production cycle.

Description

A kind of method utilizing cheap raw material to produce tetramethylpyrazine

technical field

The invention belongs to the field of biochemical industry, in particular to a method for producing tetramethylpyrazine by utilizing cheap raw materials.

Background technique

Tetramethylpyrazine (2,3,5,6-tetramethylpyrazine), also known as ligustrazine, is the main active alkaloid component of the rhizome of traditional Chinese medicine Chuanxiong. It has good effects such as dilating blood vessels, improving microcirculation, inhibiting platelet adhesion and aggregation and thrombosis. Pharmacological effects have certain curative effects in the treatment of cardiovascular and cerebrovascular diseases, respiratory system diseases and glomerular diseases, and have been widely used in clinical practice as a standing drug. In addition, tetramethylpyrazine occurs naturally in dairy products, soy products, cocoa, coffee, peanuts, hazelnuts, and liquor, and has a characteristic roasted and nutty aroma. Tetramethylpyrazine is also an important aroma compound in Chinese liquor. A certain amount of tetramethylpyrazine has been detected in traditional Chinese liquor. It not only has an important contribution to the flavor of Chinese liquor, but also endows Chinese liquor with beneficial effects. healthy function. The threshold value of tetramethylpyrazine is extremely low, and the national standard GB2760-1996 stipulates that it is an edible flavor that is allowed to be used.

The production methods of tetramethylpyrazine can be divided into plant extraction method, chemical method and biosynthesis method. The extraction method can be used to extract tetramethylpyrazine from the rhizome of the traditional Chinese medicine Chuanxiong, but the plant source of Chuanxiong is limited, and the content of tetramethylpyrazine in the rhizome of Chuanxiong is low, so large-scale commercial production cannot be realized. The reaction conditions for the chemical synthesis of tetramethylpyrazine are generally severe, and the equipment requirements are relatively high. There are generally serious environmental problems, and the product does not belong to the category of "natural" or "biosynthesis", and safety and quality issues are being affected. constant questioning. The tetramethylpyrazine produced by the biochemical method has the advantages of green and natural products, low cost, mild reaction conditions, and low environmental pollution. With the increasing demand for green products, this method has received more and more attention. However, due to the relatively late start of the research on the production of acetonitrile by microbial fermentation and its conversion into tetramethylpyrazine, the technology of producing tetramethylpyrazine by biotechnology is far from mature.

Chinese patent 201010238685.5 discloses a method for producing tetramethylpyrazine and its production strains. The screened Bacillus subtilis XZ1124, Bacillus licheniformis BL-L1, MT-6 and MT-15 use glucose as carbon source and yeast extract as nitrogen The accumulation of acetylene in the fermenter was 30-44g/L, but the period was as long as 72h, and the accumulation of by-products caused the conversion rate of glucose to be too low. The fermentation broth was centrifuged and the supernatant was added to diammonium hydrogen phosphate with 2 times the molar concentration of acetoin, and the reaction was carried out at 55-90°C in a water bath shaker (rotation speed 150rpm) for 20h, and the concentration of tetramethylpyrazine was 11-20g/L. The process of the invention is relatively simple, but the strains used are wild-type strains, and various by-products will accumulate during the fermentation process, which reduces the conversion rate of the raw materials, thereby affecting the yield of tetramethylpyrazine. During the fermentation of acetoin by microorganisms, a large amount of reduced coenzyme NADH will be produced during the glycolysis process, while the production of acetoin and tetramethylpyrazine cannot consume NADH, so the microbial cells will start lactic acid, acetic acid, ethanol, butyl The synthesis pathway of by-products such as diacids consumes excess NADH and realizes the regeneration of oxidized coenzyme NAD+. The accumulation of these by-products not only reduces the conversion rate of raw materials and the yield of tetramethylpyrazine, but also inhibits the growth and fermentation of microorganisms. .

In order to improve the yield of acetoin and tetramethylpyrazine, it is necessary to optimize the intracellular NADH/NAD+ ratio by using coenzyme engineering strategies, such as expressing NADH oxidase to directly catalyze the conversion of NADH to NAD ⁺ , and using metabolic engineering techniques to prevent interrupt or weaken the synthetic pathway of major by-products. In addition, the present invention needs to use high-priced yeast paste as the 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 the method. The composition of the medium for microbial fermentation has a great impact on the production cost, and the use of high-purity sugar (glucose) will increase the cost of raw materials. In addition, yeast powder/yeast paste is often used as a nitrogen source for fermentative production of acetoin and tetramethylpyrazine because it is rich in amino acids, vitamins, trace elements and other nutrients, but its use usually greatly increases production costs, such as In some lactic fermentations, the cost of yeast powder can account for 30% of the total fermentation cost. Therefore, it is also necessary to develop and utilize cheap nitrogen sources (such as cottonseed meal) to replace high-priced nitrogen sources such as yeast powder/yeast extract when optimizing the fermentation process. In summary, the use of coenzyme engineering to optimize the intracellular redox state, the use of metabolic engineering to block the synthesis pathway of major by-products, and the development of inexpensive raw materials that can be efficiently utilized are extremely important for the industrialized production of tetramethylpyrazine.

SUMMARY OF THE INVENTION

Aiming at the shortcomings of excessive accumulation of by-products in the production of tetramethylpyrazine, low yield of tetramethylpyrazine, too long production cycle or too high cost of raw materials, and difficulty in industrialized production, the invention provides a method for producing tetramethylpyrazine by utilizing cheap raw materials. The method of methylpyrazine. The invention utilizes metabolic engineering and coenzyme regulation to carry out reasonable genetic modification on key metabolic nodes, weakens the main by-product synthesis pathway to strengthen the acetylene synthesis pathway, effectively regulates the intracellular redox state, and increases the yield of the precursor substance acetylene , to achieve the purpose of high-yield tetramethylpyrazine. The invention also provides the production of tetramethylpyrazine by using non-grain cassava flour and cheap nitrogen sources as fermentation raw materials, instead of high-purity sugar and high-priced nitrogen sources, which can greatly reduce the cost of raw materials.

