CN109486871A - A method of utilizing bacillus licheniformis engineered strain fermenting and producing 3-hydroxy-2-butanone - Google Patents
A method of utilizing bacillus licheniformis engineered strain fermenting and producing 3-hydroxy-2-butanone Download PDFInfo
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Abstract
The invention discloses a kind of methods using bacillus licheniformis engineered strain fermenting and producing 3-hydroxy-2-butanone, it is that its glycerol dehydrogenase gdh gene and 2 are knocked out to go out bacterium germination with bacillus licheniformis (Bacillus licheniformis) 10-1-A, 3- butanediol dehydrogenation enzyme gene budC and in Thermococcus profundus DT5432 nadh oxidase gene nox construct replacing lactic acid dehydrogenase gene ldh and can be used to produce the bacillus licheniformis engineered strain of 3-hydroxy-2-butanone, then using glucose as substrate, the biofermentation bacterium, obtains 3-hydroxy-2-butanone by fermentation liquid.Experiment confirms that engineering bacteria of the invention can produce the 3-hydroxy-2-butanone of 82.14 grams per liters, and production efficiency reaches 2.28 grams per liters/hour, and the production process is at low cost, and yield is high, has good application value and considerable economic benefit.
Description
Technical field
The present invention relates to a kind of method of fermenting and producing 3-hydroxy-2-butanone more particularly to a kind of utilization bacillus licheniformis engineering bacterias
The method of strain fermenting and producing 3-hydroxy-2-butanone.
Background technique
3-hydroxy-2-butanone (3- hydroxy-2-butanone) is a kind of volatile compound, it is widely used in food addition, plant life
Long promotion and biological centrol etc..In addition, 3-hydroxy-2-butanone is also used as a series of chemical synthesis precursor substance of compounds, such as
Biacetyl and contain alkyl pyrazine, the latter one include Tetramethylpyradine.In view of the extensive purposes of 3-hydroxy-2-butanone and realization
The potentiality of industrialized production are classified as one of the platform chemicals that 30 kinds are first developed by U.S. Department of Energy.
Business is the chemical synthesis mode based on non-renewable resources with 3-hydroxy-2-butanone main source at present, and the processing step is multiple
It is miscellaneous, severe reaction conditions, seriously polluted, and application of the chemically synthesized 3-hydroxy-2-butanone in fields such as food, medicine, daily use chemicals is limited
System.Also numerous researchers are dedicated to realizing the industrialized production of 3-hydroxy-2-butanone using microbial fermentation, but compared to chemical technology
Method, the cost that microbial fermentation produces 3-hydroxy-2-butanone are still higher.Therefore, 3-hydroxy-2-butanone industry is reduced to provide non-renewable petroleum
The green production of 3-hydroxy-2-butanone is realized in the dependence in source, improves the economic competitiveness of microorganism 3-hydroxy-2-butanone zymotechnique, is had important
The meaning of economic benefit and social agency.
Retrieval discovery: existing multiple-microorganism, which is reported, can be used for 3-hydroxy-2-butanone fermenting and producing, United States Patent (USP) (US
0815840) it reports through high resistance to sugar screening and NTG mutagenesis, obtains a bacillus pumilus, 3-hydroxy-2-butanone yield reaches
63g/L.Sun Jianan et al. passes through the heterogenous expression nadh oxidase raising NAD intracellular in serratia marcescens+Concentration, with grape
Sugar is carbon source, and final 3-hydroxy-2-butanone yield reaches 75.2g/L, this is the peak of 3-hydroxy-2-butanone yield in current report.However, utilizing
The key gene (gdh and budC) of by-product 2,3-butanediol route of synthesis is knocked out, and using from Thermococcus
Nadh oxidase gene (nox) in profundus DT5432 bacterial strain replaces bacillus licheniformis lactic acid dehydrogenase gene
(ldh) throughput manner for reducing by-product lactic acid constructs bacillus licheniformis engineered strain, to realize the efficient production of 3-hydroxy-2-butanone
Method have not been reported.
Summary of the invention
For the deficiency of the prior art, the object of the present invention is to provide a kind of bacillus licheniformis engineerings using building
The method of strain fermentation production 3-hydroxy-2-butanone.
Method of the present invention using bacillus licheniformis engineered strain fermenting and producing 3-hydroxy-2-butanone, step is:
(1) it is bacterium germination with bacillus licheniformis (Bacillus licheniformis) 10-1-A, knocks out lichens gemma
Bacillus (Bacillus licheniformis) 10-1-A glycerol dehydrogenase gdh gene and 2,3- butanediol dehydrogenation enzyme gene budC
And to replace lactic dehydrogenase base from nadh oxidase gene nox in Thermococcus profundus DT5432
The bacillus licheniformis engineered strain of 3-hydroxy-2-butanone can be used to produce because ldh is constructed, which to be named as lichens gemma bar
Bacterium Bacillus licheniformis Δ gdh Δ budC Δ ldh::nox；
Wherein, starting strain bacillus licheniformis (Bacillus licheniformis) 10-1-A is in 2011
November 14 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are as follows: CGMCC
NO.5461；
(2) using glucose as substrate, biofermentation bacillus licheniformis Bacillus licheniformis Δ gdh Δ
BudC Δ ldh::nox, obtains 3-hydroxy-2-butanone by fermentation liquid；
Wherein, the fermentation condition is: cultivation temperature is 50 ± 1 DEG C, and training method is stir culture, and speed of agitator is
500 ± 50 revs/min, radius of turn is 33 ± 1 millimeters, and ventilatory capacity is 1.0 ± 0.1vvm, and incubation time is 18~36 hours；
Fermentation medium composition are as follows: 50~80 grams per liter of glucose, 12 grams per liter of yeast powder, 6.5 grams per liter of anhydrous sodium acetate,
1 grams per liter of ammonium citrate, 2 grams per liter of dipotassium hydrogen phosphate, 0.25 grams per liter of epsom salt are 10 ml ls of metal ion mother liquor, remaining
Amount is water；PH is 7.0；Metal ion mother liquor formula therein is: 2.25 grams per liter of ferrous sulfate, 0.75 grams per liter of zinc sulfate, sulphur
Sour 0.38 grams per liter of manganese.
