CN104403984A - Yield improvement method of acetoin by strengthening expression of bacillus subtilis glucose-6-phosphate dehydrogenase - Google Patents
Yield improvement method of acetoin by strengthening expression of bacillus subtilis glucose-6-phosphate dehydrogenase Download PDFInfo
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- CN104403984A CN104403984A CN201410748415.7A CN201410748415A CN104403984A CN 104403984 A CN104403984 A CN 104403984A CN 201410748415 A CN201410748415 A CN 201410748415A CN 104403984 A CN104403984 A CN 104403984A
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- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01049—Glucose-6-phosphate dehydrogenase (1.1.1.49)
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Abstract
The invention relates to a yield improvement method of acetoin by strengthening expression of bacillus subtilis glucose-6-phosphate dehydrogenase, and belongs to the fields of gene engineering and fermentation engineering. The glucose-6-phosphate dehydrogenase is expressed in high-yield acetoin wild-type bacillus subtilis B.subtillis JNA (with the preservation number of CCTCC M209309 and the patent application publication number of CN101864381A) obtained in previous selection of a laboratory through an expression vector pMA5, and finally the acetoin is produced by utilizing the fermentation of recombinant bacteria after metabolic engineering reform, so that 150 g/L of glucose can be converted into 71.4 g/L of acetoin, the production efficiency of the acetoin rises to 0.74 g/(L*h), and the yield is improved by about 84%; the content of a byproduct 2,3-butanediol is only 2.4 g/L, and is reduced by 86% compared with an original bacterial strain. The acetoin is produced by utilizing a strengthened phosphopentose oxidative pathway to reduce the concentration of NADH and the proportion of NADH/NAD+ in bacillus subtilis for the first time at home and abroad, and the purposes of improving the production efficiency and reducing NADH dependent-type byproducts are achieved.
Description
Technical field
Utilization adds strongly expressed subtilis glucose-6-phosphate dehydrogenase (G6PD) and reduces NADH dependent form by product raising acetoin output, the invention belongs to genetically engineered and field of fermentation engineering.Be specifically related to structure and the fermentative production thereof of genetic engineering bacterium.
Technical background
Acetoin is natural to be present in the numerous food products such as corn, grape, cocoa, apple, banana, cheese, meat.That one is widely used, charming flavouring agent, there is strong cream, fat, butter sample fragrance, have pleasant milk fragrance after high dilution, therefore acetoin is mainly used in the essence configuring milk flavoured type, meat odor type, strawberry odor type, or is directly used in milk preparation.China standard GB/T 2760-86 clear stipulaties its be allow use food grade spice, U.S. food and extraction association (FEMA) security number be 2008.
At present, traditional fermentation technique has become the study hotspot of China's present stage flavouring agent industry.Utilize fermentable to produce the ultimate principle of spices to be: the katalysis undertaken by the enzyme produced in the own metabolism of microorganism and microorganism growth process, through the biochemical reaction of series of complex, various organism is made to be converted into the multiple compound with aromatic odour.Research shows that some bacterium has the ability of production acetoin both at home and abroad at present, mainly comprises Klebsiella (Klebisella), enterobacter (Enterobacter), bacillus (Bacillus), serratia (Serratia) and lactococcus (Lactococcus) etc.But in most of bacterial strain metabolic process, acetoin exists as the by product of 2,3-butanediol and dimethyl diketone metabolism, and accumulated concentrations is lower, thus directly results in and be difficult to utilize these microbial strains industrial fermentation production acetoins.Deep research.
