CN107177620A - A kind of method that utilization cheap raw material produces Tetramethylpyrazine - Google Patents
A kind of method that utilization cheap raw material produces Tetramethylpyrazine Download PDFInfo
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- CN107177620A CN107177620A CN201710509031.3A CN201710509031A CN107177620A CN 107177620 A CN107177620 A CN 107177620A CN 201710509031 A CN201710509031 A CN 201710509031A CN 107177620 A CN107177620 A CN 107177620A
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- Prior art keywords
- hydroxy
- butanone
- tetramethylpyrazine
- gene
- raw material
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- FINHMKGKINIASC-UHFFFAOYSA-N Tetramethylpyrazine Chemical compound CC1=NC(C)=C(C)N=C1C FINHMKGKINIASC-UHFFFAOYSA-N 0.000 title claims abstract description 110
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- 238000000034 method Methods 0.000 title claims abstract description 32
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- WTLNOANVTIKPEE-UHFFFAOYSA-N 2-acetyloxypropanoic acid Chemical compound OC(=O)C(C)OC(C)=O WTLNOANVTIKPEE-UHFFFAOYSA-N 0.000 claims abstract description 14
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
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- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
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Abstract
The invention discloses a kind of method that utilization cheap raw material produces Tetramethylpyrazine, the nucleotide sequence of α acetolactate synthase genes, α acetolactate decarboxylases gene and nadh oxidase gene is subjected to codon optimization；Splicing obtains the gene cluster for including three genes；Gene cluster is inserted in expression vector, polycistronic expression plasmid is obtained；Host Strains are imported againE. coli, obtain the engineering strain of production 3-hydroxy-2-butanone；The inoculation of activation is subjected to fermented and cultured to the fermentation medium containing cheap raw material, the zymotic fluid containing 3-hydroxy-2-butanone is obtained；Take zymotic fluid to carry out centrifugal treating, take supernatant, add diammonium hydrogen phosphate, ferment the 3-hydroxy-2-butanone produced and NH4 +Reaction synthesis Tetramethylpyrazine.The bacterial strain that the present invention is provided can effectively utilize wide material sources, raw material production high concentration of precursor material 3-hydroxy-2-butanone with low cost, oxidized coenzyme NAD+Can effective regeneration, 3-hydroxy-2-butanone yield and production efficiency can be improved, thus effectively reduce Tetramethylpyrazine production cost, shorten the production cycle.
Description
Technical field
The invention belongs to biological chemical field, the method that specifically a kind of utilization cheap raw material produces Tetramethylpyrazine.
Background technology
Tetramethylpyrazine (2,3,5,6-tetramethylpyrazine) also known as ligustrazine, are the masters of chuanxiong rhizome
Active bio alkali composition is wanted, good with expansion blood vessel, improvement microcirculation, the aggregation of suppression platelet adhesion reaction and thrombosis etc.
Pharmacological action, has certain curative effect, conduct in terms for the treatment of cardiovascular and cerebrovascular disease, respiratory disease and renal glomerular disease
Standing medicine is widely used in clinic.In addition, Tetramethylpyrazine be naturally occurring in dairy products, bean product, cocoa, coffee, peanut,
In fibert and white wine, the peat-reek with baking and nut.It is important fragrance in China white wine that Tetramethylpyrazine, which is also,
Compound, detects a certain amount of Tetramethylpyrazine in Chinese traditional liquor, and it not only has important to Chinese liquor flavor
Contribution, but also impart the wholesome function of China white wine.Tetramethylpyrazine Low Flavour Threshold is extremely low, national standard
GB2760-1996 provides that it is the flavorant for allowing to use, and is now widely used for baked goods, cold drink, meat, breast system
The allotment of the essence such as product, cigarette.
The production method of Tetramethylpyrazine can be divided into plant extraction method, chemical method and biological synthesis process.Utilize the side of extraction
Method can extract Tetramethylpyrazine from Chinese medicine Rhizome of Ligusticum Sinense Oliv. Cv. Chuanxiong, but Ligusticum wallichii plant origin is limited, and the tetramethyl in Rhizome of Ligusticum Sinense Oliv. Cv. Chuanxiong
Base levels of pyrazine is relatively low, it is impossible to which realization is commercially produced on a large scale.The reaction condition of chemical method synthesis Tetramethylpyrazine is general
Relatively acutely, generally existing severeer environmental issue higher to equipment requirement, and product is not belonging to " natural " or " biosynthesis "
Category, security and quality problem are constantly queried.The Tetramethylpyrazine of biochemical industry method production has product
The advantages of green natural, cost are low, reaction condition is gentle, environmental pollution is small, with people's increasingly carrying to green product demand
Height, the method is received more and more attention.However, due to producing second accidental cause using microbial fermentation and being translated into tetramethyl
The correlative study of pyrazine is started late, and the technology for producing Tetramethylpyrazine by biotechnology approach is ripe far away.