In order to achieve the above purpose, the technical scheme adopted in the present invention is as follows:

A method of utilizing cheap raw materials to produce tetramethylpyrazine, comprising the following steps:

(1) carrying out codon optimization on the nucleotide sequences of α-acetolactate synthase gene, α-acetolactate decarboxylase gene and NADH oxidase gene;

(2) splicing the codon-optimized α-acetolactate synthase gene, α-acetolactate decarboxylase gene and NADH oxidase gene to obtain a gene cluster comprising the three genes;

(3) inserting the gene cluster into the expression vector to obtain a polycistronic recombinant plasmid;

(4) importing the polycistronic recombinant plasmid into host bacterium E.coli to obtain a genetically engineered strain producing acetoin;

(5) cassava flour, nitrogen source raw material are hydrolyzed to obtain fermentation medium, the activated genetically engineered strain for producing acetoin is inoculated into fermentation medium and fermented and cultivated to obtain a fermentation broth containing acetoin;

(6) Take the above fermentation broth for centrifugation, take the supernatant, add diammonium hydrogen phosphate, and react acetoin with NH ₄ ⁺ to synthesize tetramethylpyrazine.

Further, the specific steps of the codon optimization of the α-acetolactate synthase gene, the α-acetolactate decarboxylase gene and the NADH oxidase gene are to add a ribosome binding site and a spacer sequence in front of each gene. Nucleotide sequence TAAGGAGGATATACA.

In the cell, the ribosome is responsible for translating mRNA into protein. 16S rRNA is a subunit of the ribosome, which can be accurately identified with the ribosome binding site through the principle of base complementarity. Adding the TAAGGAGGATATAC sequence in front of the gene can enhance the ribosome The ability to recognize and bind to mRNA, and the appropriate spacer sequence can also improve the translation efficiency, at the same transcription level, improve the activity of α-acetolactate synthase, α-acetolactate decarboxylase and NADH oxidase, enzymatically The reaction is accelerated, and finally the synthesis capacity of acetoin is improved.

Further, the host bacterium E.coli is a mutant strain with multiple gene deletions, obtained by superimposing and knocking out the key genes for synthesizing by-products in the strain; the by-products include 2,3-butanediol, succinic acid, Lactic acid and acetate; key genes for the synthetic by-products include gldA, frdABCD, ldhA and pta.

Further, the source strains of the α-acetolactate synthase gene and the α-acetolactate decarboxylase gene are selected from Enterobacter cloacae, Klebsiella pneumoniae, Enterobacter aerogenes, Klebsiella oxytoca, Serratia marcescens and Bacillus licheniformis.

Further, the source strain of the NADH oxidase gene is selected from Lactobacillus brevis, Lactococcus lactis, Streptococcus pyogenes, Clostridium aminovalericum and Bacillus subtilis.

Further, the concrete operation steps of described step (5) are:

S1: Take tapioca flour, nitrogen source raw materials, add water, liquefaction enzyme and calcium chloride, liquefy at 80-100°C for 0.5-5h, let stand for cooling; when the temperature drops below 55°C, add saccharification enzyme and protease , saccharification at 50-60°C for 5-20h, adjust pH to 6.5-7.5, collect supernatant by centrifugation or filtration, dilute the supernatant and sterilize it at 115°C for 15min to obtain fermentation medium; based on 1000mL of water, The dosage of each component is: tapioca flour 100-200g, nitrogen source raw material 40-80g, liquefaction enzyme 0.5-2mL, calcium chloride 0.01-0.5g, saccharification enzyme 0.2-1mL, protease 0.05-0.5g; the tapioca flour It is prepared by peeling the cassava and drying it and then pulverizing it through a 50-200 mesh sieve. The nitrogen source raw material is one or more combinations of cottonseed protein powder, soybean meal powder and soybean meal powder;

S2: under aseptic conditions, add 50-100 μg/mL ampicillin to the fermentation medium, then move it to the fermenter, and use the activated acetoin-producing engineering bacteria liquid at a volume ratio of 1-10% The amount of inoculum was inoculated into the fermentation medium, and fermented for 20-72h under the conditions of temperature 35-42°C, stirring speed 200-800rpm, and ventilation rate 0.5-1.5vvm, when the concentration of acetoin in the fermentation broth was detected no longer increased. When the fermentation is completed, the fermentation broth containing acetoin is obtained.

Further, the preparation method of the activated acetoin-producing engineering bacteria bacterial liquid comprises the following steps: streaking the production acetoin genetic engineering strain to agar containing 1.8% mass-volume ratio and containing 100 μg/mL ampicillin cultured at 37°C for 10-15h; under sterile conditions, pick a single colony on the LB plate, then inoculate it into LB liquid seed medium containing 100 μg/mL ampicillin, shake at 37°C Bed shaking culture for 10-15h; the formula of the LB medium is: 10g/L peptone, 5g/L yeast powder, 10g/L sodium chloride.

Further, in the fermentation process of step S2, detect the concentration of glucose in the fermentation liquid, when the glucose concentration is reduced to 10-30g/L, add tapioca reducing sugar mother liquor so that the glucose concentration in the fermentation liquid is 40-80g/L; The glucose concentration of the cassava reducing sugar mother liquor is 500-800 g/L.

Further, the specific operation steps of the step (6) are: the fermentation broth containing acetoin is centrifuged at high speed, the precipitation is removed, the supernatant is taken to measure the content of acetoin, and then 1-2.5 times the mole of acetoin is added. The concentration of diammonium hydrogen phosphate, after dissolving, adjust the pH to 7.5, and react in a water bath shaker at 65-95 ° C for 6-20 hours to obtain tetramethylpyrazine.

Further, hydrogen peroxide with 0.1-2 times molar concentration of acetoin is added during the water bath shaking reaction process.

