CN112011474B

CN112011474B - Genetically engineered bacterium for low-yield n-propanol and application thereof

Info

Publication number: CN112011474B
Application number: CN202010933724.7A
Authority: CN
Inventors: 肖冬光; 王亚平; 张翠英; 陈叶福; 杜丽平; 郭学武
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2020-09-08
Filing date: 2020-09-08
Publication date: 2023-07-18
Anticipated expiration: 2040-09-08
Also published as: CN112011474A

Abstract

The invention provides a genetically engineered bacterium for low-yield n-propanol, which is obtained by deleting all homoserine dehydrogenase genes THR6 from saccharomyces cerevisiae. Wherein the homoserine dehydrogenase encoded by the THR6 gene is capable of catalyzing a dimeric enzyme of the third step of the threonine biosynthetic pathway, which plays a key role in the synthetic pathway of threonine in Saccharomyces cerevisiae. Threonine can be converted to 2-ketobutyric acid. 2-ketobutyric acid is an essential precursor for synthesizing n-propanol by Saccharomyces cerevisiae, and directly affects the yield of n-propanol. The genetically engineered bacterium disclosed by the invention is applied to white spirit production by a liquid fermentation method.

Description

Genetically engineered bacterium for low-yield n-propanol and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, relates to breeding of industrial microorganisms, and in particular relates to saccharomyces cerevisiae genetically engineered bacteria for low-yield n-propanol and application thereof.

Background

The flavor substances in the white spirit mainly comprise higher alcohols, esters, aldehydes and the like. Among them, higher alcohols are one of important chemical substances for forming flavor and mouthfeel of white spirits. The proper higher alcohol content has the functions of ensuring that the taste and the aroma of the white spirit are plump and the spirit body is soft and coordinated, but excessive higher alcohol is a main source of the foreign flavor of the white spirit and is also an important reason for leading the white spirit to go up after drinking. As one of important components in the higher alcohol, the normal propyl alcohol has a great influence on the style of the white wine, and a proper amount of normal propyl alcohol can bring wine fragrance and sweetness to the white wine body, and the excessive normal propyl alcohol has ether-like odor and bitter taste. Therefore, in the process of brewing white spirit, the formation of n-propanol should be controlled as much as possible. The liquid method is adopted by most of the domestic white spirit manufacturers at present to brew white spirit, and the liquid method has the advantages of high white spirit yield, low cost, small occupied area, higher degree of mechanization, labor and raw material cost saving, production condition improvement, comprehensive benefits of brewing, convenience for comprehensive utilization, automation and the like. However, the white spirit brewed by the process cannot well show the due characteristics in terms of taste, and the problem of higher content of higher alcohol often exists. Therefore, the control of the content of higher alcohols in white spirit has become a problem to be solved in the white spirit industry in China. The problem of too high n-propanol content is solved by controlling the content of higher alcohols in the white spirit by a liquid method. The normal propyl alcohol content in the liquid-state white spirit is generally more than three times that in the solid-state white spirit, and the side effect is obvious after drinking, which is one of the main reasons for inhibiting the development of the liquid-state white spirit. In order to reduce the n-propanol content in the liquor by the liquid method, the yeast inoculation amount is generally increased, and the multiplication times of yeast in the fermentation process are reduced, so that the formation of higher alcohols in the multiplication process is reduced. However, too high an inoculation amount of yeast can lead the yeast to enter into a decay period prematurely, and fermentation is stopped in advance, which leads to insufficient fermentation of the white spirit and seriously affects the formation and quality of flavor substances of the white spirit; the other way is to adjust the nutrient components in the fermentation liquor by changing the fermentation process, and the yeast growth is inhibited when the nutrient components such as nitrogen source, inorganic salt and the like are lacking in the fermentation culture medium, which also leads to insufficient fermentation, not only affects the yield of white spirit, but also affects the quality of the white spirit.

