CN112011474A

CN112011474A - Genetically engineered bacterium with low n-propanol yield and application thereof

Info

Publication number: CN112011474A
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: 2020-12-01
Anticipated expiration: 2040-09-08
Also published as: CN112011474B

Abstract

The invention provides a genetically engineered bacterium with low n-propanol production, 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 threonine biosynthesis pathway, which plays a key role in the threonine synthesis pathway of Saccharomyces cerevisiae. Threonine can be converted to 2-ketobutyric acid. 2-ketobutyric acid is an essential precursor substance for synthesizing the n-propanol by the saccharomyces cerevisiae, and directly influences the yield of the n-propanol. The genetically engineered bacterium is applied to the production of white spirit by a liquid fermentation method.

Description

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

Technical Field

The invention belongs to the technical field of bioengineering, relates to breeding of industrial microorganisms, and particularly relates to saccharomyces cerevisiae genetic engineering bacteria with low n-propanol yield 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 the important chemical substances that form the flavor and taste of white spirits. The proper content of the higher alcohol has the functions of ensuring that the white spirit has rich taste and aroma and the spirit body is soft and coordinated, but the excessive higher alcohol is the main source of the foreign flavor of the white spirit and is also the important reason for leading the white spirit to be up to the head after being drunk. As one of important components in higher alcohol, n-propanol has great influence on the style of white spirit, a proper amount of n-propanol can bring wine fragrance and sweet taste to the white spirit body, and excessive n-propanol is like ether odor and has bitter taste. Therefore, the formation of n-propanol should be controlled as much as possible during the brewing process of white spirit. At present, most domestic white spirit manufacturers brew white spirit by a liquid state method, and the white spirit brewed by the liquid state method has the advantages of high spirit yield, low cost, small occupied area, higher degree of mechanization, labor and raw material cost saving, improvement on production conditions, improvement on comprehensive benefits of brewing, convenience in comprehensive utilization, contribution to automation and the like. However, the white spirit brewed by the process cannot well show the due characteristics in taste, and the problem of high content of higher alcohol is often existed. Therefore, the control of the content of the higher alcohol in the white spirit is a problem which needs to be solved urgently in the white spirit industry of China. The problem of overhigh content of the n-propanol is firstly solved by controlling the content of the higher alcohol in the white spirit by the liquid method. The n-propanol content in the liquid-process white spirit is generally more than three times of that of the solid-process white spirit, and the side effect is obvious after drinking, which is one of the main reasons for inhibiting the development of the liquid-process white spirit. In order to reduce the content of the n-propanol in the white spirit by the liquid method, the yeast inoculation amount is generally increased in the industry, the multiplication times of the yeast in the fermentation process are reduced, and the formation of higher alcohol in the multiplication process is reduced. However, too high yeast inoculation amount can cause the yeast to enter the decline period too early and the fermentation to be terminated early, which can cause the insufficient fermentation of the white spirit and seriously affect 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 in the fermentation medium if nutrient substances such as nitrogen sources and inorganic salts are lacked, which can also cause insufficient fermentation, thereby not only affecting the yield of the white spirit, but also affecting the quality of the white spirit.