Compared with the prior art, the advantages and beneficial effects of the present invention are:

1, the bacterial strain provided by the invention can effectively utilize the raw material with a wide range of sources and low cost to produce the precursor substance acetoin, ^and the oxidized coenzyme NAD can be effectively regenerated, and the acetoin output and production efficiency can be improved, thereby effectively reducing the tetramethyl acetoin The production cost of pyrazine is shortened, the production cycle is shortened, and the problems existing in the prior art are overcome.

2. The construction method of the strain of the present invention has the advantages of simple operation, high production efficiency, low cost and the like, and is easy to realize industrial production.

3. The present invention uses metabolic engineering technology to knock out the key genes for synthesizing main by-products in the host bacteria, and expresses NADH oxidase to effectively regulate the intracellular redox state, improve the yield of the precursor substance acetylene, and finally increase the tetramethylpyridine oxazine yield.

4. The present invention utilizes non-grain cassava flour as carbon source, cottonseed protein powder, soybean meal powder or soybean meal powder as nitrogen source, and replaces the high-purity sugar (such as glucose) and yeast powder that have been reported to be used for fermentation to produce precursor substance B. Atoin and tetramethylpyrazine can not only improve the comprehensive utilization value of these cheap raw materials, but also provide cheaper raw materials for the production of tetramethylpyrazine, which can greatly reduce the cost of raw materials.

Detailed ways

The present invention will be further described in detail below with reference to the embodiments, but is not limited to the protection scope of the present invention.

Materials involved in the following examples: liquefaction enzyme, saccharification enzyme, and protease are commercial products, which can be purchased from companies such as Novozymes (China) Biotechnology Co., Ltd.; cottonseed protein powder, soybean meal powder, and soybean meal powder are commercial products. Products can be purchased from Qingdao Kerui Culture Medium Co., Ltd. and other companies.

In the following embodiment, in the process of producing tetramethylpyrazine, adopt SBA-40C analyzer to detect glucose concentration in the fermentation broth, utilize liquid chromatography to measure the concentration of main by-products, utilize gas chromatography to measure acetoin, tetramethylpyridine azine concentration.

1. Construction of genetically engineered strains producing acetoin

Example 1

Construction of the genetically engineered strain ECTMP1 to produce the precursor substance acetoin:

The nucleotide sequences of the α-acetolactate synthase gene budB from Enterobacter cloacae, the α-acetolactate decarboxylase gene budA and the NADH oxidase gene noxE from Lactobacillus brevis were codon-optimized and added in front of each gene The nucleotide sequence TAAGGAGGATATACA containing the ribosome binding site was obtained, and then the gene cluster budB-budA-noxE was obtained by artificial synthesis, and the nucleotide sequence length was 3849 bases, and the nucleotide sequence was as shown in SEQ ID NO.1 said. Using the method of double enzyme digestion and ligation, the gene cluster budB-budA-noxE was inserted behind the promoter of the plasmid pTrc99A to obtain a 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 an acetoin-producing genetically engineered strain ECTMP1.

Example 2

Construction of the genetically engineered strain ECTMP2 for the production of precursor acetoin:

Through analysis, it was found that the main by-products of the fermentation of the engineered strain ECTMP1 were 2,3-butanediol, succinic acid, lactic acid, and acetic acid, and the key genes of its synthetic pathway were gldA, frdABCD, ldhA and pta. Using the principle that the E. coli phage-derived Red recombination system can efficiently mediate homologous recombination events in bacteria, the above target genes were first replaced with antibiotic resistance genes with FRT sites on both sides, and then exogenous thermosensitive Plasmid expresses FLP recombinase to delete antibiotic resistance gene to achieve the purpose of knocking out the target gene. The specific steps are as follows:

The pKD46 plasmid was transformed into host cells to prepare electrotransformation competent cells; the target sequence (containing chloramphenicol resistance gene) was constructed by PCR using primers, and it was directly transformed into host cells containing pKD46; chloramphenicol plate screening occurred Cloning of homologous recombination; use sequencing technology to verify and select clones whose target gene is replaced by chloramphenicol resistance gene to prepare electrotransformation competent cells; electrotransform into pCP20 plasmid to delete chloramphenicol resistance gene; chloramphenicol resistance gene The deleted clones were serially streaked three times, and glycerol tubes were prepared and stored at -20°C. By stacking knockout, a mutant strain E.coli MG1655/ΔgldAΔfrdABCDΔldhAΔpta with deletion of multiple genes can be obtained, and electrotransformation competent cells are prepared. The polycistronic recombinant plasmid pTrc99A-budB-budA-noxE constructed in Example 1 was electroporated into a multi-gene deletion mutant strain to obtain an engineering strain ECTMP2.

Table 1 Nucleotide sequence list of each primer

Example 3

Construction of genetically engineered strain ECTMP3 to produce precursor substance acetoin:

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

Example 4

Construction of the genetically engineered strain ECTMP4 to produce the precursor substance acetoin:

The difference between this example and Example 2 is that the polycistronic recombinant plasmid constructed in Example 3 was introduced into a multi-gene deletion mutant strain to obtain an engineering strain ECTMP4.

Example 5

Construction of genetically engineered strain ECTMP5 producing precursor substance acetoin:

The difference between this example and Example 1 is that the source strain of α-acetolactate synthase gene and α-acetolactate decarboxylase gene is Serratia marcescens, and the source strain of NADH oxidase gene is Streptococcuspyogenes.

Example 6

Construction of genetically engineered strain ECTMP6 to produce precursor substance acetoin:

The difference between this example and Example 2 is that the polycistronic recombinant plasmid constructed in Example 5 was introduced into a multi-gene deletion mutant strain to obtain an engineering strain ECTMP6.

Example 7

Construction of the genetically engineered strain ECTMP7 that produces the precursor substance acetoin:

The difference between this example and Example 1 is: the source strain of α-acetolactate synthase gene and α-acetolactate decarboxylase gene is Enterobacter aerogenes, the source strain of NADH oxidase gene is Bacillus subtilis, and the host bacteria is E. coli BW25113.