Chinese patent 201010238685.5 discloses a kind of method for producing Tetramethylpyrazine and its production bacterial strain, screening
Bacillus subtilis XZ1124, Bacillus licheniformis BL-L1, MT-6 and MT-15, utilize grape
When sugar is that carbon source, yeast extract are nitrogen source, second accidental cause accumulation is 30-44g/L in fermentation tank, but the cycle is up to 72h, and secondary
The accumulation of product causes the conversion ratio of glucose too low.Zymotic fluid centrifuging and taking supernatant 2 times of corresponding 3-hydroxy-2-butanone molar concentrations of addition
Diammonium hydrogen phosphate, in 55-90 DEG C of shaking bath (rotating speed 150rpm) reaction 20h, the concentration of Tetramethylpyrazine is 11-20g/L.Should
Invented technology is relatively simple, but used strain is wild-type strain, can accumulate various accessory substances in fermentation process, reduce
The conversion ratio of raw material, and then have impact on the yield of Tetramethylpyrazine.When microbial fermentation produces 3-hydroxy-2-butanone, glycolytic cycle can be produced
Raw a large amount of reduced coenzyme NADH, and the generation of 3-hydroxy-2-butanone and Tetramethylpyrazine can not consume NADH, therefore microorganism is thin
Born of the same parents can start the route of synthesis of the accessory substances such as lactic acid, acetic acid, ethanol, succinic acid to consume excessive NADH, realize that oxidized form is auxiliary
Enzyme NAD+ regeneration, the accumulation of these accessory substances not only reduces the conversion ratio and Tetramethylpyrazine yield of raw material, can also suppress
The growth and fermentation of microorganism.
In order to improve the yield of 3-hydroxy-2-butanone and Tetramethylpyrazine, it is necessary to optimize intracellular NADH/NAD using coenzyme engineering strategy
+ ratio, such as expression nadh oxidase is directly catalyzed NADH and is converted into NAD+, and blocked simultaneously using metabolic engineering technological means
Or weaken the route of synthesis of Main By product.In addition, the invention needs to use the yeast extract of high price for nitrogen source, and carbon source grape
Sugar be high-purity sugar, there is also cost it is higher the problem of, reduce further the economic feasibility of the method.The culture of microbial fermentation
Based component influences very big to production cost, and the use of high-purity sugar (glucose) can increase cost of material.In addition, dusty yeast/yeast
Cream comes fermenting and producing 3-hydroxy-2-butanone and tetramethyl due to rich in nutritional ingredients such as amino acid, vitamin, trace elements, being often used as nitrogen source
Base pyrazine, but it using that would generally greatly increase production cost, such as dusty yeast cost can account for total fermentation in some lactic fermentations
The 30% of cost.Therefore, also need to develop cheap nitrogen source (such as cotton seed meal) during fermentation technology optimization to replace dusty yeast/ferment
The high price nitrogen source such as female cream.In summary, using coenzyme engineering optimization intracellular redox state, master is blocked using metabolic engineering
Want the route of synthesis of accessory substance, and the cheap raw material that exploitation can be utilized efficiently, the industrialization for realizing Tetramethylpyrazine
Production is particularly important.
The content of the invention
The byproducts build-up of the invention existed for Tetramethylpyrazine production is excessively, Tetramethylpyrazine yield is low, produce week
Phase is oversize or cost of material is too high, it is difficult to which the not enough of industrialized production produces Tetramethylpyrazine there is provided one kind using cheap raw material
Method.The present invention carries out rational genetic modification using metabolic engineering and coenzyme regulation and control to Key Metabolic node, weakens main
Accessory substance route of synthesis strengthens second accidental cause route of synthesis, and effectively adjusts intracellular redox state, improves precursor substance second
The yield of accidental cause, reaches the purpose of high yield Tetramethylpyrazine.The present invention also provide by the use of non-grain tapioca starch and cheap nitrogen source as
Fermentation raw material produces Tetramethylpyrazine, and instead of high-purity sugar and high price nitrogen source, cost of material can be greatly reduced.
In order to realize the above object the technical solution adopted by the present invention is as follows：
A kind of method that utilization cheap raw material produces Tetramethylpyrazine, comprises the following steps：
(1) by the nucleosides of α-acetolactate synthase gene, alpha -acetolactate decarboxylase gene and nadh oxidase gene
Acid sequence carries out codon optimization；
(2) by α-acetolactate synthase gene through codon optimization, alpha -acetolactate decarboxylase gene and NADH oxygen
Change enzyme gene and carry out the gene cluster that splicing acquisition includes three genes；
(3) gene cluster is inserted in expression vector, obtains polycistronic expression plasmid；
(4) polycistronic expression plasmid is imported into Host Strains E.coli, obtains the engineering strain of production 3-hydroxy-2-butanone；
(5) tapioca starch, nitrogen source raw material are hydrolyzed, obtain fermentation medium, by the gene work of the production 3-hydroxy-2-butanone of activation
Journey inoculation to fermentation medium carries out fermented and cultured, obtains the zymotic fluid containing 3-hydroxy-2-butanone；
(6) take above-mentioned zymotic fluid to carry out centrifugal treating, take supernatant, add diammonium hydrogen phosphate, 3-hydroxy-2-butanone and NH4 +Reaction
Synthesize Tetramethylpyrazine.