In the above-mentioned method using bacillus licheniformis engineered strain fermenting and producing 3-hydroxy-2-butanone, step (1) described engineering bacteria
The construction method of strain Bacillus licheniformis Δ gdh Δ budC Δ ldh::nox is:
Using bacillus licheniformis 10-1-A genome as template, PCR obtains the weight comprising gdh gene upstream and downstream homologous sequence
Group segment, is connected to after pKVM1 and is transferred to bacillus licheniformis 10-1-A through biparent cross, through resistance screening single-swap transformant；
After temperature-induced generation double crossing over, PCR screening obtains gdh gene successful knockout bacterial strain, is named as bacillus licheniformis
Bacillus licheniformisΔgdh；
Using bacillus licheniformis 10-1-A genome as template, PCR is obtained comprising budC gene upstream and downstream homologous sequence
Recombinant fragment is connected to after pKVM1 and is transferred to bacillus licheniformis licheniformis Δ gdh through biparent cross, through resistance screening
Single-swap transformant；After temperature-induced generation double crossing over, PCR screening obtains budC gene successful knockout bacterial strain, is named as lichens
Bacillus licheniformis Δ gdh Δ budC.
Using bacillus licheniformis 10-1-A genome as template, it includes ldh gene upstream and downstream homology arm sequence that PCR, which is obtained,；
Using the plasmid pUC57-nox of the carrying nadh oxidase of full genome synthesis as template, PCR obtains nox sequence, will be on ldh gene
Downstream homology arm sequence and nox sequence recombinant PCR obtain gene replacement recombinant fragment, turn after being connected to pKVM1 through biparent cross
Enter bacillus licheniformis Bacillus licheniformis Δ gdh Δ budC, through resistance screening single-swap transformant；Temperature lures
It leads after double crossing over occurs, PCR screening obtains the engineered strain that nox gene successfully replaces ldh gene, is named as bacillus licheniformis
Bacillus licheniformisΔgdhΔbudCΔldh::nox。
Above-mentioned bacillus licheniformis Bacillus licheniformis Δ gdh Δ budC Δ ldh::nox is gram sun
Property bacterium, the growth of aerobic or amphimicrobian, protein secretion ability is strong, and growth rate is fast, is a kind of generally acknowledged bio-safety bacterium.Its shape
State is rod shape, 1.5 μm~3.0 μm long, and 0.6 μm~0.7 μm of diameter, colony colour is red or white, produces gemma, VP reaction
It is positive, produces acid using glucose, sucrose, fructose, hydrolyzable casein, gelatin, Tween 80 can using citrate
It grows, can be grown under the conditions of 42~60 DEG C in the culture medium of the NaCl containing 100g/L.
In the above-mentioned method using bacillus licheniformis engineered strain fermenting and producing 3-hydroxy-2-butanone, step (2) the fermentation item
Part is preferably: cultivation temperature is 50 DEG C, and training method is stir culture, and speed of agitator is 500 revs/min, radius of turn 33
Millimeter, ventilatory capacity 1.0vvm, incubation time are 25~36 hours.
In the above-mentioned method using bacillus licheniformis engineered strain fermenting and producing 3-hydroxy-2-butanone, the detection side of substrate glucose
Method is: sample carries out after suitably diluting, and is surveyed using bio-sensing analyzer SBA-40D (Shandong Province academy sciences Biology Research Institute)
It is fixed.Measuring principle is to measure glucose content using immobilized glucose oxidase film specificity.
In the above-mentioned method using bacillus licheniformis engineered strain fermenting and producing 3-hydroxy-2-butanone, the inspection of tunning 3-hydroxy-2-butanone
Survey method is:
0.7 milliliter of isoamyl alcohol is added in every 500 milliliters of ethyl acetate, as extractant；Using the extractant, to fermentation liquid
Middle fermentation product samples carry out equal-volume extraction, are shaken 30 seconds using sample of the turbula shaker to extraction, are then allowed to stand, take
Layer sample carries out vapor detection.Specific vapor detection condition is as follows:
The model Agilent 6820 of gas chromatograph used, nitrogen is as carrier gas, the temperature of sample injector and detector
It is set as 280 DEG C；Capillary column is SupelcoBeta-DexTM120 (0.25 millimeter of internal diameter, 30 meters of length)；Detection process
Column temperature setting are as follows: 40 DEG C are kept for 3 minutes, then 80 DEG C are warming up to 1.5 DEG C of rate per minute, with 0.5 DEG C of speed per minute
Rate is warming up to 86 DEG C, is warming up to 200 DEG C with 30 DEG C of rate per minute.1.0 microlitres of sample volume, carry out vapor detection.
The features of the present invention and prominent effect are:
(1) bacillus licheniformis Bacillus licheniformis Δ gdh Δ budC Δ ldh: provided by the invention:
Nox is the bio-safety bacterial strain of high yield 3-hydroxy-2-butanone, has knocked out the key gene of by-product 2,3-butanediol route of synthesis
(gdh and budC), so that 3-hydroxy-2-butanone blocked to be converted into the metabolic fluxes of 2,3-butanediol, also with coming from Thermococcus
Nadh oxidase gene (nox) in profundus DT5432 bacterial strain replaces bacillus licheniformis lactic acid dehydrogenase gene
(ldh) to reach the yield for reducing by-product lactic acid, and the by-product route of synthesis of NADH dependent form is reduced to improve
The yield of 3-hydroxy-2-butanone.
(2) it is obtained using glucose as substrate in the case where the present invention provides fermentation condition using engineered strain provided by the invention
3-hydroxy-2-butanone maximum concentration up to 82.14 grams per liters, production efficiency is up to 2.28 grams per liters/hour；
(3) method of production 3-hydroxy-2-butanone provided by the invention makes full use of the advantage of hot fermentation process, pollution microbes
Probability is small, while realizing yield height, the big target of production intensity.