Example 8

Construction of the genetically engineered strain ECTMP8 to produce the precursor substance acetoin:

The difference between this example and Example 1 is: the source strain of α-acetolactate synthase gene and α-acetolactate decarboxylase gene is Klebsiella oxytoca, the source strain of NADH oxidase gene is Lactobacillus rhamnosus, and the host bacteria is E. coli BW25113.

Example 9

Construction of genetically engineered strain ECTMP9 to produce precursor substance acetoin:

The difference between this example and Example 1 is that the source strain of α-acetolactate synthase gene and α-acetolactate decarboxylase gene is Bacillus licheniformis, and the source strain of NADH oxidase gene is Clostridium aminovalericum.

The methods for synthesizing gene clusters in Examples 3-9 are the same as those in Example 1, or are conventional methods. Those skilled in the art can learn the nucleotide sequences of the gene clusters through the disclosure of the present application, and there is no need to worry about this. The above-mentioned Examples 3-10 correspond to the nucleotide sequences of the synthesized gene clusters.

2. Application of engineered strains in the production of precursor substance acetoin

Application Example 1

The application of engineering strains ECTMP1, ECTMP2, ECTMP3, ECTMP4, ECTMP5, ECTMP6, ECTMP7, ECTMP8, and ECTMP9 in the production of precursor substance acetoin includes the following steps:

(1) After the cassava is peeled and sun-dried, pulverized to a particle size of 100 mesh cassava flour; 9 parts of 180 g of cassava flour were weighed and added to the beaker respectively, and each part was added with 60 g of cottonseed protein powder, 1000 mL of tap water, 0.5 mL of Liquefaction enzyme and 0.2g calcium chloride, shake well, liquefy at 95°C for 1.5h, stand to cool; when the temperature drops below 55°C, add 0.5mL saccharification enzyme and 0.1g protease, saccharify at 55°C for 20h, adjust pH 7.5, collect the supernatant by centrifugation, dilute the supernatant with sterile tap water to make the reducing sugar concentration 100g/L, and sterilize at 115°C for 15min to obtain the fermentation medium;

(2) Add 100 μg/mL ampicillin to the fermentation medium, and transfer 500 mL of them to Shanghai Bailun Liulian Fermentation Tank. Under sterile conditions, take the activated ECTMP1, ECTMP2, ECTMP3, ECTMP4, ECTMP5, ECTMP6, ECTMP7, ECTMP8, and ECTMP9 engineering bacteria were inoculated into the above fermentation medium with a volume ratio of 5%, respectively, and then fermented and cultured at a temperature of 37 ° C, a stirring speed of 300 rpm, and a ventilation volume of 1 vvm, and fermented for 16h and 28h. Supplementation with 50 g/L of sugar was carried out, and the fermentation was terminated when the concentration of acetoin no longer increased to obtain the fermentation broth of acetoin. The samples were taken and the concentrations of the product acetoin and the by-products butanediol, succinic acid, lactic acid and acetic acid were determined by gas chromatography and liquid chromatography. The results are shown in Table 2.

The activation method of the engineering bacteria liquid: the ECTMP1, ECTMP2, ECTMP3, ECTMP4, ECTMP5, ECTMP6, ECTMP7, ECTMP8, ECTMP9 engineering strains that produce acetoin are respectively streaked to those containing 1.8% agar in a mass-to-volume ratio and containing On the LB plate containing 100 μg/mL ampicillin, cultivate at 37°C for 12 hours; pick a single colony on the LB plate with a toothpick, then inoculate it into LB liquid medium containing 100 μg/mL ampicillin, and cultivate at 37°C with shaking for 10 hours. ; The LB culture medium formula is: 10g/L peptone, 5g/L yeast powder, 10g/L sodium chloride.

Table 2 Fermentation products of different engineered strains

Engineered strains Acetoin (g/L) Butanediol (g/L) Succinic acid(g/L) Lactic acid (g/L) Acetic acid (g/L) ECTMP1 60.5 2.8 3.5 2.7 3.6 ECTMP2 71.2 0.4 0.1 0.4 0.3 ECTMP3 52.3 3.8 3.3 3.1 3.9 ECTMP4 63.1 0.5 0 0.3 0.5 ECTMP5 45.2 3.5 3.9 2.7 3.4 ECTMP6 53.3 0.5 0.2 0.6 0.2 ECTMP7 48.2 3.5 2.8 2.6 3.7 ECTMP8 51.8 3.6 3.2 2.2 2.8 ECTMP9 46.3 3.2 3.5 2.5 2.9

As can be seen from Table 2, the key genes gldA, frdABCD, ldhA and pta of the by-product synthesis pathway were knocked out, and the production of acetoin, the precursor substance of the engineered strains ECTMP2, ECTMP4 and ECTMP6, was different from that of the engineered strains without gene knockout. (corresponding to ECTMP1, ECTMP3, and ECTMP5, respectively) were significantly increased, and the concentrations of the by-products of acetoin, 2,3-butanediol, succinic acid, lactic acid, and acetic acid, were greatly reduced, thus knocking out the key to the synthesis pathway of by-products The gene increases the production of the precursor substance acetoin, and can reduce the production cost of tetramethylpyrazine.

Application Example 2

The application of engineering strain ECTMP2 in the production of acetoin includes the following steps:

(1) after the cassava is peeled and dried, pulverized to a particle size of 100 purpose cassava flour; take three parts of 200 g of cassava flour, add in the beaker respectively, then add 70g soybean meal powder, 70g soybean meal powder, 70g cottonseed protein powder respectively, Then add 1000mL of tap water, 1.2mL of liquefaction enzyme and 0.5g of calcium chloride, shake well, liquefy at 90°C for 2h, and let stand to cool; when the temperature drops below 50°C, add 0.3mL of saccharification enzyme and 0.2g of protease, and at 55 Saccharification at ℃ for 24h, adjusting pH to 6.5, collecting the supernatant by centrifugation, diluting the supernatant with sterile tap water to make the reducing sugar concentration 100g/L, and sterilizing at 115 ℃ for 15min to obtain the fermentation medium;

(2) In the fermentation medium, 100 μg/mL ampicillin was taken, and 500 mL was added to the Shanghai Bailun Liulian fermenter. Under aseptic conditions, the activated ECTMP2 engineering bacteria liquid was taken with a volume ratio of 10% of the inoculum. Inoculated into the above fermentation medium, fermented and cultured at a temperature of 40 ° C, a stirring speed of 600 rpm, and a ventilation volume of 1.0 vvm, fermented for 12 h and 24 h, respectively, supplemented with 40 g/L glucose, and fermented when the concentration of acetoin in the fermentation broth no longer increased. At the end, a fermentation broth containing acetoin was obtained. The yields of acetoin corresponding to soybean meal meal, soybean meal meal, and cottonseed protein meal were determined to be 59.3 g/L, 63.8 g/L, and 73.3 g/L, respectively.