Further, the α-acetolactate synthase gene, alpha -acetolactate decarboxylase gene and nadh oxidase base
The core containing ribosome bind site and intervening sequence is added in comprising the concrete steps that for the codon optimization of cause before each gene
Nucleotide sequence TAAGGAGGATATACA.
In the cell, ribosomes is responsible for mRNA translating into protein, and 16S rRNA are a ribosomal subunits, and it can
Accurately recognized by base complementrity principle with ribosome bind site, TAAGGAGGATATAC sequences are added before gene, can
To strengthen identification and the binding ability of ribosomes and mRNA, and appropriate intervening sequence can also improve translation efficiency, identical
Transcriptional level under, improve the vigor of α-acetolactate synthestase, alpha -acetolactate decarboxylase and nadh oxidase, enzymatic is anti-
It should accelerate, the final synthesis capability for improving 3-hydroxy-2-butanone.
Further, the Host Strains E.coli is the mutant strain of polygene deletion, is closed by being superimposed in knock-out bacterial strain
Obtained into the key gene of accessory substance；The accessory substance includes 2,3- butanediols, succinic acid, lactic acid and acetic acid；The synthesis
The key gene of accessory substance includes gldA, frdABCD, ldhA and pta.
Further, the source strain of described α-acetolactate synthase gene and alpha -acetolactate decarboxylase gene is selected good strains in the field for seed
From Enterobacter cloacae, Klebsiella pneumoniae, Enterobacter aerogenes,
Klebsiella oxytoca, Serratia marcescens and Bacillus licheniformis.
Further, the source strain of described nadh oxidase gene select good strains in the field for seed from Lactobacillus brevis,
Lactococcus lactis, Streptococcus pyogenes, Clostridium aminovalericum and
Bacillus subtilis。
Further, the concrete operation step of the step (5) is：
S1：Tapioca starch, nitrogen source raw material are taken, water, α-amylase and calcium chloride is added, liquefy 0.5-5h at 80-100 DEG C,
Stand cooling；When temperature is reduced to below 55 DEG C, carbohydrase and protease are added, be saccharified 5-20h, regulation at 50-60 DEG C
PH6.5-7.5, centrifuges or is collected by filtration the 15min that sterilized after supernatant, dilution supernatant under the conditions of 115 DEG C, produce fermented and cultured
Base；Based on 1000mL water, the consumption of each component is：Tapioca starch 100-200g, nitrogen source raw material 40-80g, α-amylase 0.5-2mL, chlorine
Change calcium 0.01-0.5g, carbohydrase 0.2-1mL, proteinase-10 .05~0.5g；The tapioca starch is through powder after cassava peeling is dried
Broken 50-200 mesh sieves of crossing are prepared, and the nitrogen source raw material is one or more groups in cottonseed flour, bean cake powder, beancake powder
Close；
S2：Aseptically, 50-100 μ g/mL ampicillins are added in the fermentation medium, then move to fermentation
In tank, the inoculum concentration by the engineering bacteria bacterium solution of activated production 3-hydroxy-2-butanone using volume ratio as 1-10% is inoculated into fermentation medium
In, the fermented and cultured 20-72h under the conditions of 35-42 DEG C of temperature, speed of agitator 200-800rpm, throughput 0.5-1.5vvm works as inspection
When the concentration for surveying 3-hydroxy-2-butanone in zymotic fluid is not further added by, fermentation ends obtain the zymotic fluid containing 3-hydroxy-2-butanone.
Further, the preparation method of the engineering bacteria bacterium solution of the production 3-hydroxy-2-butanone of the activation, comprises the following steps：Will be raw
Production 3-hydroxy-2-butanone engineering strain is scoring to containing mass volume ratio for 1.8% agar and containing 100 μ g/mL ampicillins
On LB flat boards, 10-15h is cultivated at 37 DEG C；Under sterile conditions, a single bacterium colony on picking LB flat boards, is then seeded into
In LB liquid seed culture mediums containing 100 μ g/mL ampicillins, 37 DEG C of shaking table concussion and cultivate 10-15h；The LB cultures
The formula of base is：10g/L peptones, 5g/L dusty yeasts, 10g/L sodium chloride.
Further, the concentration of glucose in zymotic fluid is detected in step S2 fermentation process, when concentration of glucose drop
During to 10-30g/L, adding cassava reduced sugar mother liquor makes concentration of glucose in zymotic fluid be 40-80g/L；The cassava reduced sugar
The concentration of glucose of mother liquor is 500-800g/L.
