CN104403984A - Yield improvement method of acetoin by strengthening expression of bacillus subtilis glucose-6-phosphate dehydrogenase - Google Patents

Abstract

The method for enhancing the expression of Bacillus subtilis glucose-6-phosphate dehydrogenase to increase the yield of acetoin belongs to the fields of genetic engineering and fermentation engineering. Through the expression vector pMA5, the glucose-6-phosphate dehydrogenase was obtained in the high-yield acetoin-producing wild-type Bacillus subtilis JNA (preservation number CCTCC M209309; patent application publication number CN 101864381 A) obtained by our laboratory's previous screening expression, and finally use the recombinant bacteria after metabolic engineering to ferment and produce acetoin, which can convert 150 g/L glucose into 71.4 g/L acetoin, and the production efficiency of acetoin rises to 0.74 g/(L h) , the yield increased by nearly 84%; the by-product 2,3-butanediol was only 2.4 g/L, which was 86% lower than that of the original strain. It is the first time at home and abroad to use the enhanced pentose phosphate oxidation pathway to reduce the concentration of NADH and the ratio of NADH/NAD ⁺ to ferment acetoin in Bacillus subtilis, achieving the purpose of improving production efficiency and reducing NADH-dependent by-products.

Description

Enhancing the expression of Bacillus subtilis glucose-6-phosphate dehydrogenase to increase the production of acetoin

technical field

The method utilizes enhanced expression of Bacillus subtilis glucose-6-phosphate dehydrogenase to reduce NADH-dependent by-products and increase acetoin production, and the invention belongs to the field of genetic engineering and fermentation engineering. It specifically relates to the construction of genetically engineered bacteria and its fermentation production.

technical background

Acetoin occurs naturally in corn, grapes, cocoa, apples, bananas, cheese, meat, and many other foods. It is a widely used and lovable food spice, with a strong creamy, fat, butter-like aroma, and a pleasant milky aroma after being highly diluted, so acetoin is mainly used to prepare milk-flavored, meat-flavored type, strawberry-flavored flavor, or directly used in dairy products. my country's national standard GB2760-86 clearly stipulates that it is a food-grade spice that is allowed to be used, and the safety number of the American Food and Extraction Association (FEMA) is 2008.

At present, traditional fermentation technology has become a research hotspot in the edible flavor industry in my country at this stage. The basic principle of using microbial fermentation to produce spices is: through the metabolism of microorganisms and the catalysis of enzymes produced during the growth of microorganisms, through a series of complex biochemical reactions, various organic substances are converted into a variety of compounds with aromatic odors. . At present, domestic and foreign studies have shown that some bacteria have the ability to produce acetoin, mainly including Klebsiella (Klebisella), Enterobacter (Enterobacter), Bacillus (Bacillus), Serratia (Serratia) And Lactococcus (Lactococcus) and so on. However, in the metabolic process of most strains, acetoin exists as a by-product of 2,3-butanediol and diacetyl metabolism, and the accumulation concentration is low, which directly leads to the difficulty of using these microbial strains for industrial fermentation production Acetomarry. Deep research.

There is a strain of Bacillus subtilis B. subtilis JNA (preservation number CCTCC M2093092009.12.21; patent application publication number CN 101864381A) that uses glucose as a substrate to high-yield acetoin. The pathway requires a large amount of NAD ⁺ , which will form a large amount of NADH-dependent by-products, such as lactic acid, ethanol and 2,3-butanediol, resulting in the inability to further increase the production of acetoin. Therefore, it is considered to reduce intracellular NADH levels and NADH/NAD ⁺ proportion to reduce the formation of these by-products. By strengthening the pentose phosphate oxidation pathway, thereby reducing the metabolism of the glycolytic pathway, reducing the demand for NAD ⁺ , and by strengthening the key enzyme of the pentose phosphate oxidation pathway—glucose-6-phosphate dehydrogenase, increasing the NADPH content, achieving a reduction in NADH levels and NADH/NAD ⁺ ratio. But so far, there is no report on the use of pentose phosphate oxidation pathway to increase acetoin in Bacillus subtilis.

The present invention realizes the high-efficiency expression of B.subtilis JNA glucose-6-phosphate dehydrogenase (abbreviated as zwf) through the expression vector pMA5, and utilizes the enhanced expression of Bacillus subtilis glucose-6-phosphate dehydrogenase to improve production efficiency and reduce The purpose of NADH-dependent by-products is to finally realize the high-efficiency production of acetoin in engineered strains.

Contents of the invention

The original strain used in the present invention is B. subtilis JNA, which ferments glucose in the early stage to synthesize 2,3-butanediol, and reversely transforms it into acetoin in the later stage. It has been preserved in the China Type Culture Collection Center, and the preservation number is: CTCCM 209309.