The activation method of the ECTMP2 engineering bacteria solution: streak the ECTMP2 engineering strains on LB plates containing 1.8% agar in a mass-to-volume ratio and containing 100 μg/mL ampicillin, and cultivate at 37° C. for 15 hours; under sterile conditions Then, pick a single colony on the LB plate with a toothpick, then inoculate it into the LB liquid medium containing 100 μg/mL ampicillin, and cultivate at 37°C with shaking for 12 hours; the LB medium formula is: 10g/L Peptone, 5g/L yeast powder, 10g/L sodium chloride.

Application Example 3

The application of engineering strain ECTMP4 in the production of acetoin includes the following steps:

(1) After peeling and drying the cassava, pulverize it to a particle size of 150 mesh cassava flour; weigh 185 g of cassava flour, add it to a beaker, add 25 g of cottonseed protein powder and 15 g of soybean meal powder, and then add 1000 mL of tap water, 0.8 mL of liquefied water Enzyme and 0.3g calcium chloride, shake well, liquefy at 95°C for 2h, let stand to cool; when the temperature drops below 55°C, add 0.8mL saccharification enzyme and 0.3g protease, saccharify at 55°C for 18h, adjust pH 7.0, The supernatant was collected by centrifugation, diluted with sterile tap water to make the reducing sugar concentration 100g/L, and sterilized at 115°C for 15min to obtain the fermentation medium;

(2) Add 100 μg/mL ampicillin to the fermentation medium, take 500 mL and transfer it to Shanghai Bailun Liulian fermenter, under aseptic conditions, take the activated ECTMP4 engineering bacteria liquid with a volume ratio of 8% of the inoculum Inoculated into the above fermentation medium, fermented at a temperature of 37 °C, a stirring speed of 500 rpm, and a ventilation volume of 0.7 vvm, supplemented with 50 g/L of glucose for 15 h and 28 h, respectively, and fermented for 45 h to obtain a fermentation broth containing acetoin. The yield of acetoin was determined to be 67.2 g/L.

The activation method of the ECTMP4 engineering bacteria liquid: streak the ECTMP4 engineering strains on LB plates containing 1.8% agar with a mass-to-volume ratio and containing 100 μg/mL ampicillin, and cultivate at 37° C. for 10 hours; under sterile conditions Then, pick a single colony on the LB plate with a toothpick, then inoculate it into the LB liquid medium containing 100 μg/mL ampicillin, and cultivate at 37°C with shaking for 15 hours; the LB medium formula is: 10g/L Peptone, 5g/L yeast powder, 10g/L sodium chloride.

Application Example 4

The application of engineering strain ECTMP5 in the production of acetoin includes the following steps:

(1) After the cassava is peeled and sun-dried, pulverized to a particle size of 50 mesh cassava flour; weigh 100 g of cassava flour, add it to a beaker, add 30 g of cottonseed protein powder and 30 g of soybean meal powder, and then add 1000 mL of tap water and 2.0 mL of liquefaction enzyme and 0.01g calcium chloride, shake well, liquefy at 80°C for 5h, let stand to cool; when the temperature drops below 55°C, add 0.2mL saccharification enzyme and 0.05g protease, saccharify at 60°C for 5h, adjust pH 7.0, centrifuge Collect the supernatant, dilute the supernatant with sterile tap water to make the reducing sugar concentration 100g/L, and sterilize at 115°C for 15min to obtain the fermentation medium;

(2) Add 50 μg/mL ampicillin to the fermentation medium, take 500 mL and transfer it to Shanghai Bailun Liulian fermenter, under aseptic conditions, take the activated ECTMP5 engineering bacteria liquid with a volume ratio of 1% of the inoculum Inoculated into the above fermentation medium, fermented and cultured for 16 hours at a temperature of 42°C, agitation speed of 300rpm, and aeration of 1.3vvm, and then supplemented with 40g/L of sugar. After 32h of fermentation, the same amount of tapioca reducing sugar mother liquor was added again. A fermentation broth containing acetoin was obtained. The yield of acetoin was determined to be 48.3 g/L.

The activation method of the ECTMP5 engineering bacteria liquid: streak the ECTMP5 engineering strains on LB plates containing 1.8% agar with a mass-to-volume ratio and containing 100 μg/mL ampicillin, and cultivate at 37° C. for 12 hours; under sterile conditions Then, pick a single colony on the LB plate with a toothpick, then inoculate it into the LB liquid medium containing 100 μg/mL ampicillin, and cultivate at 37°C with shaking for 12 hours; the LB medium formula is: 10g/L Peptone, 5g/L yeast powder, 10g/L sodium chloride.

