TWI690593B - Method of producing 2,3-butanediol with high yield - Google Patents

Abstract

A method is provided for producing 2,3-butanediol. An escherichia coli (E. coli) strain obtained through an innovation of genetic modification is used. In vivo of the strain, desired genes for producing 2,3-butanediol are expressed through gene transfer in a gene-recombination way. The present invention is characterized in that, during producing 2,3-butanediol, the strain does not change its yield owing to the change of oxygen concentration in the fermentation environment. The strain is capable of self-regulation by using an in-vivo metabolic pathway for dealing with the external environmental oxygen concentration. Thus, 2,3-butanediol is continuously produced with a high yield.

Description

Method for producing high-yield 2,3-butanediol

The invention relates to a method for producing high-yield 2,3-butanediol, in particular to a method capable of producing high-yield 2,3-butane by gene recombination, gene knockout, and simultaneous transfer of multiple genes to express plastids Escherichia coli strains of diols, especially those strains that have the ability to self-regulate and utilize the mechanism of metabolic pathways in the body to cope with changes in the oxygen concentration in the external environment and continue to produce high yields of 2,3-butanediol.

2,3-butanediol (2,3-butanediol) is an important chemical raw material and liquid fuel, widely used in chemical, pharmaceutical, food and aviation fields. In addition, through subsequent simple chemical reactions, many high-value derivative chemicals can be produced. For example, 2,3-butanediol is often used to prepare resins and solvents, etc.; its combustion value (27.2KJ/g) is comparable to methanol (22.1KJ/g) and ethanol (29.0KJ/g), which is a highly potential gasoline Additive; it has the characteristics of low freezing point (-60℃), and can be used as antifreeze. 2,3-Butanediol can produce methyl ethyl ketone (MEK) through dehydration reaction. This MEK is a high-priced liquid fuel additive and a low-boiling chemical solvent. It is often used in coatings, adhesives, and inks. It is also of high quality. Aviation fuel; 2,3-butanediol precursors Acetoin and 2,3-butanedione (Diacetyl), often used as a source of natural edible flavorings and flavorings for dairy products to give food a creamy taste; It can also inhibit bacterial growth as a bacteriostatic agent. 2,3-Butanediol can produce Acetone 2,3-butanediol ketal after acetalization (ketone) reaction, which is used in gasoline additives similar to methyl tert-butylether (MTBE). 2,3-Butanediol can be produced after esterification 2,3-Butanedioldiester, commonly used in medicine, cosmetics intermediates and carriers; thermoplastic polyester fiber plasticizer. One of the most important applications is the dehydration reaction of 2,3-butanediol to produce the special chemical 1,3-butandiene (1,3-butandiene), which is an important chemical raw material used to make synthetic rubber (Such as cis-butadiene rubber, neoprene rubber, styrene-butadiene rubber, nitrile rubber), plastics, resins (such as ABS, SBS, BS, MBS, etc.), elastic fiber and other synthetic rubber industries. During the Second World War, Ding Er Diene is listed as one of the important development resources by many countries. At present, the industry mainly obtains butadiene through the process of oil cracking. It is estimated that more than 12 million tons of 1,3-butadiene is used by the chemical industry to manufacture synthetic rubber, plastics, elastic fibers and other materials every year.

The production of butadiene by biological methods, in addition to the specific characteristics of strain enzymes can increase the yield of butadiene, compared with chemical methods, the production of 1,3-butadiene by biological methods can also have low The advantages of carbon footprint. At present, there is no progress in commercial mass production of biologically-produced succinate. The technological development is mainly divided into two ways. One is to directly apply biotechnology to construct and transform strains, and convert raw materials into succinate. One-step direct method, however, at this stage, there are still many technical bottlenecks in the one-step direct production of 1,3-butadiene by biological methods in the whole process of microbial strains; another way is through the biochemical processes of other biological methods (such as Ethanol, butylene glycol, etc.), followed by a two-step indirect method of chemical conversion into butadiene. Compared with ethanol, the process of converting 2,3-butanediol chemical method to butadiene is simpler and only requires a dehydration step, and the yield is relatively higher. The global market of butadiene is huge, and the bio-process has both environmental protection and carbon reduction benefits. At present, the two-step indirect bio-process butadiene process has entered the stage of trial production.