Specific embodiment
The content of present invention is described in detail combined with specific embodiments below.Example as described below be only it is of the invention compared with
Good embodiment, is not intended to limit the present invention in any form, according to the technical essence of the invention to implementation
Any simple modification that mode is made, equivalent variations and modification, belong in the range of technical solution of the present invention.
Embodiment 1: the engineering bacteria Bacillus licheniformis Δ gdh Δ budC Δ of building production 3-hydroxy-2-butanone
ldh::nox
Starting strain is bacillus licheniformis Bacillus licheniformis10-1-A, and the bacterium is in November, 2011
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number within 14th are as follows: CGMCC NO.5461.
(1) knockout of glycerol dehydrogenase gdh gene
Glycerol dehydrogenase gene gdh sequence length is 1104 bases, and nucleotide sequence is as shown in SEQ ID NO.1.
The genomic DNA of B.licheniformis 10-1-A is prepared using conventional method, which goes out with reference to science
The method of bacterial genomes prepared in a small amount, extracts bacillus licheniformis in " the fine works Molecular Biology " that version society publishes
The genomic DNA of Bacillus licheniformis10-1-A；Using primer " gdh1-f and gdh1-r " with " gdh2-f and
The upstream and downstream homology arm of gdh2-r " PCR amplification gdh encoding gene.Then " gdh1-f and gdh2-r " primer is utilized, to be obtained
Upstream and downstream homology arm be that template carries out recombinant PCR, obtain the truncated segment of gdh, both ends include the digestion of BglII and BamHI
Site.
BglII and BamHI is used to carry out double digestion, digestion products respectively the truncated segment of gdh and suicide plasmid pKVM1
After glue recycles, T is used4DNA ligase connection, obtains and knocks out plasmid pKVM1- Δ gdh.
Wherein, the design of primers for expanding recombinant fragment is as follows:
gdh1-f:5’-ATTTAGATCTAACAAGCCGCGTCATTCAAG-3’(BglII)
gdh1-r:5’-ACTTGGCGCCATTCTTCTTCGACACATCGCAAATGATA-3’
gdh2-f:5’-TATCATTTGCGATGTGTCGAAGAAGAATGGCGCCAAGT-3’
gdh2-r:5’-TACCGTGGATCCGCTTTAAG-3’(BamHI)
1) inoculation carries Escherichia coli the S17-1 λ pir and bacillus licheniformis 10-1-A for knocking out plasmid, at 37 DEG C
Bacterial strain is cultivated to OD600=1.2.By 4~7 milliliters of Escherichia coli bacteria liquids and 1 milliliter of bacillus licheniformis 6000 revs/min from
It the heart 5 minutes, is washed twice with the phosphate buffer of 50mM pH 7.5, is resuspended and is centrifuged after being mixed, then use LB culture medium
It is resuspended and drips on LB plate, be incubated overnight at 30 DEG C.After the LB liquid medium preheated with 30 DEG C collects cell, dilution 10-4
With 10-5It is coated on the LB solid plate of addition erythromycin and polymyxin B, 30 DEG C are cultivated 48~72 hours.
2) transformant is chosen to the LB culture medium for adding Erythromycinresistant, and 37 DEG C are cultivated 6~12 hours, are then washed with LB culture medium
42 DEG C culture 6~12 hours in fresh LB are forwarded to after one time, then gradient dilution to erythromycin LB plate, 42 DEG C of mistakes
42 DEG C of transformant cultures are chosen in night culture, and the single colonie of picking growth carries out PCR verifying using upstream and downstream primer, and obtaining can be simultaneously
PCR goes out the correct single-swap purpose bacterium of long segment and short-movie section.
3) transformant is chosen to LB culture medium, and 30 DEG C of non-resistant cultures were simultaneously transferred for two generations, dilution 10-5With 10-6Apply plate extremely
LB, 37 DEG C after culture 12 hours, are chosen white transformant to 50 DEG C and cultivate and carry out Molecular, carried out using upstream and downstream primer
PCR verifying, the purpose bacterium of double crossing over, energy PCR go out short-movie section (the upstream and downstream homology arm of building), select correct double crossing over bacterium,
Phenotype verifying is carried out, the engineered strain for correctly knocking out bacillus licheniformis 10-1-A glycerol dehydrogenase gdh gene is obtained
Bacillus licheniformisΔgdh。
(2) knockout of 2,3- butanediol dehydrogenase budC gene
2,3-butanediol dehydrogenase gene budC sequence length is 783 bases, nucleotide sequence such as SEQ ID NO.2
It is shown.
The knockout step of gdh, primer sequence are as follows in knockout step reference the present embodiment step (1) of gene budC:
budC1-f:5’-AAACCATGGAATAAACGAGTTGACGGAAA-3’(NcoI)
budC1-r:5’-GCAAAGCAATTGCGGTTAAATTGCATTAAAACGCTTATCC-3’
budC2-f:5’-GGATAAGCGTTTTAATGCAATTTAACCGCAATTGCTTTGC-3’
budC2-r:5’-TTTGGATCCTATGCTCGCGGTGTTCTAT-3’(BamHI)
Glycerol dehydrogenase gdh gene and 2,3- butanediol dehydrogenase budC gene in the correct knockout bacillus licheniformis
Engineered strain be named as Bacillus licheniformis Δ gdh Δ budC.
(3) Bacillus is replaced with nadh oxidase gene nox in Thermococcus profundus DT5432
Lactic acid dehydrogenase gene ldh in licheniformis Δ gdh Δ budC bacterial strain
Nadh oxidase gene nox sequence length is 1320 bases, and nucleotide sequence is as shown in SEQ ID NO.3.
Lactic dehydrogenase ldh sequence length is 960 bases, and nucleotide sequence is as shown in SEQ ID NO.4.