Further, the concrete operation step of the step (6) is：By the zymotic fluid high speed centrifugation containing 3-hydroxy-2-butanone, go
Except precipitation, take supernatant to determine the content of 3-hydroxy-2-butanone, then add the diammonium hydrogen phosphate of 1-2.5 times of molar concentration of 3-hydroxy-2-butanone, it is molten
Xie Hou, adjusts pH to 7.5,6-20h is reacted in 65-95 DEG C of shaking bath, Tetramethylpyrazine is produced.
Further, the hydrogen peroxide of 0.1-2 times of molar concentration of 3-hydroxy-2-butanone is added in shaking bath course of reaction.
Compared with prior art, advantages of the present invention and have the beneficial effect that：
1st, it is even that the bacterial strain that the present invention is provided can effectively utilize wide material sources, raw material with low cost produces precursor substance second
Relation by marriage, oxidized coenzyme NAD+Can effective regeneration, 3-hydroxy-2-butanone yield and production efficiency can be improved, thus effectively reduce tetramethyl pyrrole
The production cost of piperazine, shortens the production cycle, overcomes the problem of prior art is present.
2nd, the construction method of bacterial strain of the present invention has the advantages that easy to operate, production efficiency is high, with low cost, easily realizes
Industrialized production.
3rd, the present invention knocks out the key gene that Main By product is synthesized in Host Strains using metabolic engineering technology, and expresses
Nadh oxidase effectively adjusts intracellular redox state, improves the yield of precursor substance second accidental cause, final to improve tetramethyl pyrrole
The yield of piperazine.
4th, the present invention is carbon source using non-grain tapioca starch, and cottonseed flour, bean cake powder or beancake powder are nitrogen source, are substituted existing
Report that high-purity sugared (such as glucose), the dusty yeast that use are used for fermenting and producing precursor substance 3-hydroxy-2-butanone and Tetramethylpyrazine, not only
The comprehensive utilization value of these cheap raw materials can be improved, also more cheap raw material is provided for the production of Tetramethylpyrazine,
Cost of material can be greatly reduced.
Embodiment
The present invention is described in further detail below in conjunction with embodiment, but is not limited to protection scope of the present invention.
Involved material in following embodiments：α-amylase, carbohydrase, protease are commercially produced product, can be purchased from Novi
Believe the companies such as (China) Bioisystech Co., Ltd；Cottonseed flour, bean cake powder, beancake powder are commercially produced product, can be purchased from green grass or young crops
The companies such as Dao Kerui culture mediums Co., Ltd.
Following embodiments detect glucose in zymotic fluid during production Tetramethylpyrazine using SBA-40C analyzers
Concentration, using the concentration of liquid chromatogram measuring Main By product, using gas Chromatographic Determination 3-hydroxy-2-butanone, Tetramethylpyrazine it is dense
Degree.
First, the structure of 3-hydroxy-2-butanone engineering strain is produced
Embodiment 1
Produce precursor substance 3-hydroxy-2-butanone engineering strain ECTMP1 structure：
By from Enterobacter cloacae α-acetolactate synthase gene budB, α-acetolactic acid decarboxylation
Enzyme gene budA and from Lactobacillus brevis nadh oxidase gene noxE nucleotide sequence carry out it is close
Numeral optimizes, and nucleotide sequence TAAGGAGGATATACA, Ran Houli containing ribosome bind site are added before each gene
The method manually synthesized obtains gene cluster budB-budA-noxE, and its nucleotide sequence length is 3849 bases, nucleotides
Sequence is as described in SEQ ID NO.1.Method using double digestion with being connected, plasmid is inserted by gene cluster budB-budA-noxE
Behind pTrc99A promoter, polycistronic expression plasmid pTrc99A-budB-budA-noxE is obtained, then by recombinant plasmid
PTrc99A-budB-budA-noxE imports Host Strains E.coli MG1655, obtains the engineering strain of production 3-hydroxy-2-butanone
ECTMP1。
Embodiment 2
Produce precursor substance 3-hydroxy-2-butanone engineering strain ECTMP2 structure：
Find that the Main By product of engineered strain ECTMP1 fermentations is 2,3- butanediols, succinic acid, lactic acid, second by analysis
Acid, the key gene of its route of synthesis is gldA, frdABCD, ldhA and pta.The Red weights originated using coliphage