The metabolic pathways for synthesizing n-propanol by saccharomyces cerevisiae are mainly two: one is an alpha-keto acid catabolic pathway called the ellichi pathway, i.e., the amino acid catabolic pathway; the other is the alpha-keto acid anabolic pathway, known as the harris pathway, the higher alcohol anabolic pathway. There have been many reports on the regulation of the metabolism of n-propanol by these two pathways, and the influence of LIWEI et al (Li, W., Chen, S.J., Wang, J.H., Zhang, C.Y., Shi, Y., Guo, X.W., Chen, Y.F., & Xiao, D.G. (2018) Genetic engineering to animal tongue flux for varied bacteria high alcohol production by Saccharomyces cerevisiae 102(4), 1783-1795. applied microbiology and biotechnology, 1783-1795. htttv/doi.org/10.1007/00253-017 5-5) on the metabolism of high alcohol by using AY15 as a parent strain and studying the deletion of high alcohol metabolism genes in detail. The constructed ILV1 gene double-knockout recombinant strain has the concentration of n-propanol, active amyl alcohol and 2-phenethyl alcohol reduced by 30.33 percent, 35.58 percent and 11.71 percent respectively; the concentrations of n-butanol and isoamyl alcohol were increased by 326.39% and 57.6%, respectively. Eden et al (Eden, A., Van Nedervelde, L., Drukker, M., Benveninsty, N., & Debourg, A. (2001) investment of branched-chain amino acid amides in the production of fuse alcohol reduction in applied microbiology and biotechnology 55(3), 296-300. https:// doi.org/10.1007/s002530000506) found that deletion of the BAT2 gene encoding an aminotransferase had a greater effect on the amount of propanol produced. These breedings are mostly carried out around beer-fermenting or solid-state process white spirit-fermenting yeasts, or in the case of n-propanol reduction, lead to an increase in the content of other higher alcohols. At present, the transformation and breeding of genes related to the metabolism of higher alcohols of saccharomyces cerevisiae used for white spirit by a liquid method are rarely reported. In modern liquid-method white spirit fermentation, a plurality of process methods are used for reducing the content of high-grade alcohol, for example, people from Tang Lai et al (Tang Lai, Li Jing, Li Ling, Liu Cai Xia, Hu Xue jiao, Xiao Dong Guang, (2015) research on novel liquid-fermentation production of rice-flavor white spirit, application of an enzyme preparation in liquid-fermentation rice-flavor white spirit, brewing technology (9 th), 8-11.) through adding a proper amount of enzyme preparation in the production of rice-flavor white spirit by liquid fermentation, and regulating and controlling the formation of flavor substances in the liquid-method white spirit by saccharification and fermentation in cooperation with a traditional saccharification and fermentation agent. However, in actual production, the difference of the regulation and control effects among different batches is large, and the effect is not ideal. Therefore, the selection of excellent strains with suitable higher alcohol yield is the most fundamental way for regulating and controlling the synthesis of the n-propanol by focusing on the saccharomyces cerevisiae strains.

Disclosure of Invention

The invention aims to solve the problem that the n-propanol content of a saccharomyces cerevisiae strain is high in the production of white spirit by a liquid method, and the saccharomyces cerevisiae strain with low n-propanol yield is constructed by regulating and controlling a threonine synthesis way 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 genes THR6 by saccharomyces cerevisiae.

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

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

preferably, the Saccharomyces cerevisiae is a haploid strain α 5 of Saccharomyces cerevisiae AY 15.

Preferably, the genetically engineered bacterium is obtained by knocking out the THR6 gene of saccharomyces cerevisiae serving as an original strain by a homologous recombination method.

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

(1) taking the genome of the saccharomyces cerevisiae as a template, and carrying out PCR amplification to obtain DNA molecular fragments of upstream and downstream sequences of the THR6 gene;

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

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

(4) subculturing to lose the free plasmid introduced in the step (3) and obtaining 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 genetic engineering bacteria in the production of white spirit by a liquid fermentation method.

In a specific embodiment of the invention, the production of white spirit by the liquid fermentation method is carried out by taking the genetically engineered bacteria as production strains, taking sorghum as raw material, preparing sorghum hydrolysate as fermentation medium, inoculating the genetically engineered bacteria, and standing for fermentation.

Has the advantages that:

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

The production amount of the n-propanol of the genetic engineering bacteria is obviously reduced. After the fermentation by a liquid method, the n-propanol production of the original strain alpha 5 is 25.77mg/L, and the n-propanol production of the recombinant strain lacking the THR6 gene is 5.69mg/L, which is reduced by 77.92% compared with that of the parent strain; meanwhile, the total amount of higher alcohol 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 does not influence the growth performance of the recombinant strain or other negative conditions. Besides, the content of isobutanol, isoamyl alcohol and phenethyl alcohol of the strain is reduced to different degrees besides the normal propyl alcohol, which has important significance for controlling the content of higher alcohol in the white spirit. Particularly, the saccharomyces cerevisiae recombinant strain lacking the THR6 gene provided by the invention realizes the purposes of reducing n-propanol, improving the content of ester substances, effectively reducing the alcohol ester ratio and obviously improving the flavor of liquid-process white spirit.

Drawings

FIG. 1: the construction scheme of the recombinant strain α 5- Δ THR6-k-p is shown in the examples.