This Laboratories Accession has a strain to take glucose as subtilis B.subtilis JNA (the preserving number CCTCC M2093092009.12.21 of substrate high yield acetoin; Public announcement of a patent application CN 101864381A), find that its earlier fermentation is subject to glycolytic pathway and needs a large amount of NAD under study for action
+requirement can form a large amount of NADH dependent form by product, as lactic acid, ethanol and 2,3-butanediol, causes acetoin output to improve further, therefore considers by reducing NADH level and NADH/NAD in born of the same parents
+ratio reduces the generation of these by products.By strengthening phosphopentose oxidative pathway, thus reducing glycolytic pathway metabolism, reducing NAD
+demand, and by strengthening phosphopentose oxidative pathway key enzyme-glucose-6-phosphate dehydrogenase (G6PD), improve NADPH content, realize reducing NADH level and NADH/NAD
+ratio.But so far, have not been reported about the research utilizing phosphopentose oxidative pathway to improve acetoin in subtilis.
The present invention is by expression vector pMA5, achieve the high expression of B.subtilis JNA glucose-6-phosphate dehydrogenase (G6PD) (being called for short zwf), utilization adds strongly expressed subtilis glucose-6-phosphate dehydrogenase (G6PD), reach and enhance productivity, reduce the object of NADH dependent form by product, finally realize engineering strain High-efficient Production acetoin.
Summary of the invention
Original strain used in the present invention is B.subtilis JNA, and this bacterial strain prior fermentation glucose synthesis 2,3-butanediol, the later stage is reverse is converted into acetoin, and be preserved in China typical culture collection center, deposit number is: CTCCM 209309.
Main research of the present invention: the present invention utilizes molecular engineering to clone acetoin reductase gene (be called for short zwf) from B.subtilis JNA, build recombinant expression vector pMA5-zwf, and be converted into B.subtilis JNA, successfully construct engineering strain B.subtilis JNA/pMA5-zwf.Finally, utilization adds strongly expressed subtilis glucose-6-phosphate dehydrogenase (G6PD) fermentative production acetoin, final B.subtilis JNA fermentation 96h, consume acetoin 150g/L conversion of glucose being about 71.4g/L, by product 2,3-butyleneglycol is only 2.4g/L comparatively original strain decline 86%, and acetoin production efficiency rises to 0.74g/ (Lh), output increased nearly 84%.
Advantage of the present invention and positively effect are:
(1) reported first of the present invention reduces NADH level and NADH/NAD in born of the same parents by strengthening phosphopentose oxidative pathway
+ratio reduces the generation of NADH dependent form by product, provides certain theory possibility for fermentation of bacillus subtilis production acetoin reduces NADH dependent form by product.
(2) the present invention utilizes and adds strongly expressed subtilis glucose-6-phosphate dehydrogenase (G6PD), realize the method for High-efficient Production acetoin, final utilization adds the engineering strain fermentation 96h of strongly expressed subtilis glucose-6-phosphate dehydrogenase (G6PD), consume acetoin 150g/L conversion of glucose being about 71.4g/L, 2 of by product 2.4g/L, 3-butyleneglycol comparatively original strain declines 86%, and acetoin production efficiency rises to 0.74g/ (Lh), output increased nearly 84%.
Embodiment
Embodiment 1: the amplification of goal gene and the structure of recombined bacillus subtilis
First, with the chromosomal DNA of bacterial strain B.subtilis JNA for template, utilize primer P1 and P2, the zwf gene of nucleotide sequence as shown in SEQ ID NO:1 of one section of 1470bp size is obtained by round pcr amplification, by the zwf gene after purifying after restriction enzyme MluI and BamHI digests, connect with the same plasmid pMA5 through above-mentioned two kinds of digestion with restriction enzyme, construction recombination plasmid pMA5-zwf, at T
4the lower 16 DEG C of connections of spending the night of effect of DNA ligase, are converted in E. coli competent E.coli JM109, picking positive transformant by connecting fluid, extract the plasmid in transformant, confirm that recombinant plasmid pMA5-zwf successfully constructs through digestion verification.After double digestion checking, show this construction of recombinant plasmid success.Recombinant plasmid pMA5-zwf is converted into B.subtilis JNA with the method for chemical conversion, picking positive transformant, namely obtains recombined bacillus subtilis B.subtilis JNA/pMA5-zwf.Can find out original bacteria and recombinant bacterium intracellular protein expression analysis, gene zwf successfully realizes overexpression in recombinant bacterium.