System system can efficiently mediate the principle of homologous recombination events in bacterium, and the antibiotic resistance base in FRT sites is first carried with both sides
Reached because replacing above-mentioned target gene, then by inducing exogenous temperature-sensitive plasmid to express FLP recombinases deletion antibiotics resistance gene
To the purpose for knocking out target gene, comprise the following steps that：
PKD46 plasmids are transformed into host cell, Electroporation-competent cells are prepared；Enter performing PCR structure using primer to beat
Target sequence (contain chloramphenicol resistance gene), and it is directly transformed into the host cell containing pKD46；Chloramphenicol plate screening occurs
The clone of homologous recombination；Verified using sequencing technologies, select the clone that target gene is replaced by chloramphenicol resistance gene, prepare electricity
Transformed competence colibacillus cell；Electricity is transduceed and deletes chloramphenicol resistance gene into pCP20 plasmids；The clone that chloramphenicol resistance gene is deleted connects
Continuous line is passed on three times, prepares -20 DEG C of preservations of glycerol tube.Knocked out by being superimposed, the mutant strain of polygene deletion can be obtained
E.coli MG1655/ Δ gldA Δ frdABCD Δ ldhA Δ pta, prepare Electroporation-competent cells.Embodiment 1 is built
Polycistronic expression plasmid pTrc99A-budB-budA-noxE electricity is transduceed into polygene deletion mutant strain, obtains engineered strain
ECTMP2。
The nucleotides sequence list of each primer of table 1
Embodiment 3
Produce precursor substance 3-hydroxy-2-butanone engineering strain ECTMP3 structure：
The present embodiment is a difference in that with embodiment 1：α-acetolactate synthase gene and alpha -acetolactate decarboxylase
The source bacterial strain of gene is Klebsiella pneumoniae, and the source bacterial strain of nadh oxidase gene is Lactococcus
lactis。
Embodiment 4
Produce precursor substance 3-hydroxy-2-butanone engineering strain ECTMP4 structure：
The present embodiment is a difference in that with embodiment 2：The polycistronic expression plasmid that embodiment 3 is built imports many bases
Because of deletion mutation strain, engineered strain ECTMP4 is obtained.
Embodiment 5
Produce precursor substance 3-hydroxy-2-butanone engineering strain ECTMP5 structure：
The present embodiment is a difference in that with embodiment 1：α-acetolactate synthase gene and alpha -acetolactate decarboxylase
The source bacterial strain of gene is Serratia marcescens, and the source bacterial strain of nadh oxidase gene is Streptococcus
pyogenes。
Embodiment 6
Produce precursor substance 3-hydroxy-2-butanone engineering strain ECTMP6 structure：
The present embodiment is a difference in that with embodiment 2：The polycistronic expression plasmid that embodiment 5 is built imports many bases
Because of deletion mutation strain, engineered strain ECTMP6 is obtained.
Embodiment 7
Produce precursor substance 3-hydroxy-2-butanone engineering strain ECTMP7 structure：
The present embodiment is a difference in that with embodiment 1：α-acetolactate synthase gene and alpha -acetolactate decarboxylase
The source bacterial strain of gene is Enterobacter aerogenes, and the source bacterial strain of nadh oxidase gene is Bacillus
Subtilis, Host Strains are E.coli BW25113.
Embodiment 8
Produce precursor substance 3-hydroxy-2-butanone engineering strain ECTMP8 structure：
The present embodiment is a difference in that with embodiment 1：α-acetolactate synthase gene and alpha -acetolactate decarboxylase
The source bacterial strain of gene is Klebsiella oxytoca, and the source bacterial strain of nadh oxidase gene is Lactobacillus
Rhamnosus, Host Strains are E.coli BW25113.
Embodiment 9
Produce precursor substance 3-hydroxy-2-butanone engineering strain ECTMP9 structure：
The present embodiment is a difference in that with embodiment 1：α-acetolactate synthase gene and alpha -acetolactate decarboxylase
The source bacterial strain of gene is Bacillus licheniformis, and the source bacterial strain of nadh oxidase gene is Clostridium
aminovalericum。
The method of embodiment 3-9 synthetic gene clusters is same as Example 1, or is existing conventional techniques method, this area skill
Art personnel can learn the nucleotide sequence of the gene cluster by present disclosure, and embodiment 3-10 is not repeated to this
The nucleotide sequence of the gene cluster of correspondence synthesis.