FIG. 2: example for the validation of the electropherograms of THR6A, THR6B, loxP-KanMX-loxP fragments.

FIG. 3: example validation of the electropherogram of the recombinant strain α 5- Δ THR 6.

FIG. 4: in the examples, the electrophoretogram was verified for the recombinant strain α 5- Δ THR6-k (knock-out KanMX resistance gene).

FIG. 5: example A validated electropherogram of recombinant strain α 5- Δ THR6-k-p (discarding pSH-Zeocin plasmid).

Detailed Description

The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.

The Saccharomyces cerevisiae used in the present invention is a strain of Saccharomyces cerevisiae that can be of any origin, Saccharomyces cerevisiae alpha 5 being only one preferred embodiment, and it is derived from a haploid strain of Saccharomyces cerevisiae AY15 (accession No. CICC32315).

Example 1: construction of Saccharomyces cerevisiae Strain deficient in THR6 Gene

A recombinant gene engineering strain is constructed by a homologous recombination method by taking Saccharomyces cerevisiae alpha 5(Li W, Wang JH, Zhang CY, Ma HX, Xiao DG (2017b) Regulation of Saccharomyces cerevisiae genetic engineering on the production of acetate esters and higher alcohols along with growth of maize genetic engineering. J Ind Microbiol Biotechnol 44: 949-960) as a host bacterium.

The specific construction steps are detailed as follows:

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

(2) The plasmid pUG6 is used as a template, THR6K-F and THR6K-R are used as primers, and PCR products loxP-KanMX-loxP (1663bp) containing KanMX marker genes are obtained by PCR amplification. The KanMX marker gene can also be obtained from other plasmids containing the gene sequence or directly synthesized into DNA molecular fragments.

FIG. 2 is a check electrophoresis diagram of THR6A, THR6B, loxP-KanMX-loxP fragments. Wherein, lane M is DL5000 DNA marker; lane 1 is the result of PCR amplification using Saccharomyces cerevisiae alpha 5 genome as template and THR6A-F and THR6A-R as primer set (972bp single band); lane 2 is the result of PCR amplification using Saccharomyces cerevisiae alpha 5 genome as template and THR6B-F and THR6B-R as primer set (724bp single band); lane 3 is the result of PCR amplification using the plasmid pUG6 genome as a template and THR6K-F and THR6K-R as primer sets (1663bp single band).

(3) Through a lithium acetate chemical transformation method, the PCR product fragments THR6A and THR6B obtained in the steps (1) and (2) are connected with loxP-KanMX-loxP in the middle and are transformed into the starting strain, G418 resistant plates are used for screening transformants, yeast colonies growing on the G418 resistant plates are selected, DNA of the purified yeast strains is extracted as a template, THR6-M1-U/THR6-M1-D and THR6-M2-U/THR6-M2-D are respectively used as primers, and the PCR is used for carrying out site-directed verification on the transformants to respectively obtain correct bands with lengths of 1758bp and 1248 bp. The correct positive transformant is marked as the recombinant strain alpha 5-delta THR 6.

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

(4) Chemically transforming pSH-Zeocin plasmids into the recombinant strains in the step (3) by using a Cre/loxP recombination system through lithium acetate, respectively taking genomes of the recombinant strains alpha 5-delta THR6 and transformants as templates, taking K-F and K-R as primers to perform PCR amplification, and taking the recombinant strains alpha 5-delta THR6 as templates to obtain a fragment 1613bp obtained by PCR amplification, wherein in the PCR amplification by taking the genomes of the recombinant strains as the templates, no band exists, so that the transformants with the KanMX resistance markers removed are proved to be marked as the recombinant strains alpha 5-delta THR 6-K.

FIG. 4 is a validated electropherogram of recombinant strain α 5- Δ THR 6-k. Wherein, lane M is DL5000 DNA marker; lane 1 is a fragment (1613bp single fragment) obtained by PCR amplification using the genome of the recombinant strain α 5- Δ THR6 as a template and K-F and K-R as primer pairs; lane 2 is the result of PCR amplification using the genome of α 5- Δ THR6-K as a template and K-F and K-R as primer sets.