The genomic DNA of B.licheniformis 10-1-A is prepared using conventional method, which goes out with reference to science
The method of bacterial genomes prepared in a small amount, extracts bacillus licheniformis in " the fine works Molecular Biology " that version society publishes
The genomic DNA of Bacillus licheniformis10-1-A；With the gene of Bacillus licheniformis10-1-A
Group DNA is that template utilizes primer " ldh::nox1-f and ldh::nox1-r " and " ldh::nox3-f and ldh::nox3-r " PCR
Expand the upstream and downstream homology arm of replacement gene nox.In the Thermococcus profundus DT5432 of full genome synthesis
The gene of nadh oxidase nox is that template utilizes primer " ldh::nox2-f and ldh::nox2-r " to amplify interlude replacement base
Because of nox.Then " gdh1-f and gdh2-r " primer is utilized, using upstream homology arm obtained and interlude nox gene as template
It carries out recombinant PCR and obtains 1-2 segment." gdh1-f and gdh3-r " primer is finally utilized, it is homologous with the 1-2 segment of acquisition and downstream
Arm pieces section is the gene replacement segment that template carries out that recombinant PCR obtains Δ ldh::nox, and both ends include the digestion of BamHI and SmaI
Site.
BamHI and SmaI is used to carry out double enzymes respectively the gene replacement segment of Δ ldh::nox and suicide plasmid pKVM1
It cuts, digestion products use T after glue recycles4DNA ligase connection, obtains gene replacement plasmid pKVM1- Δ ldh::nox.
Wherein, the design of primers for expanding recombinant fragment is as follows:
ldh::nox1-f:5’-AATGGATCCTAGCGTAGCAAGCAGTGTTCCGCTGTT-3’(BamHI)
ldh::nox1-r:5’-TGACGACTCTTTTGCGTTCCATGACTCATCATTCCTTTGCCG-3’
ldh::nox2-f:5’-CGGCAAAGGAATGATGAGTCATGGAACGCAAAAGAGTCGTCA-3’
ldh::nox2-r:5’-AGTATCTTCATGGTGTTCAGTTAAAATTTCAGGACGCGCG-3’
ldh::nox3-f:5’-CGCGCGTCCTGAAATTTTAACTGAACACCATGAAGATACT-3’
ldh::nox3-r:5’-AATCCCGGGCGGTGACCAAGCGTTCATAAT-3’(SmaI)
It is de- that nadh oxidase gene nox replaces the lactic acid in Bacillus licheniformis Δ gdh Δ budC bacterial strain
Knockout step of the gene replacement step of hydrogen enzyme ldh referring to gdh in the present embodiment step (1), final acquisition recombination lichens gemma
Bacillus Bacillus licheniformis Δ gdh Δ budC Δ ldh::nox.
Experiment confirms: above-mentioned Bacillus licheniformis Δ gdh Δ budC Δ ldh::nox genetic engineering bacterium is
Gram-positive bacteria, optimum culturing temperature are
Further, above-mentioned bacillus licheniformis Bacillus licheniformis Δ gdh Δ budC Δ ldh::nox
The growth of aerobic or amphimicrobian, protein secretion ability is strong, and growth rate is fast, is a kind of generally acknowledged bio-safety bacterium.Its form is
Rod shape, 1.5 μm~3.0 μm long, 0.6 μm~0.7 μm of diameter, colony colour is red or white, produces gemma, VP reaction is in sun
Property, acid is produced using glucose, sucrose, fructose, hydrolyzable casein, gelatin, Tween 80 can contain using citrate
It grows, can be grown under the conditions of 42~60 DEG C in the culture medium of 100g/L NaCl.
Embodiment 2: bacillus licheniformis Bacillus licheniformis Δ gdh Δ budC Δ ldh::nox bacterial strain exists
The rotational speed optimization of 1L fermentation cylinder for fermentation production 3-hydroxy-2-butanone
It is bacterium germination with wild type bacillus licheniformis Bacillus licheniformis 10-1-A referring to embodiment 1
Strain knocks out glycerol dehydrogenase gdh gene and 2,3-butanediol dehydrogenase gene budC, expresses Thermococcus profundus
Nadh oxidase gene nox replaces lactic dehydrogenase ldh in DT5432, is named as bacillus licheniformis Bacillus
licheniformisΔgdhΔbudCΔldh::nox。
It (1) will recombination bacillus licheniformis Bacillus licheniformis Δ gdh Δ budC Δ ldh::nox scribing line
To containing mass volume ratio be 1.5~1.8% agar LB culture medium flat plate on, it is small in 50 ± 1 DEG C of shaking table shaken cultivations 12 ± 1
When；
(2) under sterile conditions, with the single colonie on sterile toothpick picking step (1) plate, it is then seeded into 5mL
LB culture medium in, in 50 ± 1 DEG C shaking table shaken cultivation 12 ± 1 hours, obtain recombination bacillus licheniformis first order seed；
(3) under sterile conditions, the resulting first order seed bacterium solution of step (2) is taken, the inoculation for being 2~4% with volume ratio
Amount be inoculated into the LB culture medium of two bottles of 100mL, in 50 ± 1 DEG C shaking table shaken cultivation 12 ± 1 hours, obtain recombination lichens gemma
Bacillus secondary seed bacterium solution；
(4) under sterile conditions, the resulting secondary seed bacterium solution of step (3) is taken, is connect with the inoculum concentration that volume ratio is 5%
Kind is into three 1 liter of fermentors equipped with 0.8 liter of sterilized fermentation medium, and under the conditions of 50 DEG C, ventilatory capacity is
1.0vvm, fermentor speed of agitator are respectively 400 revs/min, 500 revs/min and 600 revs/min, are fermented 18~36 hours.
It was sampled in fermentation every 3 hours, detects OD600nm.With 8,000 × g is centrifuged 10 minutes sample, and it is even to detect second in supernatant
The concentration of relation by marriage and glucose adds glucose dry powder according to concentration of glucose, concentration of glucose is made to maintain 20~50 grams per liters；
The concentration for carrying out 3-hydroxy-2-butanone in gas chromatographic analysis measurement fermentation liquid to supernatant stops when the concentration of 3-hydroxy-2-butanone is not further added by
Fermentation.
Testing result shows Bacillus lichenformis Δ gdh Δ budC Δ after 36 hours
The optimum speed condition of ldh::nox fermenting and producing 3-hydroxy-2-butanone is 500 revs/min.