2nd, the application of engineered strain body material 3-hydroxy-2-butanone before manufacture
Application Example 1
Engineered strain ECTMP1, ECTMP2, ECTMP3, ECTMP4, ECTMP5, ECTMP6, ECTMP7, ECTMP8,
The application of ECTMP9 body material 3-hydroxy-2-butanones before manufacture, comprises the following steps：
(1) after cassava peeling is dried, it is crushed to the tapioca starch that particle diameter is 100 mesh；Nine parts of tapioca starch 180g is weighed, respectively
Add in beaker, every part is added into 60g cottonseed flours, 1000mL running water, 0.5mL α-amylases and 0.2g calcium chloride again,
After fully shaking up, 95 DEG C of liquefaction 1.5h stand cooling；When temperature is reduced to less than 55 DEG C, 0.5mL carbohydrase and 0.1g eggs are added
White enzyme, be saccharified 20h at 55 DEG C, adjusts pH 7.5, supernatant is collected by centrifugation, and makes concentration of reduced sugar with aseptic water dilution supernatant
For 100g/L, sterilize 15min under the conditions of 115 DEG C, produces fermentation medium；
(2) 100 μ g/mL ampicillins are added in the fermentation medium, take 500mL to be transferred to the logical sequence six of Shanghai hundred respectively
Fermentation tank, aseptically, take the ECTMP1 activated, ECTMP2, ECTMP3, ECTMP4, ECTMP5, ECTMP6,
ECTMP7, ECTMP8, ECTMP9 engineering bacteria bacterium solution are inoculated into above-mentioned fermented and cultured respectively by 5% inoculum concentration of volume ratio respectively
In base, the then fermented and cultured under the conditions of 37 DEG C of temperature, speed of agitator 300rpm, throughput 1vvm, fermentation 16h, 28h are mended respectively
Sugared 50g/L, terminates fermentation when not being further added by 3-hydroxy-2-butanone concentration, obtain the zymotic fluid of 3-hydroxy-2-butanone.Sample and utilize gas-chromatography
With liquid chromatogram measuring product 3-hydroxy-2-butanone and accessory substance butanediol, succinic acid, lactic acid, acetic acid concentration, as a result as shown in table 2.
The activation method of the engineering bacteria bacterium solution：To produce the ECTMP1 of 3-hydroxy-2-butanone, ECTMP2, ECTMP3, ECTMP4,
It is 1.8% agar that ECTMP5, ECTMP6, ECTMP7, ECTMP8, ECTMP9 engineered strain are scoring to containing mass volume ratio respectively
And on the LB flat boards containing 100 μ g/mL ampicillins, 37 DEG C culture 12h；With a single bacterium on toothpick picking LB flat boards
Fall, be then seeded into the LB fluid nutrient mediums containing 100 μ g/mL ampicillins, 37 DEG C of shaking table concussion and cultivate 10h；It is described
LB culture medium prescriptions be：10g/L peptones, 5g/L dusty yeasts, 10g/L sodium chloride.
The tunning of the different engineered strains of table 2
| Engineered strain | 3-hydroxy-2-butanone (g/L) | Butanediol (g/L) | Succinic acid (g/L) | Lactic acid (g/L) | Acetic acid (g/L) |
| ECTMP1 | 60.5 | 2.8 | 3.5 | 2.7 | 3.6 |
| ECTMP2 | 71.2 | 0.4 | 0.1 | 0.4 | 0.3 |
| ECTMP3 | 52.3 | 3.8 | 3.3 | 3.1 | 3.9 |
| ECTMP4 | 63.1 | 0.5 | 0 | 0.3 | 0.5 |
| ECTMP5 | 45.2 | 3.5 | 3.9 | 2.7 | 3.4 |
| ECTMP6 | 53.3 | 0.5 | 0.2 | 0.6 | 0.2 |
| ECTMP7 | 48.2 | 3.5 | 2.8 | 2.6 | 3.7 |
| ECTMP8 | 51.8 | 3.6 | 3.2 | 2.2 | 2.8 |
| ECTMP9 | 46.3 | 3.2 | 3.5 | 2.5 | 2.9 |
It is known that knocking out key gene gldA, frdABCD, ldhA and pta of accessory substance route of synthesis, work from table 2
Journey bacterial strain ECTMP2, ECTMP4, ECTMP6 precursor substance 3-hydroxy-2-butanone yield, (divide with the engineered strain without gene knockout
Dui Yingyu ECTMP1, ECTMP3, ECTMP5) compared to significantly improving, accessory substance 2,3-butanediol, succinic acid, the breast of 3-hydroxy-2-butanone
The concentration of acid and acetic acid is substantially reduced, therefore the key gene of knockout accessory substance route of synthesis improves the production of precursor substance 3-hydroxy-2-butanone
Amount, can reduce the production cost of Tetramethylpyrazine.
Application Example 2
Engineered strain ECTMP2 comprises the following steps in the application of production 3-hydroxy-2-butanone：
(1) after cassava peeling is dried, it is crushed to the tapioca starch that particle diameter is 100 mesh；Tri- parts of tapioca starch 200g is weighed, respectively
Add in beaker, then be separately added into 70g bean cake powders, 70g beancake powders, 70g cottonseed flours, add 1000mL running water,
1.2mL α-amylases and 0.5g calcium chloride, after fully shaking up, 90 DEG C of liquefaction 2h stand cooling；When temperature is reduced to less than 50 DEG C,
Add 0.3mL carbohydrase and 0.2g protease, be saccharified 24h at 55 DEG C, adjusts pH 6.5, supernatant is collected by centrifugation, with it is sterile originally
Water dilution supernatant makes concentration of reduced sugar be 100g/L, and sterilize 15min under the conditions of 115 DEG C, produces fermentation medium；
(2) 100 μ g/mL ampicillins in the fermentation medium, take 500mL to be fermented added to the logical sequence six of Shanghai hundred respectively
Tank, aseptically, the ECTMP2 engineering bacterias bacterium solution for activation of learning from else's experience are inoculated into above-mentioned hair by 10% inoculum concentration of volume ratio
In ferment culture medium, the fermented and cultured under the conditions of 40 DEG C of temperature, speed of agitator 600rpm, throughput 1.0vvm, ferment 12h and 24h
The fermentation ends when concentration for mending 3-hydroxy-2-butanone in 40g/L glucose, zymotic fluid respectively is not further added by, obtain the fermentation containing 3-hydroxy-2-butanone
Liquid.It is 59.3g/L, 63.8g/L, 73.3g/L respectively to determine bean cake powder, beancake powder, the corresponding 3-hydroxy-2-butanone yield of cottonseed flour.