The metabolic pathways for synthesizing n-propanol by Saccharomyces cerevisiae are mainly two: one is the alpha-keto acid catabolic pathway known as the ellich pathway, the amino acid catabolic pathway; the other is the alpha-keto acid anabolic pathway, known as the harris pathway, i.e., the higher alcohol anabolic pathway. There have been many related reports on the regulation of metabolism of n-propanol by these two pathways, li Wei et al (Li, W., chen, S.J., wang, J.H., zhang, C.Y., shi, Y., guo, X.W., chen, Y.F., and/or Xiao, D.G. (2018) Genetic engineering to alter carbon flux for various higher alcohol productions by Saccharomyces cerevisiae for Chinese Baijiu reference.applied microbiology and biotechnology,102 (4), 1783-1795.Https:// doi.org/10.1007/s 00253-017-8715-5) using white spirit yeast AY15 as a parent strain, and the effect of ILV1 gene deletion on higher alcohol metabolism was studied in detail. The concentration of the constructed ILV1 gene double knockout recombinant strain of n-propanol, active amyl alcohol and 2-phenethyl alcohol is respectively reduced by 30.33%, 35.58% and 11.71%; the concentrations of n-butanol and isoamyl alcohol are respectively increased by 326.39% and 57.6%. Eden et al (Eden, A., van Nedervelde, L., drukker, M., benvenisty, N., & Debourg, A. (2001) Involvement of branched-chain amino acid aminotransferases in the production of fusel alcohols during fermentation in yeast. Applied microbiology and biotechnology,55 (3), 296-300.Https:// doi. Org/10.1007/s 002530000506) found that the BAT2 gene-deleted mutant strain encoding aminotransferase had a large effect on the production of n-propanol. Most of these breedings are performed around beer-fermenting or solid-state white spirit-fermenting yeasts, or in the case of reducing n-propanol, result in an increase in the content of other higher alcohols. At present, transformation and breeding of genes related to higher alcohol metabolism of saccharomyces cerevisiae for liquor by a liquid method are freshly reported. There are many technological methods in modern liquid process white spirit fermentation to reduce the content of higher alcohols, such as Tang Qulai et al (Tang Qulai, li Jingjing, li Lingling, liu Caixia, hu Xuejiao, sho Dong guang (2015) research (I) on the production of rice-flavor white spirit by novel liquid fermentation, namely the application of enzyme preparation in the liquid fermentation of rice-flavor white spirit, brewing technology (9 stage), 8-11.) by adding proper amount of enzyme preparation in the liquid fermentation to produce rice-flavor white spirit, and synergistic saccharification and fermentation with traditional saccharification and fermentation agents to regulate the formation of flavor substances in the liquid process white spirit. However, in actual production, the difference of the regulation and control effects among different batches is large, and the effect is not ideal. Therefore, focusing on Saccharomyces cerevisiae strains, the selection of excellent strains with proper higher alcohol production is the most fundamental way to regulate and control the synthesis of n-propanol.

Disclosure of Invention

The invention aims to solve the problem that the content of n-propanol produced by a saccharomyces cerevisiae strain in the production of white spirit by a liquid method is high, and constructs the saccharomyces cerevisiae strain with low n-propanol production by regulating and controlling the threonine synthesis path of the saccharomyces cerevisiae.

In order to solve the technical problems, the technical scheme of the invention is as follows:

the invention provides a genetically engineered bacterium, which is obtained by deleting all homoserine dehydrogenase gene THR6 from saccharomyces cerevisiae.

Wherein the homoserine dehydrogenase encoded by the THR6 gene is capable of catalyzing a dimeric enzyme of the third step of the threonine biosynthetic pathway, which plays a key role in the synthetic pathway of threonine in Saccharomyces cerevisiae. Threonine can be converted to 2-ketobutyric acid. 2-ketobutyric acid is an essential precursor for synthesizing n-propanol by Saccharomyces cerevisiae, and directly affects the yield of n-propanol.

Preferably, the nucleotide sequence of the THR6 gene is shown as SEQ ID NO. 1. The Gene ID of the THR6 Gene which can be queried in NCBI is: 853604.

preferably, the Saccharomyces cerevisiae is a haploid strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae) AY15, α5.

Preferably, the genetically engineered bacterium is obtained by taking saccharomyces cerevisiae as an original strain and knocking out THR6 genes by a homologous recombination method.

In a specific embodiment of the present invention, the genetically engineered bacterium may be obtained by:

(1) PCR amplification is carried out by taking a saccharomyces cerevisiae genome as a template to obtain DNA molecular fragments of upstream and downstream sequences of THR6 genes;

(2) Transforming the DNA molecular fragments of the upstream and downstream sequences and the screening marker gene in the step (1) into the saccharomyces cerevisiae by a lithium acetate chemical transformation method, and obtaining a recombinant strain knocked out of the THR6 gene by homologous recombination;

(3) Knocking out the screening marker gene in the recombinant strain in the step (2) by using a Cre-loxP recombination system;

(4) Subculturing to lose the episomal plasmid introduced in the step (3) to obtain the saccharomyces cerevisiae recombinant strain.