The production process of 2,3-butanediol by chemical method is mainly based on the four-carbon hydrocarbons produced by the cracking of petroleum, which is obtained by hydrolysis under high temperature and high pressure reaction conditions, but the chemical synthesis method is complicated, expensive and commercial. Chemistry is currently limited to biochemical approaches. The biological method of producing 2,3-butanediol by microbial fermentation can not only overcome the difficulties of the chemical method, but also reduce the traditional dependence on non-renewable petrochemical resources as raw materials, and convert it into a biological refining process that uses renewable biological materials as raw materials. , In line with the development trend of the international trend of green industrial process and low-carbon economy. In nature, many microbial strains can use monosaccharides as raw materials to metabolize 2,3-butanediol. The typical metabolic production pathway mainly passes through three key enzymes, acetolactate synthase (ALS), ethyl acetate Acetylactate decarboxylase (ALDC), and 2,3-butanediol dehydrogenase (2,3-BDH) or acetoin reductase (AR) ), the final conversion to 2,3-butanediol. Accompanying the production of acetic acid, lactic acid, ethanol, succinic acid, formic acid and other by-products in the process of metabolic metabolism. Native strains currently producing a chemical yield of 2,3-butanediol and which has a production scale than the potential by only including Klebsiella (Klebsiella), Enterobacter (Enterobacter), Bacillus (Bacillus ) And Paenibacillus genus ( Paenibacillus ) this few genus. Among them, Klebsiella and Enterobacter genus have more industrial production potential. These 2 genus are 2,3-butanediol producing native strains, which have many advantages such as high yield and wide range of fermentation substrate utilization, and can be quickly used on cheap substrates. It can also use non-grain fiber sources to produce 2,3-butanediol. However, these bacteria belong to the genus of microorganisms with microbial safety level 2. This is a genus of microorganisms with a risk of pathogenic infection, so it is used in There are serious environmental safety concerns when industrial production is scaled up and mass-produced.

In addition, because the chemical structural formula of 2,3-butanediol has two chiral centers, there are three forms of isomers (RR, meso, and SS isomers) in nature, of which RR and SS are different. The structure has optical activity and therefore has high application value in pharmaceutical raw materials. Generally speaking, different strains of native species will produce different forms of 2,3-butanediol isomers, and most strains themselves can produce more than two kinds of 2,3-butanediol isomer mixed products, because If we want to produce a single 2,3-butanediol optical isomer with higher purity, we must rely on the modified strain to achieve it.

There are many strains of bacteria that can be used for the fermentation production of 2,3-butanediol in nature. Although it provides a larger choice of strains, because 2,3-butanediol is a secondary product of strain metabolism, its production is limited. And the metabolic production process will also produce many by-products at the same time, thus reducing the target product and purity. In recent years, genetic engineering editing techniques have been used to modify genes in microorganisms, or to colonize foreign genes for expression. This not only strengthens the production of target products in host cells, but also allows for the expansion of the source availability of host strains. With the development of synthetic biological technology, it is possible to biosynthesize specific product metabolic pathways, and use Escherichia coli or Saccharomyces cerevisiae to produce non-native 2,3-butanediol that is easy to cultivate and has clear genetic information. The strain is a host cell, which constructs a synthetic 2,3-butanediol production metabolic pathway in the strain body, eliminates or strengthens key enzymes, regulates cofactors related to the metabolic pathway, and other strategies for high yield. 2, 3-Butanediol production.

The efficiency of chemical production strains is related to the industrialization process of biomass chemicals. The efficiency of the strains is expressed in the product concentration (Titer, g/L), production rate (Rate, g/L/h), and product yield (Yield) , g/g) and other aspects, the product concentration will affect the process equipment and energy consumption requirements, the product production rate will affect the fermentation tank setting and the scale of the commercial plant, and the product yield is related to the cost of the biomass source, and the three performance indicators are closely related Escherichia coli is widely used as a host strain for the production of qualitative chemicals due to its fast growth, clear information on strain metabolic pathways and mechanisms, and easy cultivation and other advantages. Through metabolic engineering and synthetic biotechnology, and according to industrial needs To achieve high efficiency 2,3-butanediol production strain construction.