Above-mentioned fermentation medium composition are as follows: 50~80 grams per liter of glucose, 12 grams per liter of yeast powder, 6.5 grams of anhydrous sodium acetate/
It rises, 1 grams per liter of ammonium citrate, 2 grams per liter of dipotassium hydrogen phosphate, 0.25 grams per liter of epsom salt, 10 ml ls of metal ion mother liquor,
Surplus is water；PH is 6~7.
Wherein, above-mentioned metal ion mother liquor formula is: 2.25 grams per liter of ferrous sulfate, 0.75 grams per liter of zinc sulfate, manganese sulfate
0.38 grams per liter.
Embodiment 3: bacillus licheniformis Bacillus licheniformis Δ gdh Δ budC Δ ldh::nox bacterial strain exists
1L fermentation cylinder for fermentation produces the ventilatory capacity optimization of 3-hydroxy-2-butanone
It is bacterium germination with wild type bacillus licheniformis Bacillus licheniformis 10-1-A referring to embodiment 1
Strain knocks out glycerol dehydrogenase gdh gene and 2,3-butanediol dehydrogenase gene budC, expresses Thermococcus profundus
Nadh oxidase gene nox replaces lactic dehydrogenase ldh in DT5432, is named as bacillus licheniformis Bacillus
licheniformisΔgdhΔbudCΔldh::nox。
It (1) will recombination bacillus licheniformis Bacillus licheniformis Δ gdh Δ budC Δ ldh::nox scribing line
To containing mass volume ratio be 1.5~1.8% agar LB culture medium flat plate on, it is small in 50 ± 1 DEG C of shaking table shaken cultivations 12 ± 1
When；
(2) under sterile conditions, with the single colonie on sterile toothpick picking step (1) plate, it is then seeded into 5mL
LB culture medium in, in 50 ± 1 DEG C shaking table shaken cultivation 12 ± 1 hours, obtain recombination bacillus licheniformis first order seed；
(3) under sterile conditions, the resulting first order seed bacterium solution of step (2) is taken, the inoculation for being 2~4% with volume ratio
Amount be inoculated into the LB culture medium of two bottles of 100mL, in 50 ± 1 DEG C shaking table shaken cultivation 12 ± 1 hours, obtain recombination lichens gemma
Bacillus secondary seed bacterium solution；
(4) under sterile conditions, the resulting secondary seed bacterium solution of step (3) is taken, is connect with the inoculum concentration that volume ratio is 5%
Kind is into three 1 liter of fermentors equipped with 0.8 liter of sterilized fermentation medium, and under the conditions of 50 DEG C, fermentor stirring turns
Speed is 500 revs/min, and ventilatory capacity is respectively 0.5vvm, 1.0vvm and 1.5vvm, is fermented 36 hours.
It was sampled in fermentation every 3 hours, detects OD600nm.With 8,000 × g is centrifuged 10 minutes sample, and it is even to detect second in supernatant
The concentration of relation by marriage and glucose adds glucose dry powder according to concentration of glucose, concentration of glucose is made to maintain 20~50 grams per liters；
The concentration for carrying out 3-hydroxy-2-butanone in gas chromatographic analysis measurement fermentation liquid to supernatant stops when the concentration of 3-hydroxy-2-butanone is not further added by
Fermentation.
Testing result shows Bacillus lichenformis Δ gdh Δ budC Δ after 36 hours
The best ventilatory capacity condition of ldh::nox fermenting and producing 3-hydroxy-2-butanone is 1.0vvm.
Above-mentioned fermentation medium composition are as follows: 50~80 grams per liter of glucose, 12 grams per liter of yeast powder, 6.5 grams of anhydrous sodium acetate/
It rises, 1 grams per liter of ammonium citrate, 2 grams per liter of dipotassium hydrogen phosphate, 0.25 grams per liter of epsom salt, 10 ml ls of metal ion mother liquor,
Surplus is water；PH is 6~7.
Wherein, above-mentioned metal ion mother liquor formula is: 2.25 grams per liter of ferrous sulfate, 0.75 grams per liter of zinc sulfate, manganese sulfate
0.38 grams per liter.
Embodiment 4: bacillus licheniformis Bacillus licheniformis Δ gdh Δ budC Δ ldh::nox bacterium is utilized
Strain produces 3-hydroxy-2-butanone in 5L fermentation cylinder for fermentation with fermentation medium
It is bacterium germination with wild type bacillus licheniformis Bacillus licheniformis 10-1-A referring to embodiment 1
Strain knocks out glycerol dehydrogenase gdh gene and 2,3-butanediol dehydrogenase gene budC, expresses Thermococcus profundus
Nadh oxidase gene nox replaces lactic dehydrogenase ldh in DT5432, is named as bacillus licheniformis Bacillus
licheniformisΔgdhΔbudCΔldh::nox。
It (1) will recombination bacillus licheniformis Bacillus licheniformis Δ gdh Δ budC Δ ldh::nox scribing line
To containing mass volume ratio be 1.5~1.8% agar LB culture medium flat plate on, it is small in 50 ± 1 DEG C of shaking table shaken cultivations 12 ± 1
When；
(2) under sterile conditions, with the single colonie on sterile toothpick picking step (1) plate, it is then seeded into 5mL
LB culture medium in, in 50 ± 1 DEG C shaking table shaken cultivation 12 ± 1 hours, obtain recombination bacillus licheniformis first order seed；
(3) under sterile conditions, the resulting first order seed bacterium solution of step (2) is taken, the inoculation for being 2~4% with volume ratio
Amount be inoculated into the LB culture medium of two bottles of 100mL, in 50 ± 1 DEG C shaking table shaken cultivation 12 ± 1 hours, obtain recombination lichens gemma
Bacillus secondary seed bacterium solution；
(4) under sterile conditions, the resulting secondary seed bacterium solution of step (3) is taken, with the inoculum concentration of percent by volume 5%
It is seeded in 5 liters of fermentors equipped with 4 liters of sterilized fermentation mediums, under the conditions of 50 DEG C, fermentor speed of agitator 500
Rev/min, ventilatory capacity 1.0vvm ferments 36 hours.