Preferably, the selectable marker gene is a KanMX resistance gene.

The invention also aims to provide the application of the genetically engineered bacterium in the production of white spirit by a liquid fermentation method.

In a specific embodiment of the invention, the liquid fermentation method is to take the genetically engineered bacterium as a production strain, take sorghum as a raw material, prepare sorghum hydrolysate as a fermentation medium, inoculate the genetically engineered bacterium, and perform standing fermentation.

The beneficial effects are that:

the low-yield n-propanol saccharomyces cerevisiae strain provided by the invention can inhibit the expression of homoserine dehydrogenase and regulate and control the threonine synthesis path in yeast metabolism on the premise of keeping good fermentation performance, achieves the effect of obviously reducing n-propanol, and lays a theoretical foundation for brewing liquid-process white spirit with good flavor and unique taste.

The production amount of n-propanol of the genetically engineered bacterium is obviously reduced. After fermentation by a liquid method, the yield of n-propanol of the original strain alpha 5 is 25.77mg/L, and the yield of n-propanol of the recombinant strain lacking the THR6 gene obtained by the invention is 5.69mg/L, which is reduced by 77.92% compared with a parent strain; meanwhile, the total amount of higher alcohols of the recombinant strain is reduced by 19.89 percent compared with that of the parent strain.

The genetically engineered bacterium has good fermentation performance and growth performance, and the growth performance of the recombinant strain is not affected or other negative conditions are not caused. In addition, the strain has the advantages that the content of isobutanol, isopentanol and phenethyl alcohol is reduced to different degrees besides n-propanol, and the strain has important significance in regulating the content of higher alcohol in white spirit. Particularly, the saccharomyces cerevisiae recombinant strain lacking the THR6 gene provided by the invention can reduce n-propanol and simultaneously improve the content of esters, effectively reduce the alcohol-ester ratio and remarkably improve the flavor of liquid white spirit.

Drawings

Fig. 1: the construction scheme of recombinant strain alpha 5-DeltaTHR 6-k-p in the examples.

Fig. 2: verification of THR6A, THR, B, loxP-KanMX-loxP fragment in examples.

Fig. 3: verification of recombinant strain α5- Δthr6 in examples electrophoretogram.

Fig. 4: verification of recombinant strain α5- Δthr6-k (KanMX resistance gene knocked out) in examples.

Fig. 5: verification of recombinant strain α5-. DELTA.THR6-k-p (pSH-Zeocin plasmid was discarded) electrophoresis pattern in examples.

Detailed Description

The invention is described below by means of specific embodiments. The technical means used in the present invention are methods well known to those skilled in the art unless specifically stated. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention.

Saccharomyces cerevisiae, a preferred embodiment of which Saccharomyces cerevisiae alpha 5 is only one, may be used in the present invention as any source of Saccharomyces cerevisiae strain, derived from a haploid strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae) AY15 (accession number No. CICC 32315).

Example 1: construction of Saccharomyces cerevisiae Strain lacking THR6 Gene

Saccharomyces cerevisiae alpha 5 (Li W, wang JH, zhang CY, ma HX, and Xiao DG (2017 b) Regulation of Saccharomyces cerevisiae genetic engineering on the production of acetate esters and higher alcohols during Chinese Baijiu reference.J Ind Microbiol Biotechnol 44:949-960) is used as host bacteria, and a recombinant genetic engineering strain is constructed by a homologous recombination method.

The specific construction steps are detailed as follows:

(1) Taking the genome of the original strain Saccharomyces cerevisiae alpha 5 as a template, taking THR6A-F and THR6A-R as primers, and performing PCR amplification to obtain an upstream DNA molecular fragment THR6A (972 bp) of the THR6 gene; the downstream DNA molecular fragment THR6B (724 bp) of the THR6 gene is obtained by PCR amplification with the Saccharomyces cerevisiae alpha 5 genome as a template and THR6B-F and THR6B-R as primers.

(2) PCR was performed using the plasmid pUG6 as a template and THR6K-F and THR6K-R as primers to obtain a PCR product loxP-KanMX-loxP (1663 bp) containing the KanMX marker gene. The KanMX marker gene can also be obtained from other plasmids containing the gene sequence or can be directly synthesized into DNA molecular fragments.