The main research contents of the present invention: the present invention has cloned the acetoin reductase gene (zwf for short) from B. subtilis JNA by molecular technology, constructed the recombinant expression vector pMA5-zwf, and transformed it into B. subtilis JNA, successfully A genetic engineering strain B. subtilis JNA/pMA5-zwf was constructed. Finally, acetoin was fermented by enhanced expression of Bacillus subtilis glucose-6-phosphate dehydrogenase, and finally B. subtilis JNA was fermented for 96 hours, consuming 150 g/L glucose into about 71.4 g/L acetoin, the by-product 2,3-butanediol is only 2.4g/L, which is 86% lower than that of the original strain, and the production efficiency of acetoin rises to 0.74g/(L·h), and the yield increases by nearly 84%.

Advantage and positive effect of the present invention are:

(1) The present invention reports for the first time that by strengthening the pentose phosphate oxidation pathway to reduce the intracellular NADH level and NADH/NAD ⁺ ratio to reduce the generation of NADH-dependent by-products, and to reduce NADH-dependent by-products for the fermentation of Bacillus subtilis to produce acetoin The product provides a certain theoretical possibility.

(2) The present invention utilizes the enhanced expression of Bacillus subtilis glucose-6-phosphate dehydrogenase to realize the method for efficiently producing acetoin, and finally utilizes the engineering strain that strengthens the expression of Bacillus subtilis glucose-6-phosphate dehydrogenase to ferment for 96 hours, Consumption converts 150g/L glucose into about 71.4g/L of acetoin, the by-product 2.4g/L of 2,3-butanediol drops by 86% compared with the original strain, and the production efficiency of acetoin rises to 0.74g/( L·h), the yield increased by nearly 84%.

Detailed ways

Embodiment 1: Amplification of target gene and construction of recombinant Bacillus subtilis

First, using the chromosomal DNA of the bacterial strain B.subtilis JNA as a template, using primers P1 and P2, a 1470bp nucleotide sequence was amplified by PCR to obtain a nucleotide sequence of 1470bp such as the zwf gene shown in SEQ ID NO: 1, and the purified After the zwf gene was digested with restriction endonucleases MluI and BamHI, it was ligated with the plasmid pMA5 that had also been digested with the above two restriction endonucleases to construct the recombinant plasmid pMA5-zwf, and it was placed overnight at 16°C under the action of T ₄ DNA ligase For connection, the connection solution was transformed into Escherichia coli competent E.coli JM109, the positive transformants were picked, the plasmids in the transformants were extracted, and the recombinant plasmid pMA5-zwf was confirmed to be successfully constructed by enzyme digestion. After verification by double enzyme digestion, it indicated that the recombinant plasmid was constructed successfully. The recombinant plasmid pMA5-zwf was transformed into B. subtilis JNA by chemical transformation method, and positive transformants were selected to obtain recombinant B. subtilis JNA/pMA5-zwf. Analysis of the intracellular protein expression of the original bacteria and the recombinant bacteria showed that the gene zwf was successfully overexpressed in the recombinant bacteria.

Using the total genomic DNA of B. subtilis JNA as a template, two primers were designed, and the primers for PCR amplification were designed as follows:

P1: 5'-AGGC GGATCC GTGAAAACAAACCAACAACC-3' (BamH I)

P2: 5'-ACCG ACGCGT TTATATGTTCCACCAGTGTA-3' (Mlu I)

Embodiment 2: Determination of activity of recombinant bacteria B.subtilis JNA/pMA5-zwf glucose-6-phosphate dehydrogenase

The recombinant bacteria B. subtilis JNA/pMA5-zwf constructed in Example 1 and the starting strain B. subtilis JNA were inoculated in 10 mL of LB medium containing kanamycin, cultured with shaking at 37°C overnight, and the next day at 4% The inoculum amount was transferred to LB medium, cultured at 37°C for 24 hours, the fermentation broth was taken at 4°C, centrifuged at 10000r/min for 10min, washed 3 times with pH 7.0 sodium phosphate buffer, and the cells were resuspended in pH 6.5 sodium phosphate buffer Then, the liquid was placed in an ultrasonic breaker to treat the cells for 20 minutes, centrifuged at 15000r·min ^-1 for 30 minutes, and the supernatant was the crude enzyme solution. Add 20 μl of crude enzyme solution into the enzyme activity assay buffer system and immediately detect the change of A ₃₄₀ absorbance value.

Determination of glucose-6-phosphate dehydrogenase activity: the enzyme reaction system is 1mL, containing 50mmol/L potassium phosphate (pH 6.5), 10mmol/L glucose-6-phosphate and 1mmol/L NADP ⁺ ; Immediately after the enzyme solution, the enzyme activity unit (IU) is defined as the amount of enzyme required to reduce 1 μmol of NADP ⁺ per minute at 25°C.