Cell-based biological conversion processes often need to undergo a large number of complex reactions and require specific cofactors to participate, such as nicotineamide adenine dinucleotide (NADH) or nicotineamide adenine dinucleotide Nicotinamide adenine dinucleoside phosphate (NADPH), in which the supply of cofactors is insufficient, it is easy to cause redox imbalance in the metabolic process, and it also affects biotransformation. Therefore, regulating the metabolic cycle of redox cofactors is an important strategy to optimize biotransformation metabolic engineering one. In the past research, in order to obtain more cofactor NADH in the reaction in order to increase the production of 2,3-butanediol, the production strain-Bacillus subtilis ( Bacillus subtilis ) was transformed with a gene encoding udhA Transhydrogenase (transhydrogenase), on the one hand, restricts fermentation to low dissolved oxygen conditions and additional reducing substances to achieve the final production of strains (R,R)-2,3-butanediol up to 49g/L.

The construction strategy of common non-natural 2,3-butanediol production strains (such as E. coli or yeast) is to transfer the metabolic pathway of complete heterologous 2,3-butanediol into the host, except for the necessary The key in the performance path is to produce enzymes ALS and ALDC to produce 2,3-butanediol precursor acetoin, and also to display AR/2,3-BDH, also known as secondary alcohol dehydrogenase (secondary alcohol dehydrogenase, sADH), 2,3-BDH The purpose of this enzyme is to reduce oxidation of acetoin to the target product of 2,3-butanediol through the cofactor (commonly used NADH) to assist oxidation. In addition, 2,3-butanediol production line belongs to fermentation and metabolic pathways. In order to increase production, on the one hand, it is necessary to avoid the formation of other byproducts of fermentation and metabolism (such as ethanol, lactate, succinate, etc.) ), so that the carbon flux is concentrated to the production path of 2,3-butanediol, on the other hand, the oxygen concentration in the production environment must be controlled in a microaerobic state (microaerobic) to facilitate the growth of the strain, and at the same time, it can take into account 2,3-Butanediol production. In addition, E. coli under different oxygen culture conditions, the carbon flux of glycolysis and the five-carbon sugar metabolic pathway will change, causing the concentration ratio of NAD ⁺ /NADH and NADP ⁺ /NADPH to change in response to changes in ambient oxygen concentration. It has a specific selective enzyme catalytic ability for cofactors, which affects the target product yield. In the low dissolved oxygen state, 2,3-butanediol is produced as the only fermentation product. The concentration of cofactor and the redox balance of the reaction will be an important limiting factor affecting the production of 2,3-butanediol.

Current research literature shows that the 2,3-butanediol-producing strains modified by metabolic engineering only exhibit 2,3-BDH that prefers a single specific cofactor (NADH or NADPH) enzyme to reduce acetoin to 2,3-butane Glycol, therefore there is still room for improvement in production. In order to cope with the different oxygen concentration changes in the environment during the cultivation and production of the strains, to avoid changes in the concentration and proportion distribution of cofactors, which affects the yield of the target substance that originally used the metabolic pathway. It is really necessary to seek additional strategies and methods that can improve the influence of cofactors on regulating metabolism. Therefore, general users cannot meet the needs of users in actual use.

The main purpose of the present invention is to overcome the above-mentioned problems encountered in the conventional art and provide a high-yield production that can maintain the flexibility of the application of the strain's own cofactor under different oxygen concentration environments to maintain good 2,3-butanediol production. , 3-butanediol method.

The secondary objective of the present invention is to provide a method that can avoid the accumulation of 2,3-butanediol precursor acetoin, thus indirectly increase the yield of 2,3-butanediol and produce (R,R)- 2,3-Butanediol production method of high yield 2,3-butanediol.