FIG. 2 is a validated electrophoresis of THR6A, THR, B, loxP-KanMX-loxP fragment. Wherein lane M is DL5000 DNA marker; lane 1 is the result of PCR amplification using Saccharomyces cerevisiae α5 genome as template, THR6A-F and THR6A-R as primer pair (972 bp single band); lane 2 is the result of PCR amplification (724 bp single band) using Saccharomyces cerevisiae α5 genome as template, THR6B-F and THR6B-R as primer pair; lane 3 is the result of PCR amplification (1663 bp single band) using the plasmid pUG6 genome as template and THR6K-F and THR6K-R as primer pairs.

(3) Transferring the PCR product fragment THR6A, THR B obtained in the steps (1) and (2) into the original strain by a lithium acetate chemical conversion method, screening transformants by using a G418 resistance plate, selecting yeast colonies growing on the G418 resistance plate, extracting DNA of the purified yeast strains as templates, respectively using THR6-M1-U/THR6-M1-D and THR6-M2-U/THR6-M2-D as primers, and carrying out fixed point verification of the transformants by using PCR to obtain correct bands with lengths of 1758bp and 1248 bp. Namely, the correct positive transformant was designated as recombinant strain α5- Δthr6.

FIG. 3 is a validated electrophoretogram of recombinant strain α5- ΔTHR6. Wherein lane M is DL5000 DNA marker; lane 1 is a fragment (1758 bp single fragment) obtained by PCR amplification with the genome of the recombinant strain alpha 5-DeltaTHR 6 as a template and THR6-M1-U and THR6-M1-D as primer pairs; lane 2 is a fragment (1248 bp single fragment) obtained by PCR amplification using the genome of the recombinant strain alpha 5-DeltaTHR 6 as a template and THR6-M2-U and THR6-M2-D as primer pairs; lane 3 is the result of PCR amplification using the α5 genome as the template and THR6-M1-U and THR6-M1-D as primer pairs; lane 4 shows the results obtained by PCR amplification using the α5 genome as a template and THR6-M2-U and THR6-M2-D as primer pairs.

(4) Using Cre/loxP recombination system, chemically transforming pSH-Zeocin plasmid into the recombinant strain in the step (3) by using lithium acetate, using genome of recombinant strain alpha 5-DeltaTHR 6 and genome of transformant as templates, respectively using K-F and K-R as primers and using PCR amplification, using recombinant strain alpha 5-DeltaTHR 6 as template to make PCR amplification to obtain fragment 1613bp, and in PCR amplification using genome of transformant as template, there is no band, and obtaining the transformant with KanMX resistance marker removed, and marking the transformant as recombinant strain alpha 5-DeltaTHR 6-K.

FIG. 4 is a validated electrophoresis diagram of recombinant strain α5- ΔTHR6-k. Wherein lane M is DL5000 DNA marker; lane 1 is a fragment (1613 bp single fragment) obtained by PCR amplification with the genome of the recombinant strain alpha 5-DeltaTHR 6 as a template and K-F and K-R as primer pairs; lane 2 shows the result of PCR amplification using the α5- ΔTHR6-K genome as a template and K-F and K-R as primer pairs.

(5) Subculturing the recombinant strain alpha 5-delta THR6-k obtained in the step (4) to discard free pSH-Zeocin plasmid, selecting strains of 4 th generation to 5 th generation and more, extracting yeast plasmid from the strain, taking the yeast plasmid as a template, carrying out PCR amplification by taking Zn-F and Zn-R as primers, and extracting pSH-Zeocin plasmid as the template and carrying out PCR amplification by taking Zn-F and Zn-R as primers, wherein 1172bp bands appear on the PCR result, and taking genome of the strain after passage as the template without bands. The recombinant strain, which was successfully discarded from the pSH-Zeocin plasmid, was demonstrated to be designated as α5- ΔTHR6-k-p.

FIG. 5 is a validated electrophoretogram of recombinant strain α5- ΔTHR6-k-p. Wherein lane M is DL5000 DNA marker; lane 1 is a fragment (1172 bp single fragment) obtained by PCR amplification with pSH-Zeocin plasmid as template and Zn-F and Zn-R as primer pair; lane 2 shows the result of PCR amplification using the α5- ΔTHR6-k-p genome as template and Zn-F and Zn-R primer pairs.

The nucleotide sequences of the PCR primers used in this example are shown in Table 1.