It was sampled in fermentation every 3 hours, detects OD600nm.With 8,000 × g is centrifuged 10 minutes sample, and it is even to detect second in supernatant
The concentration of relation by marriage and glucose adds glucose dry powder according to concentration of glucose, concentration of glucose is made to maintain 20~50 grams per liters；
The concentration for carrying out 3-hydroxy-2-butanone in gas chromatographic analysis measurement fermentation liquid to supernatant stops when the concentration of 3-hydroxy-2-butanone is not further added by
Fermentation.
Testing result is shown after 36 hours: the concentration of 3-hydroxy-2-butanone reaches 57.98 grams per liters, production efficiency be 1.61 grams per liters/
Hour.
Above-mentioned fermentation medium composition are as follows: 50~80 grams per liter of glucose, 12 grams per liter of yeast powder, 6.5 grams of anhydrous sodium acetate/
It rises, 1 grams per liter of ammonium citrate, 2 grams per liter of dipotassium hydrogen phosphate, 0.25 grams per liter of epsom salt, 10 ml ls of metal ion mother liquor,
Surplus is water；PH is 6~7.
Wherein, above-mentioned metal ion mother liquor formula is: 2.25 grams per liter of ferrous sulfate, 0.75 grams per liter of zinc sulfate, manganese sulfate
0.38 grams per liter.
Embodiment 5: bacillus licheniformis Bacillus licheniformis Δ gdh Δ budC Δ ldh::nox bacterium is utilized
Strain produces 3-hydroxy-2-butanone in 50L fermentation cylinder for fermentation with fermentation medium
It is bacterium germination with wild type bacillus licheniformis Bacillus licheniformis 10-1-A referring to embodiment 1
Strain knocks out glycerol dehydrogenase gdh gene and 2,3-butanediol dehydrogenase gene budC, expresses thermophilic coccus Thermococcus
Nadh oxidase gene nox replaces lactic dehydrogenase ldh in profundus DT5432, is named as bacillus licheniformis
Bacillus licheniformisΔgdhΔbudCΔldh::nox。
It (1) will recombination bacillus licheniformis Bacillus licheniformis Δ gdh Δ budC Δ ldh::nox scribing line
To containing mass volume ratio be 1.5~1.8% agar LB culture medium flat plate on, it is small in 50 ± 1 DEG C of shaking table shaken cultivations 12 ± 1
When；
(2) under sterile conditions, with the single colonie on sterile toothpick picking step (1) plate, it is then seeded into 5mL
LB culture medium in, in 50 ± 1 DEG C shaking table shaken cultivation 12 ± 1 hours, obtain recombination bacillus licheniformis first order seed；
(3) under sterile conditions, the resulting first order seed bacterium solution of step (2) is taken, the inoculation for being 2~4% with volume ratio
Amount be inoculated into the LB culture medium of 50mL, in 50 ± 1 DEG C shaking table shaken cultivation 12 ± 1 hours, obtain recombination bacillus licheniformis
Secondary seed bacterium solution；
(4) under sterile conditions, the resulting secondary seed bacterium solution of step (3) is taken, the inoculation for being 2~4% with volume ratio
Amount be inoculated into the LB culture medium of two bottles of 1L, in 50 ± 1 DEG C shaking table shaken cultivation 12 ± 1 hours, obtain recombination lichens gemma bar
Bacterium three-level seed bacterium solution；
(5) under sterile conditions, the resulting three-level seed bacterium solution of step (4) is taken, with the inoculum concentration of percent by volume 5%
It is seeded in 50 liters of fermentors equipped with 40 liters of sterilized fermentation mediums, under the conditions of 50 DEG C, fermentor speed of agitator
It 500 revs/min, ventilatory capacity 1.0vvm, ferments 36 hours.
It was sampled in fermentation every 3 hours, detects OD600nm.With 8,000 × g is centrifuged 10 minutes sample, and it is even to detect second in supernatant
The concentration of relation by marriage and glucose adds glucose dry powder according to concentration of glucose, concentration of glucose is made to maintain 20~50 grams per liters；
The concentration for carrying out 3-hydroxy-2-butanone in gas chromatographic analysis measurement fermentation liquid to supernatant stops when the concentration of 3-hydroxy-2-butanone is not further added by
Fermentation.
Testing result is shown after 36 hours: the concentration of 3-hydroxy-2-butanone reaches 82.14 grams per liters, production efficiency be 2.28 grams per liters/
Hour.
Above-mentioned fermentation medium composition are as follows: 50~80 grams per liter of glucose, 12 grams per liter of yeast powder, 6.5 grams of anhydrous sodium acetate/
It rises, 1 grams per liter of ammonium citrate, 2 grams per liter of dipotassium hydrogen phosphate, 0.25 grams per liter of epsom salt, 10 ml ls of metal ion mother liquor,
Surplus is water；PH is 6~7.
Wherein, above-mentioned metal ion mother liquor formula is: 2.25 grams per liter of ferrous sulfate, 0.75 grams per liter of zinc sulfate, manganese sulfate
0.38 grams per liter.