(5) Subculturing the recombinant strain alpha 5-delta THR6-k obtained in the step (4) to discard free pSH-Zeocin plasmids, selecting strains of 4-5 generations and more than 5 generations, extracting yeast plasmids from the strains as templates, performing PCR amplification by using Zn-F and Zn-R as primers, extracting pSH-Zeocin plasmids as templates, performing PCR amplification by using Zn-F and Zn-R as primers, wherein the PCR result shows a 1172bp band, and the genome of the strains after the subculture is used as a template, so that no band exists. The recombinant strain that successfully discarded the pSH-Zeocin plasmid was demonstrated to be obtained and is designated α 5- Δ THR 6-k-p.

FIG. 5 is a validated electrophoretogram of recombinant strain α 5- Δ THR 6-k-p. Wherein, lane M is DL5000 DNA marker; lane 1 is a fragment (1172bp single fragment) obtained by PCR amplification using pSH-Zeocin plasmid as a template and Zn-F and Zn-R as primer pairs; lane 2 shows the results of PCR amplification using the genome of α 5- Δ THR6-k-p as a template and Zn-F and Zn-R as primer sets.

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

TABLE 1 PCR primer sequence Listing

Example 2: liquid-process white spirit fermentation experiment of recombinant strain alpha 5-delta THR6-k-p

(1) The fermentation process route is as follows:

sorghum grains → pulverization → liquefaction, saccharification → addition of acid protease → cooling → filtration → adjustment of sugar content of sorghum juice → split charging → sterilization → inoculation, fermentation → distillation;

(2) the main process conditions are as follows:

and (3) crushing conditions: the grinding degree is proper to the sorghum which is not subjected to whole grain, and the grinding degree is not easy to be too fine so as to avoid causing too high filtering pressure;

liquefying and saccharifying conditions: adding the pulverized sorghum into warm water of 30 ℃ according to the ratio of the material to the water of 1:4, fully stirring uniformly, placing in a constant-temperature water bath kettle, keeping the temperature at 90 ℃ for 60min, and liquefying. Adjusting the temperature of the water bath to 60 deg.C, maintaining for 30min, and saccharifying. Fully stirring once every 5min in the liquefaction and saccharification processes; after saccharification is finished, adjusting the temperature of a water bath to 40 ℃, adding acid protease, stirring uniformly, and keeping for 16 hours to enable the protease to fully play a role.

And (3) filtering conditions: filtering the sorghum hydrolysate by using double-layer gauze, and adjusting the sugar degree of the sorghum hydrolysate to 18 degrees.

And (3) sterilization conditions: subpackaging the sorghum hydrolysate into triangular flasks, and sterilizing at 115 deg.C for 20 min. Cooling to room temperature to obtain the fermentation medium.

(3) Fermentation experiment:

respectively inoculating seed liquids of the saccharomyces cerevisiae starting strain alpha 5 and the recombinant strain alpha 5-delta THR6-k-p which are activated under the same experimental conditions into the fermentation culture medium prepared in the step (2) (the inoculation amount is 5 multiplied by 10)⁶CFU/mL), standing in an incubator at 30 ℃ to perform a white spirit fermentation experiment by a liquid method; oscillating and weighing every 12h during fermentation, and recording weight loss; when the fermentation is carried out for 96 hours, the weight loss of fermentation liquor of the starting strain alpha 5 and the recombinant strain alpha 5-delta THR6-k-p is not reduced any more, and the culture is stopped after the fermentation is considered to be finished; and (4) determining the weight loss, alcoholic strength, residual sugar and main aroma component content of the fermentation liquor. The comprehensive properties of the wine are represented by weight loss, alcohol content and residual sugar, and the results are shown in table 2. The results of the main aroma content are shown in table 3.

4) GC analysis to determine higher alcohols and esters content: distilling the fermentation liquor, and carrying out gas chromatography analysis on the liquor sample, wherein the chromatographic conditions are as follows: capillary chromatographic column LZP-930, 50m × 320 μm × 1.0 μm, carrier gas is nitrogen with purity of 99.99%, and split ratio is 1: 10. The injection port temperature is 200 ℃, the detector temperature is 200 ℃, and the injection amount is 1 mu L. The temperature is raised by adopting a program, the temperature is kept at 50 ℃ for 8min, the temperature is raised by 5 ℃/min, the temperature is raised to 150 ℃, and the temperature is kept for 15 min. To maintain the accuracy of the data, each sample was injected twice and the average was taken. Under the same chromatographic condition, the retention time of chromatographic peaks of known higher alcohols and esters standard substances is compared with the retention time of chromatographic peaks of higher alcohols in the sample for analysis.