With B.subtilis JNA genome DNA for template, design two primers, pcr amplification design of primers is as follows:
P1：5’-AGGC
GGATCCGTGAAAACAAACCAACAACC-3’(BamH I)
P2：5’-ACCG
ACGCGTTTATATGTTCCACCAGTGTA-3’(Mlu I)
Embodiment 2: recombinant bacterium B.subtilis JNA/pMA5-zwf glucose-6-phosphate dehydrogenase (G6PD) vitality test
The recombinant bacterium B.subtilis JNA/pMA5-zwf that embodiment 1 is built, 10mL is inoculated in respectively containing in the LB substratum of kantlex with starting strain B.subtilis JNA, 37 DEG C of shaking culture are spent the night, next day by 4% inoculum size transfer in LB substratum, cultivate 24h for 37 DEG C, get fermented liquid in 4 DEG C, the centrifugal 10min of 10000r/min, the sodium phosphate buffer of pH7.0 cleans 3 times, cell is resuspended in pH 6.5 sodium phosphate buffer, cell 20min is processed, 15000rmin under subsequently this liquid being placed in sonicator
-1centrifugal 30min, supernatant is crude enzyme liquid.20 μ l crude enzyme liquids are joined in enzyme activity determination buffer system and detects A immediately
340the change of light absorption value.
Glucose-6-phosphate dehydrogenase (G6PD) vitality test: enzyme reaction system is 1mL, containing 50mmol/L potassiumphosphate (pH 6.5), 10mmol/L G-6-P and 1mmol/L NADP
+; Enzymatic reaction starts immediately after adding a certain amount of enzyme liquid, and enzyme activity unit (IU) is defined as under 25 DEG C of conditions, and per minute reduces the NADP of 1 μm of ol
+required enzyme amount.
Result show glucose-6-phosphate dehydrogenase (G6PD) that recombinant bacterium B.subtilis JNA/pMA5-zwf expresses more alive than enzyme be 4.46U/mg, more alively than enzyme than the nadh oxidase of starting strain B.subtilis JNA improve 90 times, show successfully to build B.subtilisJNA/pMA5-zwf.
Embodiment 3:B.subtilis JNA/pMA5-zwf fermentative production acetoin, 2,3-butanediol and by product Performance Detection
(1) seed culture
From activation flat board, picking list colony inoculation is in seed culture medium, seed culture temperature 37 DEG C, shaking speed 160r/min, incubation time is about 12h, and seed culture medium forms: yeast extract 5g/L, Tryptones 10g/L, NaCl 10g/L, glucose 40g/L.
(2) fermentation culture
Preliminary fermentation volume of culture is 2L, and the fermentation medium components of employing is as follows:
Fermentation medium components: beef extract 5g/L, corn steep liquor 20g/L, urea 2g/L, glucose 100g/L.The NaOH of above-mentioned fermention medium 5mol/L is regulated its pH to 6.5, high-temperature sterilization 30min at 121 DEG C.
Fermentation condition: above-mentioned cultured seed liquor be inoculated in fermention medium by 5% inoculum size and carry out fermentation culture, leavening temperature 37 DEG C, air flow quantity is 120m
3/ hm
3substratum, mixing speed is 300r/min.Timing sampling measures the output of cell concn, acetoin and 2,3-butanediol and other NADH dependent form by product.After fermentation ends, in fermented liquid, product 2,3-butanediol and acetoin are with gas Chromatographic Determination (GC-1690J gas chromatograph, Hangzhou Ke Xiao chemical industry instrument company).Chromatographic condition is as follows: capillary column, and in 30m × 0.32mm chromatographic column, stationary liquid is AT.SE-30, detector is FID, column temperature 150 DEG C, and the temperature of vaporizing chamber and detector is 250 DEG C, and carrier gas is N
2, flow velocity 0.1Mpa, sample size 2 μ L, adopts quantified by external standard method.