TABLE 1 PCR primer sequence listing

Example 2: recombinant strain alpha 5-delta THR6-k-p liquor fermentation experiment

(1) The fermentation process route is as follows:

sorghum grain, crushing, liquefying, saccharifying, adding acid protease, cooling, filtering, adjusting sugar degree of sorghum juice, subpackaging, sterilizing, inoculating, fermenting and distilling;

(2) The main process conditions are as follows:

crushing conditions: the crushing degree is suitable for sorghum without whole grains, and the crushing degree is not easy to be too fine so as not to cause excessive filtering pressure;

liquefaction and saccharification conditions: adding 30 ℃ warm water into crushed sorghum according to the ratio of 1:4, fully and uniformly stirring, placing the mixture into a constant-temperature water bath kettle, and keeping the temperature of 90 ℃ for 60min for liquefaction. Regulating the temperature of the water bath kettle to 60 ℃, maintaining for 30min, and saccharifying. Fully stirring every 5min in the liquefaction and saccharification processes; after saccharification is completed, the temperature of the water bath kettle is regulated to 40 ℃, acid protease is added and stirred uniformly, and the temperature is kept for 16 hours, so that the protease fully plays a role.

Filtration conditions: the sorghum hydrolysate is filtered by using double-layer gauze, and the sugar degree of the sorghum hydrolysate is adjusted to be 18 degrees.

Sterilization conditions: packaging the jowar hydrolysate into triangular bottles, and sterilizing at 115deg.C for 20min. Cooling to room temperature to obtain the fermentation medium.

(3) Fermentation experiment:

inoculating seed solutions of Saccharomyces cerevisiae original strain alpha 5 and recombinant strain alpha 5-DeltatHR 6-k-p activated under the same experimental condition into the hair prepared in (2)In the fermentation medium (inoculum size 5X 10) ⁶ CFU/mL), and standing in an incubator at 30 ℃ to perform a liquid-process white spirit fermentation experiment; oscillating and weighing every 12h during fermentation, and recording the weightlessness; when fermentation is carried out for 96 hours, the weight loss of fermentation liquid of the original strain alpha 5 and the recombinant strain alpha 5-delta THR6-k-p is not reduced any more, and the fermentation is considered to be ended, and the culture is stopped; and measuring the weight loss, the alcohol content, the residual sugar and the content of main aroma components of the fermentation liquid. The overall performance was characterized by weight loss, alcoholicity, and residual sugar, and the results are shown in Table 2. The results of the content of the main aroma components are shown in Table 3.

4) GC analysis to determine higher alcohols and ester content: after the fermentation liquor is distilled, the wine sample is subjected to gas chromatography, and the chromatographic conditions are as follows: the capillary chromatographic column LZP-930, 50m×320 μm×1.0 μm, and the carrier gas is nitrogen with a purity of 99.99% and a split ratio of 1:10. The temperature of the sample inlet is 200 ℃, the temperature of the detector is 200 ℃, and the sample inlet amount is 1 mu L. Heating to 50deg.C for 8min, heating to 150deg.C at 5deg.C/min, and maintaining for 15min. To maintain accuracy of the data, each sample was sampled twice and averaged. Under the same chromatographic conditions, the retention time of the chromatographic peaks of the known higher alcohols and esters standard substances is used for analysis by comparing with the retention time of the chromatographic peaks of the higher alcohols in the sample.

TABLE 2 fermentation Performance of sorghum raw Material liquid method liquor fermentation

Note that: the data shown are the average of three parallel test results, p <0.05, p <0.01.

Table 2 shows that: in the fermentation experiment of the liquor by the liquid method, compared with the original strain, the saccharomyces cerevisiae recombinant strain obtained by the invention has no obvious change of fermentation performance. This demonstrates that the THR6 gene knockout in the present invention has no effect on the fermentation performance of Saccharomyces cerevisiae alpha 5.