The results showed that the specific enzyme activity of glucose-6-phosphate dehydrogenase expressed by the recombinant strain B.subtilis JNA/pMA5-zwf was 4.46U/mg, which was 90 times higher than that of the starting strain B.subtilis JNA. It indicated that B.subtilisJNA/pMA5-zwf was constructed successfully.

Example 3: Production of acetoin, 2,3-butanediol and performance testing of by-products by fermentation of B.subtilis JNA/pMA5-zwf

(1) Seed cultivation

Pick a single colony from the activated plate and inoculate it in the seed medium. The seed culture temperature is 37°C, the shaker speed is 160r/min, and the culture time is about 12h. The composition of the seed medium: yeast extract 5g/L, tryptone 10g/min L, NaCl 10g/L, glucose 40g/L.

(2) Fermentation culture

The initial fermentation culture volume is 2L, and the composition of the fermentation medium used is as follows:

Fermentation medium components: beef extract 5g/L, corn steep liquor 20g/L, urea 2g/L, glucose 100g/L. The above fermentation medium was adjusted to pH 6.5 with 5 mol/L NaOH, and sterilized at 121° C. for 30 minutes.

Fermentation conditions: Inoculate the above-mentioned cultivated seed liquid into the fermentation medium with 5% inoculum amount for fermentation and cultivation, the fermentation temperature is 37°C, the air flow rate is 120m ³ /h·m ³ medium, and the stirring speed is 300r/min. Samples were taken at regular intervals to determine cell concentration, production of acetoin and 2,3-butanediol, and other NADH-dependent by-products. After the fermentation, the products 2,3-butanediol and acetoin in the fermentation broth were determined by gas chromatography (GC-1690J gas chromatograph, Hangzhou Kexiao Chemical Instrument Co., Ltd.). The chromatographic conditions are as follows: capillary column, fixed liquid in 30m×0.32mm chromatographic column is AT.SE-30, detector is FID, column temperature is 150°C, temperature of vaporization chamber and detector is both 250°C, carrier gas is N _2. The flow rate is 0.1Mpa, the injection volume is 2μL, and the external standard method is used for quantification.

After 72 hours of final fermentation, 100 g/L glucose was converted into about 51.3 g/L acetoin, and the yield reached 0.71 g/(L·h). The main by-product 2,3-butanediol is only 1.7g/L.

(3) Determination of intracellular cofactor levels of recombinant strains

The intracellular cofactor levels of the recombinant strains were detected using the AAT Bioquest reagent kit. NADH, NAD ⁺ concentration and NADH/NAD ⁺ ratio were detected at Ex/Em=540/590 nm by a fluorescent microplate reader. In addition, the detection of NADPH concentration was carried out at OD _450nm using the reagent detection kit of BioVision Company.

The results showed that the intracellular NADH concentration and NADH/NAD ⁺ ratio decreased by 32.4% and 40.7%, respectively, in the recombinant strain B.subtilis JNA/pMA5-zwf compared with the original strain B.subtilis JNA, which was more conducive to the production of acetoin.

(4) Fed-batch fermentation culture of recombinant strains

Seed medium fermentation culture condition is identical with embodiment 3 (2), and glucose final concentration is controlled between 10g/L to 15g/L, and final B.subtilis JNA/pMA5-zwf ferments 96h, consumes and converts 150g/L glucose into about The acetoin of 71.4g/L and 2,3-butanediol of 2.4g/L decreased by 86% compared with the original strain, the production efficiency of acetoin rose to 0.74g/(L h), and the yield increased by nearly 84% .

Claims (2)

1. the recombined bacillus subtilis that subtilis glucose-6-phosphate dehydrogenase (G6PD) is expressed is strengthened in a strain, it is characterized in that: the subtilis G 6 PD gene mutations zwf shown in SEQ ID NO:1 being cloned into shuttle vectors pMA5 is configured to restructuring shuttle expression plasmid pMA5-zwf and is converted into deposit number is in the subtilis B.subtilis JNA of CCTCC NO:M209309.

2. the application of bacterial strain according to claim 1 in fermentative production acetoin, it is characterized in that: bacterial strain B.subtilis JNA/pMA5-zwf is inoculated in containing in kantlex 10mL LB substratum, after 37 DEG C of shaking culture 7-9h, get 1ml transfer in 2 bottles of 50ml containing 40g/L glucose LB substratum in 37 DEG C of shaking culture, when being cultured to OD ₆₀₀during=5.0-6.0, (formula of this fermention medium is: beef extract 5g/L to add 1.90L fermention medium, corn steep liquor 20g/L, urea 2g/L, glucose 100g/L, deionized water configures, pH 6.5) 5L fermentation reactor in cultivate, glucose final concentration controls between 10g/L to 15g/L, final fermentation 96h, consume acetoin 150g/L conversion of glucose being about 71.4g/L, acetoin production efficiency rises to 0.74g/ (Lh), and acetoin output comparatively original strain improves 84%; By product 2,3-butanediol is only 2.4g/L comparatively original strain decline 86%.