Application Example 5

The application of engineering strain ECTMP6 in the production of acetoin includes the following steps:

(1) after the cassava is peeled and dried, pulverized to a particle size of 200 mesh cassava flour; weigh 155 g of cassava flour, add it to a beaker, add 40 g of soybean meal powder, 20 g of cottonseed protein powder and 20 g of soybean meal powder, and then add 1000 mL of tap water, 1.5mL of liquefaction enzyme and 0.5g of calcium chloride, shake well, liquefy at 100°C for 1h, and let stand to cool; when the temperature drops below 55°C, add 1mL of saccharification enzyme and 0.5g of protease, saccharify at 50°C for 20h, and adjust the pH 7.0, collect the supernatant by centrifugation, dilute the supernatant with sterile tap water to make the reducing sugar concentration 100g/L, and sterilize at 115°C for 15min to obtain the fermentation medium;

(2) Add 80 μg/mL ampicillin to the fermentation medium, take 500 mL and transfer it to Shanghai Bailun Liulian fermenter, under aseptic conditions, take the activated ECTMP7 engineering bacteria liquid with a volume ratio of 5% of the inoculum Inoculated into the above fermentation medium, fermented and cultured for 14h under the conditions of temperature 37°C, stirring speed of 700rpm, and aeration of 0.5vvm, supplemented with 50g/L of sugar, and added 30g/L of cassava reducing sugar mother liquor after fermentation for 28h and 45h, respectively, and fermented. After 60h, the fermentation broth containing acetoin was obtained. The yield of acetoin was determined to be 68.5g/L.

The activation method of the ECTMP6 engineering bacteria liquid: streak the ECTMP6 engineering strains on LB plates containing 1.8% agar with a mass-to-volume ratio and containing 100 μg/mL ampicillin, and cultivate at 37° C. for 10 hours; under sterile conditions Then, pick a single colony on the LB plate with a toothpick, then inoculate it into the LB liquid medium containing 100 μg/mL ampicillin, and cultivate at 37°C with shaking for 15 hours; the LB medium formula is: 10g/L Peptone, 5g/L yeast powder, 10g/L sodium chloride.

Application Example 6

The application of engineering strain ECTMP7 in the production of acetoin includes the following steps:

(1) After peeling and drying the cassava, pulverize it to a particle size of 100 mesh cassava flour; weigh 150 g of cassava flour, add it to a beaker, add 40 g of cottonseed protein powder and 30 g of soybean meal powder, and then add 1000 mL of tap water and 1.0 mL of liquefied Enzyme and 0.2g calcium chloride, shake well, liquefy at 90°C for 3h, and let stand to cool; when the temperature drops below 55°C, add 0.5mL saccharification enzyme and 0.25g protease, saccharify at 55°C for 12h, adjust pH 7.0, The supernatant was collected by centrifugation, diluted with sterile tap water to make the reducing sugar concentration 100g/L, and sterilized at 115°C for 15min to obtain the fermentation medium;

(2) Add 100 μg/mL ampicillin to the fermentation medium, take 500 mL and transfer it to Shanghai Bailun Liulian Fermentation Tank, under aseptic conditions, take the activated ECTMP7 engineering bacteria liquid with a volume ratio of 8% of the inoculum Inoculated into the above fermentation medium, after fermenting and culturing for 16 hours at a temperature of 38 °C, a stirring speed of 500 rpm, and a ventilation volume of 1.2 vvm, the glucose concentration in the fermentation broth dropped to 20.3 g/L, and the sugar supplemented 40 g/L, and the fermentation was performed for 32 h and 46 h. Then, the same amount of cassava reducing sugar mother liquor was added again, and after 60h fermentation, the fermentation broth containing acetoin was obtained. The yield of acetoin was determined to be 60.3 g/L.

The activation method of the ECTMP7 engineering bacteria liquid: streak the ECTMP7 engineering strains on LB plates containing 1.8% agar in a mass-to-volume ratio and containing 100 μg/mL ampicillin, and cultivate at 37° C. for 15 hours; under sterile conditions Then, pick a single colony on the LB plate with a toothpick, then inoculate it into the LB liquid medium containing 100 μg/mL ampicillin, and cultivate at 37°C with shaking for 10 hours; the LB medium formula is: 10 g/L Peptone, 5g/L yeast powder, 10g/L sodium chloride.

3. Synthesis of Tetramethylpyrazine by Reaction of Precursor Acetoin with NH ₄ ⁺

Application Example 7

The effect of diammonium hydrogen phosphate concentration on the conversion of acetoin to tetramethylpyrazine:

Get the fermentation broth containing acetoin in Application Example 3, centrifuge at 8000rpm for 10min, remove the precipitation, get the supernatant and measure the concentration of acetoin to be 67.2g/L, respectively take 100mL and add in 3 beakers, then add ethyl acetate respectively. Atoin 1, 2.0, and 2.5 times molar concentration of diammonium hydrogen phosphate, after dissolving, adjust the pH to 7.5, add 20 mL of each into a 100 mL conical flask, and react at 65 ° C and 150 rpm in a water bath shaker for 20 hours. NH ₄ ⁺ reaction to synthesize tetramethylpyrazine. The yields of tetramethylpyrazine corresponding to different concentrations of diammonium hydrogen phosphate were determined to be 12.9 g/L, 17.9 g/L and 18.3 g/L, respectively.

Application Example 8

The effect of adding hydrogen peroxide on the conversion of acetoin to tetramethylpyrazine:

Take the fermentation broth containing acetoin in Application Example 5, centrifuge at 8000rpm for 10min, remove the precipitation, take the supernatant to measure the concentration of acetoin to be 68.5g/L, get 400mL and add it to the beaker, then add 2.0 times of acetoin Molar concentration of diammonium hydrogen phosphate, after dissolving, adjust the pH to 7.5, respectively take six 20mL solutions into 100mL conical flasks, react in a water bath shaker at 90°C and 100rpm, and add acetoin after 3 hours of reaction. Molar concentration of 0, 0.1, 0.5, 1.0, 1.5, 2.0 times hydrogen peroxide, and then react for 3h, acetoin and NH ₄ ⁺ react to synthesize tetramethylpyrazine. The corresponding tetramethylpyrazine yields were determined to be 18.7, 20.8, 22.6, 25.3, 23.7, and 21.9 g/L.