Sequence table
<110>Shandong University Shenzhen Shandong University, research institute
<120>a kind of method using bacillus licheniformis engineered strain fermenting and producing 3-hydroxy-2-butanone
<141> 2018-12-05
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1104
<212> DNA
<213>bacillus licheniformis (Bacillus licheniformis) 10-1-A
<221>glycerol dehydrogenase gene gdh sequence
<400> 1
ggaagcgtcc gtttgttgag catcgtctcg gcggagacca tgaccccttc atcaacaagc 60
gtccggctgt tttcgatcat tttttcgaaa tagcgcgccc gctgtgcaaa cgtcggcttt 120
ttatccatca tagcgaagcc gatttctgca aagtcttcga ccgttcccca gttgtgggaa 180
atatgaagca catccaggta ggggatgatc agctcgtagc ggccgatatc gagcgtcaag 240
tttgagttga tctgcgtccg cacgccgcgt tcatgagcgt attttaataa aggaacgaca 300
tattccttta cagattttaa tgagagcatc ggctcgccgc ccgtaatgct taaagaacga 360
aggcggggaa tctcatcaag ccgtttcaac agaaggctga ctggcagcgc gtccgggtcc 420
ttcgtctgga gcgtataccc gacggcgcag tgctcgcagc gcatattgca aagagtcgtc 480
gtcgtaaatt caacgttcgt cagctgtata tctccaaatt ggtcaacgtc catgtaggct 540
tcccacggat catattccgg ggtgatcgga cgagcctgca ttttttctgt catgacatat 600
ctaactcctt tatgatagga catactaata tagcccaact attcatttta gacctgatcc 660
ttgaggcatg caagtcatgg atgattgaaa aaaggccgtt tataggctac gataaaggaa 720
caaaaggagg agaatcatgg gtaacgcagt acatgacaaa gaacagcaag tcaattattt 780
gaaaaacaga ttggatatgt ttatgtcagt catcgattct ttagacccgg aatcgaccga 840
ccttgaagat attgacagac tgatcagcat gctcgacgat ttggaagcca aatacgagcg 900
ctttaaaaaa gactggaaat aaaaccgcgc attctctgcc gcggtttttt ttgttttcca 960
agtgttgctt ggtatgaaat gctcagccct tctgcacaat aagaaaaaat aaaactggga 1020
gcgagaagat gatgaaacga atctgtgcca tatgctgcgg attcctgctg acgctggcgt 1080
tcagcggcaa tgctgaagcg attt 1104
<210> 2
<211> 783
<212> DNA
<213>bacillus licheniformis (Bacillus licheniformis) 10-1-A
<221>2,3- butanediol dehydrogenation enzyme gene budC sequence
<400> 2
atgagtaaag tatctggaaa aattgctttt gttactggcg gcggtcaagg aattggagaa 60
gcaatctgca aacgattggc agaggacgga ttcgcagttg cagttgccga ttataatgta 120
gaaactgcaa cacaagttgc tgaggacatc aataagctta acggcaaagc aattgcggtt 180
aaagtggatg ttgctgatcg cgatgatgtt tttaaagctg tcgatgaaac agtaaaacgt 240
cttggcggtc ttgatgtggt gattaataat gcgggtcttg gaccaaccac ccctattgaa 300
agcattacat atgaagatta tcggaaagtc tatgatgtta acgttggcgg tacttattgg 360
ggaatacaag cagctgtaaa agcctttaaa gaacttggac acggcgggaa aatcattaat 420
gcatcttctc aagccggcca agtcggcaac ccgggcttag cggtttacgg aggaacaaag 480
ttcgctgttc gcgggattac ccaaactgcg gcaaaagatc tagctgaatt aggtattact 540
gtaaacgcct tttgtccggg tatcgttaaa actcctatga tgatggggat tgcacagcaa 600
accgctgatg aagcaggcaa gccgtttgaa tggggcatgg aacaattcgc taaaaatatt 660
gcattaaaac gcttatccga gccggaagat gtagcagcat gcgtttctta ccttgcaggg 720
ccagattcag attatatgac tggtcaagct cttatcattg atggcggaat ggtatttaat 780
taa 783
<210> 3
<211> 1320
<212> DNA
<213> Thermococcus profundus DT5432
<221>nadh oxidase gene nox sequence
<400> 3
caagtccgtg gagagaaacg aacccttcca cgtccggttt gagtccaaac tcctccggcg 60
agtgcctcag gatataggcc atcagcttgc ttaccctcgt cctctttgat cccatagtaa 120
aaagttgggg agaaaagcta aaaacctcac ggaagctcca ccagagccca caccggcctg 180
tggtctgaaa cctccacgtc acaaaggcag ccgtagtctt taatttcagc cggccagttc 240
ttcttaagca ggatgtagtc tatgttctct ttgtccctga cgccgttcct ctcccagagg 300
aaggtgtaag gcggcctctg ctcaaatgca tccctgtact cgcgggtgag tatctctatc 360
gctttctcgt ccggctcggc gttcgtgtcg ccgagtatta tctccgctat gggtccgctc 420
tccgcgaact tgaggagctc ctcggcctgc atcgccctct cctcttcgct caaacccata 480
tggacgttca cgagggtgag acccaactcc tcgaagctaa ccttctgggc tggcctggcc 540
tgaccgacgc tcttgaggtt gagctcgccc tccgtcttca tgtgccagtg ggagaagacc 600
gctattccgt aggtgccctc cactgcaggc ttgtactcgt aaacgtagcc gaggtaggcc 660
gagagcatga gcgggacgtc ctggtagccg ttgcctatca tcccaccgac gacctcctgg 720
gacgcccaga tgtcaggttt ctgttctttt aggagattaa caagctcgta gccgttgaac 780
ttcccgtcgt aaggcccgaa accctggtgg acgttgtagg tccagatgag aacctctttt 840
ttggccggtt cgtagaccgg agaggcgttg aagatggcta ggacgattat agaggccagg 900
aggaggcccc cgagggaagc cgctatctcc cttacgcttg gaaccctgat ctccaccgac 960
tttccatagg cgctcatggc gtagactacg gatgccgcaa ggatgagggc ctcaagcctg 1020
tcctccatga aagctaaacc gatgtccctg cccacatagg ccccgagggc aagggtcgcg 1080
accaggaaga gatagaccgc tccgatgact cctcccctgc tgcccttgga gctttcgacg 1140
agagctattg aagatgccag cgcaagcggg aggccgatta aagcggccgg ccttacgaag 1200
agcgccgcag agccaaggat cagcaggagt gcggctatgc caggcttttt gcccagatat 1260
gggccgagga ggatagccag tgcgacgacg aacgagaagc cgacgaactg tgggaggtag 1320
<210> 4
<211> 960
<212> DNA
<213>bacillus licheniformis (Bacillus licheniformis) Δ gdh Δ budC
<221>lactic acid dehydrogenase gene ldh sequence
<400> 4
gtgacgcacg gttatttcta tcaaagggcg cgggaaagcg aaaggcgttt ggcacgcagg 60
gcggtcaacg gggagcggct ttggccggca aagttcagtt tgtggtattt cagaccggcg 120
ggggcgtgtc ccgcacagtg gtacaatcag ccgcatgtcg gccgctttaa atcgcattgt 180
ttttacgagc cgacagcgga agagtgtgag aatgtctata atacgttcta acaaaaacat 240
cccttttcaa cggtttttga aaagggatgt ttttcgttca aaataattct gccagtcttt 300
cgtaagactg cttcttatct tcaaaccggt agacgttagt taaaatcata aactcatctg 360
tttgatatcg ttcagaaagc tcgagcagtt ttcttttgac tgtttgagga gacccgatca 420