Final fermentation 72h, is about the acetoin of 51.3g/L by 100g/L conversion of glucose, productive rate reaches 0.71g/ (Lh).Main By product 2,3-butanediol is only 1.7g/L.
(3) cofactor level determination in recombinant bacterial strain born of the same parents
In recombinant bacterial strain born of the same parents, cofactor level detection utilizes AAT Bioquest company enzyme linked detecting kit.By fluorescence microplate reader, under Ex/Em=540/590nm, detect NADH, NAD
+concentration and NADH/NAD
+ratio.NADPH Concentration Testing utilizes BioVision company enzyme linked detecting kit at OD in addition
450nmdetect.
Result shows, recombinant bacterial strain B.subtilis JNA/pMA5-zwf compared with starting strain B.subtilis JNA, NADH concentration and NADH/NAD in born of the same parents
+ratio declines 32.4% and 40.7% respectively, is more conducive to production acetoin.
(4) recombinant bacterial strain feed supplement fed-batch cultivation is cultivated
Seed culture medium fermentation culture conditions is identical with embodiment 3 (2), glucose final concentration controls between 10g/L to 15g/L, final B.subtilis JNA/pMA5-zwf fermentation 96h, consume and 150g/L conversion of glucose is about the acetoin of 71.4g/L and 2 of 2.4g/L, 3-butyleneglycol comparatively original strain declines 86%, acetoin production efficiency rises to 0.74g/ (Lh), output increased 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%.
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| CN201410748415.7A CN104403984A (en) | 2014-12-09 | 2014-12-09 | Yield improvement method of acetoin by strengthening expression of bacillus subtilis glucose-6-phosphate dehydrogenase |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1831115A (en) * | 2005-03-10 | 2006-09-13 | 味之素株式会社 | Purine-derived substance-producing bacillus and a method for producing |
| CN101864381A (en) * | 2010-05-10 | 2010-10-20 | 江南大学 | Breeding of microbial strain for producing 3-hydroxy-2-butanone by fermenting substrate glucose |
| CN103981141A (en) * | 2014-06-05 | 2014-08-13 | 江南大学 | Strategy for producing acetoin through efficient fermentation based on AR/BDH enzymatic properties of bacillus subtilis |
| CN104017758A (en) * | 2014-06-05 | 2014-09-03 | 江南大学 | Method for producing acetoin through high-efficiency fermentation by appropriately expressing novel bacillus subtilis NADH oxidizing enzyme |
| CN104017764A (en) * | 2014-06-05 | 2014-09-03 | 江南大学 | Bacillus subtilis NAD+regeneration system is used to effectively biotransform 2, 3-butanediol to produce acetoin |
-
2014
- 2014-12-09 CN CN201410748415.7A patent/CN104403984A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1831115A (en) * | 2005-03-10 | 2006-09-13 | 味之素株式会社 | Purine-derived substance-producing bacillus and a method for producing |
| CN101864381A (en) * | 2010-05-10 | 2010-10-20 | 江南大学 | Breeding of microbial strain for producing 3-hydroxy-2-butanone by fermenting substrate glucose |
| CN103981141A (en) * | 2014-06-05 | 2014-08-13 | 江南大学 | Strategy for producing acetoin through efficient fermentation based on AR/BDH enzymatic properties of bacillus subtilis |
| CN104017758A (en) * | 2014-06-05 | 2014-09-03 | 江南大学 | Method for producing acetoin through high-efficiency fermentation by appropriately expressing novel bacillus subtilis NADH oxidizing enzyme |
| CN104017764A (en) * | 2014-06-05 | 2014-09-03 | 江南大学 | Bacillus subtilis NAD+regeneration system is used to effectively biotransform 2, 3-butanediol to produce acetoin |
Non-Patent Citations (1)
| Title |
|---|
| YUN XIA DUAN ET AL.: "Overexpression of glucose-6-phosphate dehydrogenase enhances riboflavin production in Bacillus subtilis", 《APPL MICROBIOL BIOTECHNOL》 * |
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