SEQUENCE LISTING

<110> Nanning Zhongnuo Biological Engineering Co., Ltd., Guangxi Academy of Sciences

<120> Construction method and application of genetically engineered strain for producing (R)-acetoin

<130> 3849

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 3849

<212> DNA

<213> Synthetic

<400> 1

taaggaggat atacatatga actcggagaa acagtcgcgc caatgggcgc atggcgccga 60

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gaaaattgac aaggtgtttg actcgctgct ggatagcagc attgagatca ttcctgtgcg 180

ccatgaggcg aacgccgcct ttatggccgc cgcggttggt cgtctgaccg gtaaggccgg 240

cgtggcctta gtgaccagcg gtcctggttg ttctaatctg attaccggca ttgcgaccgc 300

gaactcggag ggcgatcctg tggtggcctt aggcggtgcg gttaagcgcg cggataaagc 360

caaactggtg caccagagca tggataccgt ggcgatgttt agcccggtga cgaagtacgc 420

cgttgaagtt agctcgccgg acgcgattgc ggaggtggtt agcaacgcgt ttcgtgccgc 480

ggaacatggc cgtcctggtg gcgcgttcgt ttcgctgccg caggacattg ttgatcagcc 540

tgcgaccggt gcgatcttac cggccagcgg tcctgccctg atgggtccgg cccctgaaag 600

cgcgatcaat gatgttgcga aattaattga caatgcgaaa aacccggtga ttctgctggg 660

cttaatggcc tcgcagcctg ccaatagcgc ggcgttacgc aaactgctgg agaagagccg 720

tattccggtg acttctactt accaagccgc cggcgcggtg aatcaggaac actttacccg 780

ctttgccggt cgcgtgggtc tgttcaacaa tcaagcgggt gaccgcctgt tacacctggc 840

ggatctgatc atttgcattg gctacagccc ggttgaatac gaaccgagca tgtggaacag 900

cggcgatgcc acgctggtgc atatcgatgt tctgcctgcg tacgaggaac gcaattatgt 960

gccggatatc gagctggtgg gtgacattgc cgcgaccctg aatttactgg cctcgcgtat 1020

tgaccacaag ctggaactga gccaacgcgc gtcggagatt ctggtggacc gccaacatca 1080

gcgcgattta ttagaccgcc gtggtgcgtc gctgaaccag tttgcgctgc atcctctgcg 1140

cattgttcgc gcgatgcagg atatcgtgaa caacgatgtg accctgaccg tggacatggg 1200

cagcttccat atctggatcg cgcgttatct gtacagcttc cgcgcccgtc aggtgatgat 1260

cagcaacggc cagcagacta tgggtgttgc cttaccgtgg gcgatcggcg cgtggctggt 1320

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ggagctggaa acggccgtgc gcctgaacgc caacgtgtta catattattt gggtggataa 1440

cggctataac atggtggcca tccaagagga gaagaagtat cagcgcctga gcggtgttgc 1500

gtttggcccg gtggatttca aggcctatgc ggatgccttt ggcgcccgtg gcttcgccgt 1560

ggaaagcgcc gatgcgttag aaagcaccct gcgcgcggct atggatgtta atggtccggc 1620

ggttgttgcg attccggtgg attacagcga taacccgctg ctgatgggcc agctgcatct 1680

gtcgcagatt ctgtaataag gaggatatac atatgatgca cagctcggcg tgcgattgtg 1740

aagcgtcgct gtgcgaaacc ctgcgcggct ttagcgccaa acatccggac agcgttatct 1800

accagacctc gctgatgagc gcgctgctgt cgggcgttta cgaaggcgac accacgatcg 1860

ccgatctgct ggctcatggt gatttcggtc tgggcacctt caacgaactg gacggcgaaa 1920

tgattgcctt ttcttctcag gtgtaccagt tacgcgccga cggcagcgcg cgcgcggcca 1980

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cgtttgatgc cccggtttcg cgtcagcaga ttcatgacgt gatcgatcag cagattccga 2100

gcgacaacct gttctgcgcg ctgcgcattg acggcaattt tcgccacgcc cacacccgca 2160

cggttcctcg ccaaacgccg ccgtaccgtg ccatgaccga tgtgctggac gaccagccgg 2220

tgtttcgttt taaccaacgc gaaggcgtgc tggttggctt ccgcacgccg cagcacatgc 2280

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tgatgatcga cctgccggcc gacagcgcct tcttacaagc gaatctgcac ccgagcaacc 2460