The activation method of the ECTMP2 engineering bacterias bacterium solution：ECTMP2 engineered strains are scoring to containing quality volume respectively
Than for 1.8% agar and on LB flat boards containing 100 μ g/mL ampicillins, 37 DEG C of culture 15h；Under sterile conditions,
With a single bacterium colony on toothpick picking LB flat boards, the LB fluid nutrient mediums containing 100 μ g/mL ampicillins are then seeded into
In, 37 DEG C of shaking table concussion and cultivate 12h；Described LB culture medium prescriptions are：10g/L peptones, 5g/L dusty yeasts, 10g/L chlorinations
Sodium.
Application Example 3
Engineered strain ECTMP4 comprises the following steps in the application of production 3-hydroxy-2-butanone：
(1) after cassava peeling is dried, it is crushed to the tapioca starch that particle diameter is 150 mesh；Tapioca starch 185g is weighed, beaker is added
In, 25g cottonseed flours and 15g beancake powders are added, 1000mL running water, 0.8mL α-amylases and 0.3g calcium chloride is added,
After fully shaking up, 95 DEG C of liquefaction 2h stand cooling；When temperature is reduced to less than 55 DEG C, 0.8mL carbohydrase and 0.3g albumen are added
Enzyme, be saccharified 18h at 55 DEG C, adjusts pH 7.0, supernatant is collected by centrifugation, and makes the concentration of reduced sugar be with aseptic water dilution supernatant
100g/L, sterilize 15min under the conditions of 115 DEG C, produces fermentation medium；
(2) 100 μ g/mL ampicillins are added in the fermentation medium, take 500mL to be transferred to the fermentation of logical sequence six of Shanghai hundred
Tank, aseptically, the ECTMP4 engineering bacterias bacterium solution for activation of learning from else's experience are inoculated into above-mentioned fermentation by 8% inoculum concentration of volume ratio
In culture medium, fermented under the conditions of 37 DEG C of temperature, speed of agitator 500rpm, throughput 0.7vvm, 15h and 28h are mended respectively
After 50g/L glucose, fermentation 45h, the zymotic fluid containing 3-hydroxy-2-butanone is obtained.It is 67.2g/L by determining 3-hydroxy-2-butanone yield.
The activation method of the ECTMP4 engineering bacterias bacterium solution：ECTMP4 engineered strains are scoring to containing quality volume respectively
Than for 1.8% agar and on LB flat boards containing 100 μ g/mL ampicillins, 37 DEG C of culture 10h；Under sterile conditions,
With a single bacterium colony on toothpick picking LB flat boards, the LB fluid nutrient mediums containing 100 μ g/mL ampicillins are then seeded into
In, 37 DEG C of shaking table concussion and cultivate 15h；Described LB culture medium prescriptions are：10g/L peptones, 5g/L dusty yeasts, 10g/L chlorinations
Sodium.
Application Example 4
Engineered strain ECTMP5 comprises the following steps in the application of production 3-hydroxy-2-butanone：
(1) after cassava peeling is dried, it is crushed to the tapioca starch that particle diameter is 50 mesh；Tapioca starch 100g is weighed, beaker is added
In, 30g cottonseed flours and 30g bean cake powders are added, 1000mL running water, 2.0mL α-amylases and 0.01g calcium chloride is added,
After fully shaking up, 80 DEG C of liquefaction 5h stand cooling；When temperature is reduced to less than 55 DEG C, 0.2mL carbohydrase and 0.05g eggs are added
White enzyme, be saccharified 5h at 60 DEG C, adjusts pH 7.0, supernatant is collected by centrifugation, and makes concentration of reduced sugar with aseptic water dilution supernatant
For 100g/L, sterilize 15min under the conditions of 115 DEG C, produces fermentation medium；
(2) 50 μ g/mL ampicillins are added in the fermentation medium, take 500mL to be transferred to the fermentation of logical sequence six of Shanghai hundred
Tank, aseptically, the ECTMP5 engineering bacterias bacterium solution for activation of learning from else's experience are inoculated into above-mentioned fermentation by 1% inoculum concentration of volume ratio
In culture medium, sugar 40g/L is mended after fermented and cultured 16h under the conditions of 42 DEG C of temperature, speed of agitator 300rpm, throughput 1.3vvm,
Added again after fermentation 32h after equivalent cassava reduced sugar mother liquor, fermentation 48h, obtain the zymotic fluid containing 3-hydroxy-2-butanone.By determining
3-hydroxy-2-butanone yield is 48.3g/L.