ccatacggct gcggttttgg gcaagcttct ttttatcggc agctgtgtaa gcggccgatt 480
tcgcctcttc aatgcttggg acaaggctgt ccagcccttt ttcaacccgg agaagccata 540
aatcttggct caagcagagc tcctcggctt tttcatcgga ctccgcacaa acgacgaaaa 600
cggcggccaa tgattggggg cgtttaaaga aaggggaagg ccgaaagctt tcacggtatg 660
attgaaaggc gttctttcct ctcgcgggag agatgaaatg gccgaatacg tatcccgctc 720
ccatgctacc ggccaatttt gcgctgtttt ctcccaggcc taaaagccac acctccgggg 780
gcgaatccgt caaaggggag gctttaattc cgaagtaatc atgtccggaa ggcacggaat 840
cagtcagaaa gctaaccaag tcttgtaact gtctcgggaa ttcgtgcagg cttttatgaa 900
ccccgtctgt cagcgccagc ctcgttttag ttgtgcctcc cggagagcgg ccgatcccca 960
Claims (3)
1. a kind of method using bacillus licheniformis engineered strain fermenting and producing 3-hydroxy-2-butanone, step is:
(1) it is bacterium germination with bacillus licheniformis (Bacillus licheniformis) 10-1-A, knocks out bacillus licheniformis
(Bacillus licheniformis) 10-1-A glycerol dehydrogenase gdh gene and 2,3- butanediol dehydrogenation enzyme gene budC and
To replace lactic acid dehydrogenase gene ldh from nadh oxidase gene nox in Thermococcus profundus DT5432
The bacillus licheniformis engineered strain that can be used to produce 3-hydroxy-2-butanone is constructed, which is named as bacillus licheniformis
Bacillus licheniformis ΔgdhΔbudCΔldh::nox；
Wherein, starting strain bacillus licheniformis (Bacillus licheniformis) 10-1-A is in November, 2011
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number within 14th are as follows: CGMCC NO.5461；
(2) using glucose as substrate, biofermentation bacillus licheniformis Bacillus licheniformis Δ gdh Δ budC
Δ ldh::nox obtains 3-hydroxy-2-butanone by fermentation liquid；
Wherein, the fermentation condition is: cultivation temperature is 50 ± 1 DEG C, and training method is stir culture, speed of agitator is 500 ±
50 revs/min, radius of turn is 33 ± 1 millimeters, and ventilatory capacity is 1.0 ± 0.1vvm, and incubation time is 18~36 hours；
Fermentation medium composition are as follows: 50~80 grams per liter of glucose, 12 grams per liter of yeast powder, 6.5 grams per liter of anhydrous sodium acetate, lemon
Sour 1 grams per liter of ammonium, 2 grams per liter of dipotassium hydrogen phosphate, 0.25 grams per liter of epsom salt, 10 ml ls of metal ion mother liquor, surplus is
Water；PH is 7.0；Metal ion mother liquor formula therein is: 2.25 grams per liter of ferrous sulfate, 0.75 grams per liter of zinc sulfate, manganese sulfate
0.38 grams per liter.
2. the method for utilizing bacillus licheniformis engineered strain fermenting and producing 3-hydroxy-2-butanone according to claim 1, feature exist
In the construction method of step (1) the engineered strain Bacillus licheniformis Δ gdh Δ budC Δ ldh::nox
It is:
Using bacillus licheniformis 10-1-A genome as template, PCR obtains the recombination piece comprising gdh gene upstream and downstream homologous sequence
Section, is connected to after pKVM1 and is transferred to bacillus licheniformis 10-1-A through biparent cross, through resistance screening single-swap transformant；Temperature
After double crossing over occurs for induction, PCR screening obtains gdh gene successful knockout bacterial strain, is named as bacillus licheniformis Bacillus
licheniformis Δgdh；
Using bacillus licheniformis 10-1-A genome as template, PCR obtains the recombination comprising budC gene upstream and downstream homologous sequence
Segment is connected to after pKVM1 and is transferred to bacillus licheniformis licheniformis Δ gdh through biparent cross, through resistance screening list
Exchange transformant；After temperature-induced generation double crossing over, PCR screening obtains budC gene successful knockout bacterial strain, is named as lichens bud
Spore bacillus Bacillus licheniformis Δ gdh Δ budC.
Using bacillus licheniformis 10-1-A genome as template, it includes ldh gene upstream and downstream homology arm sequence that PCR, which is obtained,；With complete
The plasmid pUC57-nox of the carrying nadh oxidase of gene chemical synthesis is template, and PCR obtains nox sequence, by ldh gene upstream and downstream
Homology arm sequence and nox sequence recombinant PCR obtain gene replacement recombinant fragment, are connected to after pKVM1 and are transferred to ground through biparent cross
Clothing bacillus licheniformis Δ gdh Δ budC, through resistance screening single-swap transformant；It is temperature-induced
After double crossing over occurs, PCR screening obtains the engineered strain that nox gene successfully replaces ldh gene, is named as bacillus licheniformis
Bacillus licheniformis ΔgdhΔbudCΔldh::nox。
3. the method for utilizing bacillus licheniformis engineered strain fermenting and producing 3-hydroxy-2-butanone according to claim 1, feature exist
In step (2) described fermentation condition is: cultivation temperature is 50 DEG C, and training method is stir culture, and speed of agitator is 500 revs/min
Clock, radius of turn are 33 millimeters, ventilatory capacity 1.0vvm, and incubation time is 25~36 hours.
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