Another object of the present invention is to provide a 2,3-butanediol produced in a batch feed mode of the fermentation tank process. After 56 hours of the fermentation process, the 2,3-butanediol can reach a high of 92g/L Yield generation, the performance of this strain in producing 2,3-butanediol is better than the yield of 2,3-butanediol produced by the non-native native species based strains currently published in the literature, whether it is in 2,3-butanediol Yield or production rate are the highest yield methods for producing high yield 2,3-butanediol. In order to achieve the above purpose, the present invention is a method for producing high-yield 2,3-butanediol, which is based on gene recombination, gene knockout and simultaneous transfer of multiple genes, which can be optimized using different cofactors. 2, 3-butanediol dehydrogenase metabolic gene expression quantity method, constructing high yield An E. coli strain producing 2,3-butanediol, and the deposit number of the strain in the Biological Resources Preservation and Research Center of the Food Industry Development Institute is BCRC 940664.

In the above embodiments of the present invention, the strain is of the genus Escherichia coli, can grow with glucose as the main carbon source, and can ferment glucose to convert to 2,3-butanediol.

In the above embodiment of the present invention, the constructed strain can express the enzymes acetolactate synthase (gene alsS ), acetolactate decarboxylase (gene alsD ) and two different 2,3-butanes required for the production of 2,3-butanediol in vivo. Diol dehydrogenase.

In the above embodiment of the present invention, the acetSactase synthase gene alsS line is derived from Bacillus subtilis .

In the above-described embodiments of the invention, the acetolactate decarboxylase gene alsD lines derived from Bacillus subtilis.

In the above embodiments of the present invention, one of the 2,3-butanediol dehydrogenases is NADH-dependent budC from Klebsiella pneumoniae .

In the above embodiments of the present invention, one of the 2,3-butanediol dehydrogenases is NADPH-dependent adh from Clostridium beijerinckii .

In the above embodiment of the present invention, the method for expressing these 2,3-butanediol dehydrogenases by this strain is to simultaneously transfer two different 2,3-butanediol dehydrogenase genes into the strain.

In the above embodiment of the present invention, the foreign gene line transferred into the strain body is expressed on the plastid and then transferred into the strain.

In the above embodiments of the present invention, the glucose source is selected from lignocellulosic raw materials and sugar liquids derived from the treatment of lignocellulosic raw materials.

Figure 1 is a schematic diagram of the effect of the present invention on the production of 2,3-butanediol by E. coli JCL166 with different cofactor-dependent BDH production under different oxygen conditions.

Figure 2 is a schematic diagram of the relevant changes of the present invention in the production of 2,3-butanediol with a batch feed strategy of fermentation tank.

The present invention is a method for producing high-yield 2,3-butanediol. The main feature is that it can construct 2,3-butanediol with high yield by gene recombination, gene knockout, and simultaneous transfer of multiple genes to express plastids. Escherichia coli ( E. coli ) strains. In addition to the gene alsS (acetolactate synthase (ALS) derived from Bacillus subtilis ( BS )) and alsD (in addition to the gene alsS (acetolactate synthase (ALS)) required for the production of 2,3-butanediol in Escherichia coli strain YCW3 Acetylactate decarboxylase (ALDC), sourced from Bacillus subtilis ), the most important technical feature is the common performance of 2,3-butanediol dehydrogenase that can utilize different cofactors NADP and NADPH (acetoin reductase, AR or 2,3-butanediol dehydrogenase, 2,3-BDH), the host strain co-transforms in the body to show NADH-dependent budC (source Klebsiella pneumoniae ), and NADPH- dependent adh (source: Clostridium beijerinckii ) 2 enzymes, so that the strain does not affect the yield due to changes in the culture and fermentation environment during the production of 2,3-butanediol, the strain can be flexible The ratio of NADH and NADPH in the body is used to adjust the ratio to produce high-yield 2,3-butanediol.

The detailed technical content and part of the specific implementation of the present invention will be described in the following content for those with ordinary knowledge in the field to which the present invention belongs to understand the characteristics of the present invention.