TABLE 2 fermentation Performance of Chinese liquor fermentation by liquid sorghum raw materials

Note: data shown are the average of three replicates, # p <0.05, # p < 0.01.

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

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

Table 3 shows that: from the production of n-propanol, the production of n-propanol by the original strain α 5 was 25.77mg/L, and the production of n-propanol by the recombinant strain α 5- Δ THR6-k-p of the present invention was 5.69 mg/L. The reduction was 77.92% compared to the parent strain. The recombinant strain can reduce the content of the n-propanol in the white spirit prepared by the liquid method to a great extent. Further, the production of several higher alcohols other than n-propanol also showed a decrease in the amount of the higher alcohols to various degrees. The total high-grade alcohol production amount of the THR6 gene knockout recombinant strain alpha 5-delta THR6-k-p obtained by the invention is 358.85mg/L, and compared with the total high-grade alcohol content of a parent strain, the total high-grade alcohol content is reduced by 19.89%. The strain obtained by the invention can reduce the content of n-propanol and total high-grade alcohol 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 the saccharomyces cerevisiae alpha 5 is respectively 14.00mg/L and 15.56mg/L, and the total amount is 29.56 mg/L. The contents of ethyl acetate and isoamyl acetate of the recombinant strain alpha 5-delta THR6-k-p with THR6 gene knocked out are 22.14mg/L and 17.62mg/L respectively, the total amount is 39.76mg/L, and the alpha 5 content is improved by 34.51 percent compared with the original strain. It should be noted that too high alcohol ester ratio of the liquid process white spirit can have a bad influence on the taste of the white spirit. In the brewing of white spirit, the generation amount of higher alcohol is properly reduced to improve the alcohol ester ratio, and the taste of the white spirit can be obviously improved. On the basis, if the content of the ester substances can be continuously maintained or increased, the taste of the white spirit can be further improved. According to the recombinant strain alpha 5-delta THR6-k-p with THR6 gene knocked out, the content of ester substances is increased while higher alcohol is reduced, and the flavor of liquid-process white spirit is obviously improved.

In addition, the recombinant strain alpha 5-delta THR6-k-p with THR6 gene knockout has certain application value. In the actual production of the white spirit by the liquid method, mixed fermentation can be considered between the recombinant strain and the original strain or other saccharomyces cerevisiae strains/non-saccharomyces cerevisiae strains which have high n-propanol yield and high ester yield according to a certain proportion, so that the aims 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, in the technology 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 wine-like ester to alcohol reaches 1:1, and the quality of white wine is obviously improved.

Therefore, the genetically engineered bacterium which is deficient in THR6 gene and has low n-propanol yield, obtained by the invention, has strong practicability in the aspects of effectively regulating and controlling the content and flavor of higher alcohol in actual production, and provides a production strain with strong potential for a white spirit fermentation process.

Although the present invention has been disclosed in the form of preferred embodiments, it is not intended to limit the present invention, and those skilled in the art may make various changes, modifications, substitutions and alterations in form and detail without departing from the spirit and principle of the present invention, the scope of which is defined by the appended claims and their equivalents.

SEQUENCE LISTING

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Claims

1. The genetically engineered bacterium is characterized in that the genetically engineered bacterium is obtained by deleting all homoserine dehydrogenase genes THR6 from saccharomyces cerevisiae.

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 saccharomyces cerevisiae is a haploid strain α 5 of saccharomyces cerevisiae AY 15.

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

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

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

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

8. The use of claim 7, wherein the liquor production by the liquid fermentation method is carried out by taking the genetically engineered bacteria as production strains, taking sorghum as raw materials, preparing sorghum hydrolysate as fermentation medium, inoculating the genetically engineered bacteria, and standing for fermentation.

9. The use according to claim 8, wherein the genetically engineered bacterium is inoculated in an amount of 5 x 10⁶CFU/mL, standing and fermenting at 30 ℃.