tggacgccgc gattcgttcg gtggaaaatt aataaggagg atatacatat gaaaatcgtt 2520

gttatcggta ccaaccacgc tggtatcgct accgctaaca ccctgctgga acagtacccg 2580

ggtcacgaaa tcgttatgat cgaccgtaac tctaacatgt cttacctggg ttgcggtacc 2640

gctatctggg ttggtcgtca gatcgaaaaa ccggacgaac tgttctacgc taaagctgaa 2700

gacttcgaag ctaaaggtgt taaaatcctg accgaaaccg aagtttctga aatcgacttc 2760

gctaacaaaa aagtttacgc taaaaccaaa tctgacgacg aaatcatcga agcttacgac 2820

aaactggttc tggctaccgg ttctcgtccg atcatcccga acctgccggg taaagacctg 2880

aaaggtatcc acttcctgaa actgttccag gaaggtcagg ctatcgacgc tgaattcgct 2940

aaagaaaaag ttaaacgtat cgctgttatc ggtgctggtt acatcggtac cgaaatcgct 3000

gaagctgcta aacgtcgtgg taaagaagtt ctgctgttcg acgctgaaaa cacctctctg 3060

gcttcttact acgacgaaga attcgctaaa ggtatggacg aaaacctggc tcagcacggt 3120

atcgaactgc acttcggtga actggctaaa gaattcaaag ctaacgaaga aggttacgtt 3180

tctcagatcg ttaccaacaa agctacctac gacgttgacc tggttatcaa ctgcatcggt 3240

ttcaccgcta actctgctct ggcttctgac aaactggcta ccttcaaaaa cggtgctatc 3300

aaagttgaca aacaccagca gtcttctgac ccggacgttt acgctgttgg tgacgttgct 3360

accatctact ctaacgctct gcaggacttc acctacatcg ctctggcttc taacgctgtt 3420

cgttctggta tcgttgctgg tcacaacatc ggtggtaaag aactggaatc tgttggtgtt 3480

cagggttcta acggtatctc tatcttcggt tacaacatga cctctaccgg tctgtctgtt 3540

aaagctgcta aaaaactggg tctggaagtt tctttctctg acttcgaaga caaacagaaa 3600

gcttggttcc tgcacgaaaa caacgactct gttaaaatcc gtatcgttta cgaaaccaaa 3660

tctcgtcgta tcatcggtgc tcagctggct tctaaatctg aaatcatcgc tggtaacatc 3720

aacatgttct ctctggctat ccaggaaaaa aaaaccatcg acgaactggc tctgctggac 3780

ctgttcttcc tgccgcactt caactctccg tacaactaca tgaccgttgc tgctctgaac 3840

gctaaataa 3849

Claims (6)

1. a method utilizing cheap raw material to produce tetramethylpyrazine, is characterized in that: may further comprise the steps: (1) Codon optimization of the nucleotide sequences of α-acetolactate synthase gene, α-acetolactate decarboxylase gene and NADH oxidase gene; (2) splicing the codon-optimized α-acetolactate synthase gene, α-acetolactate decarboxylase gene and NADH oxidase gene to obtain a gene cluster comprising the three genes; the nucleotide sequence of the gene cluster As shown in SEQ ID NO.1; (3) Insert the gene cluster into the expression vector to obtain a polycistronic recombinant plasmid; (4) The polycistronic recombinant plasmid is introduced into the host strain E. coli to obtain a genetically engineered strain producing acetoin; the host strain E. coli is a mutant strain with multiple gene deletions, and the synthetic paraffin is synthesized by stacking knockout strains. The key genes of the product are obtained; the by-products include 2,3-butanediol, succinic acid, lactic acid and acetic acid; the key genes of the synthetic by-products include gldA , frdABCD , ldhA and pta ; (5) hydrolyzing the tapioca flour and nitrogen source raw materials to obtain a fermentation medium, inoculating the activated genetically engineered strain for producing acetoin into the fermentation medium for fermentation culture, and obtaining a fermentation broth containing acetoin; (6) Take the above fermentation broth for centrifugation, take the supernatant, add diammonium hydrogen phosphate, and under heating conditions, acetoin reacts with NH ₄ ⁺ to synthesize tetramethylpyrazine. 2. the method for utilizing cheap raw material to produce tetramethylpyrazine according to claim 1 is characterized in that: the concrete operation steps of described step (5) are: S1: Take tapioca flour, nitrogen source raw materials, add water, liquefaction enzyme and calcium chloride, liquefy at 80-100 °C for 0.5-5 h, and let stand for cooling; when the temperature drops below 55 °C, add saccharification enzyme and calcium chloride. Protease, saccharify at 50-60 °C for 5-20 h, adjust pH to 6.5-7.5, collect supernatant by centrifugation or filtration, dilute the supernatant and sterilize it at 115 °C for 15 min to obtain fermentation medium; press 1000 In terms of mL water, the dosage of each component is: tapioca flour 100-200 g, nitrogen source raw material 40-80 g, liquefaction enzyme 0.5-2 mL, calcium chloride 0.01-0.5 g, saccharification enzyme 0.2-1 mL, protease 0.05~ 0.5 g; the cassava flour is prepared by peeling the cassava and drying it and passing through a 50-200 mesh sieve, and the nitrogen source raw material is one or more of cottonseed protein powder, soybean meal powder, and soybean meal powder combination; S2: Under aseptic conditions, add 50-100 μg/mL ampicillin to the fermentation medium, then move it to the fermenter, and add the activated acetoin-producing engineering bacteria liquid at a volume ratio of 1-10 % of the inoculum was inoculated into the fermentation medium, and fermented for 20-72 h under the conditions of a temperature of 35-42 °C, a stirring speed of 200-800 rpm, and a ventilation rate of 0.5-1.5 vvm. When the concentration of acetoin in the fermentation broth was detected When the increase is no longer, the fermentation is completed, and the fermentation broth containing acetoin is obtained. 3. the method for utilizing cheap raw material to produce tetramethylpyrazine according to claim 2, is characterized in that: the preparation method of the engineering bacteria bacterial liquid of described activated production acetoin, comprises the following steps: The genetically engineered strains producing acetoin were streaked onto LB plates containing 1.8% mass-volume agar and 100 μg/mL ampicillin, and cultured at 37°C for 10-15 h; A single colony on the LB plate is then inoculated into the LB liquid seed medium containing 100 μg/mL ampicillin, and shaken at 37°C for 10-15 h; the formula of the LB medium is: 10 g/L Peptone, 5 g/L yeast powder, 10 g/L sodium chloride. 4. the method for utilizing cheap raw material to produce tetramethylpyrazine according to claim 2 is characterized in that: in the fermentation process of step S2, detect the concentration of glucose in the fermentation broth, when the glucose concentration is reduced to 10-30 g/L When the cassava reducing sugar mother liquor is added, the glucose concentration in the fermentation broth is 40-80 g/L; the glucose concentration of the cassava reducing sugar mother liquor is 500-800 g/L. 5. The method for producing tetramethylpyrazine using cheap raw materials according to claim 2, wherein the specific operation steps of the step (6) are: centrifuging the fermentation broth containing acetoin, removing the precipitation, taking Measure the content of acetoin in the supernatant, then add diammonium hydrogen phosphate with 1-2.5 times molar concentration of acetoin, after dissolving, adjust the pH to 7.5, react in a water bath shaker at 65-95 °C for 6-20 h, That is, tetramethylpyrazine. 6. the method for utilizing cheap raw material to produce tetramethylpyrazine according to claim 5 is characterized in that: the hydrogen peroxide of 0.1-2 times molar concentration of acetoin is added in the water bath shaking table reaction process.