[Example 1] Construction of 2,3-butanediol production strain of E. coli- YCW3

Table 1 shows the strains and plastids used in the experiment. The E. coli strain BW25113 was designed from the wild type. The genetic characteristics of this strain are ( rrnBT14△lacZWJ16hsdR514△araBADAH33△rhaBADLD78 ). The XL-1 Blue strain was purchased from Stratagene and used to store all the plastids required for the preparation experiment. Strain JCL16 line BW25113, through conjugation, delivers F'plastids with lacIq. The preparation of JCL166 is a strain that eliminates the three genes of adhE, ldhA, and frdBC . The gene knockout method uses the strain purchased from Keio collection as a donor strain, and passes through the λ P1 phage transformation method (P1 transduction). Gene homology exchange, you can replace the target gene of the host with kanamycin (kanamycin) antibiotic gene, and then the temperature-sensitive plastid (pCP20) with FLP recombinase (Flp recombinase), into the host by electroporation and Performance, so that it can identify the FRT position and kick the kanamycin antibiotic gene out of the host, and then inactivate pCP20, and confirm with colony PCR (colony PCR), that is, the target gene deletion of the host is completed.

The method of constructing plastids is mainly based on the Gibson isothermal DNA assembly method. The gene fragments to be assembled are first prepared, purified, and measured by PCR, and then the volume of each fragment is calculated according to the size and concentration of each fragment. , Then add AMM reaction reagents containing exonuclease, polymerase, and ligase to react, and the assembled plastids are transformed into XL- by heat shock method (heart shock transformation) 1 Save strain and confirm plastid sequencing.

Table 2 shows the plastids required for the experiment and the primers required to prepare the target gene. The construction of individual plastids is described below:

pKM5: The backbone fragment was obtained by PCR with pPC150 (p15A ori, Specr) and primers K3 and K4; the Bs-alsS-alsD gene fragment was obtained by PCR with pZE12-alsS-alsD-BudC as the template and primers K9 and K10.

pKM9: The backbone fragment was obtained by PCR with pPC150 (p15A ori, Specr) and primers K3 and K4; the Bs-alsS gene fragment was obtained by PCR with pZE12-alsS-alsD-BudC as the template and primers K9 and K11.

pKM11: The backbone segment was obtained by PCR with pCS180 (pUC ori, Kanr) and primers K5 and K6; the Kp-budC gene fragment was modeled with pZE12-alsS-alsD-BudC (codon optimized) and primers K12, K13 Perform PCR.

pKM12: The backbone fragment was obtained by PCR with pCS180 (pUC ori, Kanr) and primers K5 and K6; the Cb-adh gene fragment was obtained by PCR with pZE12-alsS-alsD-CBADH (codon optimized) as the template and primers K14 and K15.

In order to produce a higher yield of 2,3-butanediol, the expression host (JCL166) was used as the original mother strain, and other possible competitive metabolic pathway genes were successively eliminated, including pflB (pyruvate formate-lyase) and ilvC (acetohydroxy acid isomeroreductase) to produce a new mutant strain YCW3 ( △adhE△ldhA△frdBC△pflB△ilvC ).

The abbreviations in the table represent gene sources: BS, Bacillus subtilis ; KP, Klebsiella pneumoniae MGH78578; CB, Clostridium beijerinckii NRRL B593.

[Embodiment 2] Enzymes with both NADH and NADPH cofactor-dependent functions can be adapted to produce 2,3-butanediol under different oxygen concentrations

Please refer to "Figure 1", which is a schematic diagram of the effect of the present invention on the production of 2,3-butanediol by BDH of Escherichia coli JCL166 which exhibits different cofactor dependence. As shown in the figure: In order to adapt the strain to various oxygen concentrations in the environment, to avoid changes in the concentration and ratio distribution of cofactors, affecting the yield of the target substance that originally used these cofactor metabolic pathways. In order to solve the above-mentioned problems, the present invention transformed the plastid pKM5 containing the enzymes acetolactate synthase (ALS) and acetolactate decarboxylase (ALDC) derived from Bacillus subtilis in E. coli JCL166 ( △adhE△ldhA△frdBC ) as a production 2, 3-butanediol precursor-Acetoin (Acetoin) required enzymes, and also co-transformed plastid pKM11 (with Klebsiella pneumonias NADH-dependent BDH, KpBudC), pKM12 (with Clostridium beijerinkiis NADPH-dependent A total of 3 strains of BDH, CbAdh) and pKM9 (with KpBudC and CbAdh, Combo) were tested for the effect of oxygen concentration on the production of 2,3-butanediol under aerobic and anaerobic conditions, respectively.