TABLE 3 content of major aroma components (mg/L) of sorghum raw material liquid fermentation

Table 3 shows that: from the viewpoint of the amount of n-propanol produced, the original strain α5 had an n-propanol production of 25.77mg/L and the recombinant strain α5-. DELTA.THR6-k-p of the present invention had an n-propanol production of 5.69mg/L. Compared with the parent strain, the strain is reduced by 77.92 percent. This shows that the recombinant strain of the invention can reduce the content of n-propanol in the liquor by the liquid method to a great extent. Further, the production of several higher alcohols other than n-propanol also showed a decrease to a different extent. The total higher alcohol yield of the recombinant strain alpha 5-delta THR6-k-p with the THR6 gene knocked out is 358.85mg/L, and the total higher alcohol content is reduced by 19.89 percent compared with that of a parent strain. The strain obtained by the invention can reduce the content of n-propanol and total higher alcohols in the liquid-process white spirit to a great extent, and lays a theoretical foundation for brewing the liquid-process white spirit with good flavor and unique taste.

In addition, the content of ethyl acetate and isoamyl acetate produced by Saccharomyces cerevisiae alpha 5 is 14.00mg/L and 15.56mg/L respectively, and the total amount is 29.56mg/L. The content of ethyl acetate and isoamyl acetate of the recombinant strain alpha 5-delta THR6-k-p with THR6 gene knocked out in the invention is respectively 22.14mg/L and 17.62mg/L, the total amount is 39.76mg/L, and the content is improved by 34.51% compared with the original strain alpha 5. It should be noted that too high alcohol-to-ester ratio of the liquid white spirit will adversely affect the taste of the white spirit. In the brewing process of the white spirit, the generation amount of the higher alcohol is properly reduced to improve the alcohol-ester ratio, so that the taste of the white spirit can be obviously improved. On the basis, if the content of the ester substances is kept or increased, the taste of the white spirit is further improved. The recombinant strain alpha 5-delta THR6-k-p with THR6 gene knocked out in the invention realizes that the content of ester substances is improved while the content of higher alcohol is reduced, and the flavor of the liquid white spirit is obviously improved.

In addition, the recombinant strain alpha 5-delta THR6-k-p with THR6 gene knocked out has certain application value. In the actual production of the white spirit by the liquid method, the recombinant strain and the original strain or other saccharomyces cerevisiae strains/non-saccharomyces cerevisiae strains which produce n-propanol and ester in high yield can be considered to be mixed for fermentation according to a certain proportion, so that the purposes of accurately regulating and controlling the n-propanol content in the white spirit and improving the aroma components of the white spirit can be achieved. For example Meng Jinming (Meng Jinming. Research on liquid fermentation process for producing barley wine [ D ] Tianjin technology university, 2016.) in the process for producing barley wine by liquid fermentation, a mixed fermentation mode of sequential inoculation of Saccharomyces cerevisiae and Hansenula is adopted, the ratio of the obtained alcohol to the ester in the wine sample reaches 1:1, and the quality of the white wine is obviously improved.

Therefore, the genetic engineering bacteria of low-yield n-propanol lacking THR6 gene obtained by the invention has strong practicability in the aspect of effectively regulating and controlling the content of higher alcohols and the flavor in actual production, and provides a production strain with strong potential for the white spirit fermentation process.

Although the present invention has been described with reference to preferred embodiments, it is not intended to be limited to the embodiments shown, but rather, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations in form and details can be made therein without departing from the spirit and principles of the invention, the scope of which is defined by the appended claims and their equivalents.

SEQUENCE LISTING

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Claims

1. A genetically engineered bacterium is characterized in that the genetically engineered bacterium is obtained by deleting all homoserine dehydrogenase genes THR6 from a haploid strain alpha 5 of Saccharomyces cerevisiae AY 15.

2. The genetically engineered bacterium of claim 1, wherein the nucleotide sequence of the THR6 gene is shown in SEQ ID No. 1.

3. The genetically engineered bacterium of claim 1, wherein the genetically engineered bacterium is obtained by knocking out THR6 gene of saccharomyces cerevisiae as an original strain by a homologous recombination method.

4. The genetically engineered bacterium of claim 3, wherein the genetically engineered bacterium is obtained by:

5. The genetically engineered bacterium of claim 4, wherein the selectable marker gene is a KanMX resistance gene.

6. Use of the genetically engineered bacterium of any one of claims 1-5 in the production of white spirit by liquid fermentation.

7. The use according to claim 6, wherein the liquid fermentation method is characterized in that the genetic engineering bacteria are used as production strains, sorghum is used as raw materials, sorghum hydrolysate is prepared as a fermentation medium, and the genetic engineering bacteria are inoculated for standing fermentation.

8. The use according to claim 7, wherein the genetic engineering bacteria is inoculated in an amount of 5X 10 ⁶ CFU/mL, and standing and fermenting at 30 ℃.