The results are shown in Figure 1. (A) in the figure is differently transformed strains, under aerobic environmental conditions, in a 250mL shake flask with 20mL culture medium (M9, 30g/L glucose, 5g/L yeast extract ), the result of producing 2,3-butanediol for 48 hours. The results showed that only strains expressing KpBudC, Mainly produces 5.6g/L meso-2,3-butanediol, while accumulating a large amount of acetoin (3.8g/L), another strain that only shows CbAdh, its butanediol production can reach 10.5g/L, of which 75 % Is (R,R)-2,3-butanediol, 25% is meso-2,3-butanediol, without any accumulation of acetoin, while showing NADH-dependent KpBudC and NADPH-dependent CbAdh strains (Combo ), the highest yield of 2,3-butanediol is 12g/L, and almost all butanediols are (R, R) isomers.

The strain (B) in the picture is cultivated in 20mL culture solution (M9, 15g/L glucose, 5g/L yeast extract) plus 5g/L acetoin, and cultivated under anaerobic environment for 24 hours to produce 2,3-butane Alcohol results. The results show that only the strain with KpBudC can produce 6.43g/L meso-2,3-butanediol, but only the strain of CbAdh. Due to the anaerobic environment, this GM strain accumulates a large amount of NADH and cannot be produced by glycolysis NADH, plus the specific NADPH-dependent CbAdh shown, also cannot use NADH as a cofactor, and excessive acetoin can be used in the reduction reaction to accumulate too much NADH. Therefore, the strain could not recycle NAD ⁺ /NADH at the end, and glycolysis could not proceed, so the strain hardly grew in an anaerobic environment, and no 2,3-butanediol was produced. In contrast, the YCW 3 strains simultaneously exhibited KpBudC and CbAdh, and had flexible regulation of NADH and NADPH cofactors, so the strain produced meso-2,3-butanediol and a small amount of (R,R)-2,3-butane alcohol.

It can be seen from the above results that when the strain only expresses a single NADH-dependent or NADPH-dependent BDH enzyme, the production of 2,3-butanediol is restricted due to the supply of specific cofactors and the stagnation in a specific metabolic state; when the strains are simultaneously When expressing the enzymes of KpBudC and CbAdH, no matter what kind of environment, the strain can adapt to the dynamic changes of NADH and NADPH in the bacteria because of the adaptability of NADH and NADPH cofactors. Under different oxygen concentration conditions, the strain can adjust itself to use in vivo The cofactors of NADH and NADPH, synergistic transformation, promote the production of 2,3-butanediol at high yield.

[Embodiment 3] Production of 2,3-butanediol by batch feeding in fermentation tank

Please refer to the "Figure 2", which is a schematic diagram of the relevant changes of the present invention for producing 2,3-butanediol with a batch feeding strategy of a fermentation tank. As shown in the figure: YCW3 strain was fermented in 1.5 liter fermentation tank and fed-batch strategy for 92 hours to produce 2,3-butanediol. The results are shown in Figure 2 (A) It is the result of changes in the production of 2,3-butanediol and acetoin. The results show that the strain can fully reduce acetoin to 2,3-butanediol under this fermentation condition to avoid the accumulation of the precursor of 2,3-butanediol, so there is a high yield of 2,3-butanediol. And produce (R,R)-2,3-butanediol with high purity.

(B) in the figure is the result of the change in the sugar concentration of the fermentation broth and the growth of the strain. The results showed that the fermentation was carried out in a stable and rapid state to 44 hours to produce 80g/L of 2,3-butanediol, with an average yield of 1.8g/L/h; when fermented to 56 hours, 2,3-butanediol The product is 92g/L up to the plateau period of its total fermentation process. When the fermentation experiment is stopped at 92 hours, the total output of 2,3-butanediol is 107.92g/L, and the productivity is 1.17g/L/h, of which more than 90% The 2,3-butanediol product is (R,R)-form isomer type (98.56g/L) with a purity of 91.32%. During fermentation, the dissolved oxygen value of the culture solution in the tank is controlled at a stirring rate to maintain 10%. It is expected that the strain can maintain basic growth and physiological metabolic activity to continuously produce 2,3-butanediol, but the strain has a strong growth and metabolism. The rate of oxygen consumption is very fast, so that the dissolved oxygen value of the culture solution in the tank is 0% for a long time, and the strain still has a yield of about 100g/L. Compared with the production conditions in the same fermentation tank, the use of KpBudC or The yield of CbAdh enzyme strain is significantly lower than that of strains that simultaneously exhibit KpBudC and CbAdh enzyme. The results show that although the dissolved oxygen in the tank environment varies greatly during the fermentation process, the strains that simultaneously express NADH/NADPH-dependent BDH can adjust and use different cofactors in response to changes in the environmental oxygen concentration. High output performance.

In summary, the present invention is a method for producing high-yield 2,3-butanediol, which can be effectively improved Due to various shortcomings of the practice, the constructed E. coli 2,3-butanediol production strain YCW3 has the flexibility to self-adjust the 2,3-butanediol production metabolic pathway cofactor NADP/NADPH concentration ratio in the body, and is optimally fermented. High-yield and high-yield 2,3-butanediol is produced under the process conditions. In addition, the strain constructed in the present invention also has no 2,3-butanediol native strains and must be considered for the pathogenicity of its biological safety level. 2,3-Butanediol has the potential for industrial mass production and application, thereby making the invention more advanced, practical, and more in line with the needs of users. It has indeed met the requirements of the invention patent application, and the patent application has been filed in accordance with the law.

However, the above are only preferred embodiments of the present invention, which should not be used to limit the scope of implementation of the present invention; therefore, simple equivalent changes and modifications made according to the scope of the patent application of the present invention and the content of the invention description , Should still fall within the scope of this invention patent.

【Biological Material Storage】

TW Republic of China Food Industry Development Institute

2018/03/07 BCRC 940664

<110> Nuclear Energy Research Institute, Atomic Energy Commission, Executive Yuan

<120> Method for producing high-yield 2,3-butanediol

<140> 107109789

<141> 2018-3-22

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<223> As a reverse primer for PCR amplification of pKM5 plastid constructed skeleton fragment gene

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Claims (5)

A method for producing high-yield 2,3-butanediol, which uses gene recombination, gene knockout, and simultaneous transmutation of multiple genes to perform together and can use different cofactors to optimize 2,3-butanediol. Hydrogenase (acetoin reductase, AR or 2,3-butanediol dehydrogenase, 2,3-BDH) metabolic gene expression method to construct an E. coli strain capable of producing 2,3-butanediol with high yield, and the strain is used in food The deposit number of the Biological Resources Conservation and Research Center of the Industrial Development Institute is BCRC 940664; the constructed strain can express two different 2,3-butanediol dehydrogenations required for the production of 2,3-butanediol in vivo The enzymes are NADH-dependent budC from Klebsiella pneumoniae and NADPH-dependent adh from Clostridium beijerinckii . The method for producing high-yield 2,3-butanediol according to item 1 of the patent application scope, wherein the strain is of the genus Escherichia coli, can grow with glucose as the main carbon source, and can ferment glucose into 2,3 -Butanediol. According to the method for producing high-yield 2,3-butanediol described in item 1 of the scope of the patent application, wherein the method for expressing the 2,3-butanediol dehydrogenase by the strain is to combine the two 2, The 3-butanediol dehydrogenase gene was transferred into the strain. The method for producing high-yield 2,3-butanediol according to item 3 of the scope of the patent application, wherein the foreign gene transferred into the strain body is expressed on the plastid and then transferred into the strain. The method for producing high-yield 2,3-butanediol according to item 2 of the scope of the patent application, wherein the glucose source is selected from lignocellulosic raw materials and sugar liquids derived from lignocellulosic raw materials after treatment.

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