The activation method of the ECTMP5 engineering bacterias bacterium solution：ECTMP5 engineered strains are scoring to containing quality volume respectively
Than for 1.8% agar and on LB flat boards containing 100 μ g/mL ampicillins, 37 DEG C of culture 12h；Under sterile conditions,
With a single bacterium colony on toothpick picking LB flat boards, the LB fluid nutrient mediums containing 100 μ g/mL ampicillins are then seeded into
In, 37 DEG C of shaking table concussion and cultivate 12h；Described LB culture medium prescriptions are：10g/L peptones, 5g/L dusty yeasts, 10g/L chlorinations
Sodium.
Application Example 5
Engineered strain ECTMP6 comprises the following steps in the application of production 3-hydroxy-2-butanone：
(1) after cassava peeling is dried, it is crushed to the tapioca starch that particle diameter is 200 mesh；Tapioca starch 155g is weighed, beaker is added
In, add 40g bean cake powders, 20g cottonseed flours and 20g beancake powders, add 1000mL running water, 1.5mL α-amylases and
0.5g calcium chloride, after fully shaking up, 100 DEG C of liquefaction 1h stand cooling；When temperature is reduced to less than 55 DEG C, 1mL carbohydrase is added
With 0.5g protease, be saccharified 20h at 50 DEG C, adjusts pH 7.0, supernatant is collected by centrifugation, and is made also with aseptic water dilution supernatant
Raw sugar concentration is 100g/L, and sterilize 15min under the conditions of 115 DEG C, produces fermentation medium；
(2) 80 μ g/mL ampicillins are added in the fermentation medium, take 500mL to be transferred to the fermentation of logical sequence six of Shanghai hundred
Tank, aseptically, the ECTMP7 engineering bacterias bacterium solution for activation of learning from else's experience are inoculated into above-mentioned fermentation by 5% inoculum concentration of volume ratio
In culture medium, sugar 50g/L is mended after fermented and cultured 14h under the conditions of 37 DEG C of temperature, speed of agitator 700rpm, throughput 0.5vvm,
Added respectively after fermentation 28h, 45h after 30g/L cassava reduced sugar mother liquors, fermentation 60h, obtain the zymotic fluid containing 3-hydroxy-2-butanone.Through
Measure 3-hydroxy-2-butanone yield is crossed for 68.5g/L.
The activation method of the ECTMP6 engineering bacterias bacterium solution：ECTMP6 engineered strains are scoring to containing quality volume respectively
Than for 1.8% agar and on LB flat boards containing 100 μ g/mL ampicillins, 37 DEG C of culture 10h；Under sterile conditions,
With a single bacterium colony on toothpick picking LB flat boards, the LB fluid nutrient mediums containing 100 μ g/mL ampicillins are then seeded into
In, 37 DEG C of shaking table concussion and cultivate 15h；Described LB culture medium prescriptions are：10g/L peptones, 5g/L dusty yeasts, 10g/L chlorinations
Sodium.
Application Example 6
Engineered strain ECTMP7 comprises the following steps in the application of production 3-hydroxy-2-butanone：
(1) after cassava peeling is dried, it is crushed to the tapioca starch that particle diameter is 100 mesh；Tapioca starch 150g is weighed, beaker is added
In, 40g cottonseed flours and 30g beancake powders are added, 1000mL running water, 1.0mL α-amylases and 0.2g calcium chloride is added,
After fully shaking up, 90 DEG C of liquefaction 3h stand cooling；When temperature is reduced to less than 55 DEG C, 0.5mL carbohydrase and 0.25g eggs are added
White enzyme, be saccharified 12h at 55 DEG C, adjusts pH 7.0, supernatant is collected by centrifugation, and makes concentration of reduced sugar with aseptic water dilution supernatant
For 100g/L, sterilize 15min under the conditions of 115 DEG C, produces fermentation medium；
(2) 100 μ g/mL ampicillins are added in the fermentation medium, take 500mL to be transferred to the fermentation of logical sequence six of Shanghai hundred
Tank, aseptically, the ECTMP7 engineering bacterias bacterium solution for activation of learning from else's experience are inoculated into above-mentioned fermentation by 8% inoculum concentration of volume ratio
In culture medium, after fermented and cultured 16h under the conditions of 38 DEG C of temperature, speed of agitator 500rpm, throughput 1.2vvm, Portugal in zymotic fluid
Grape sugar concentration drops to 20.3g/L, adds equivalent cassava reduced sugar mother liquor again after mending sugar 40g/L, fermentation 32h, 46h, ferments
After 60h, the zymotic fluid containing 3-hydroxy-2-butanone is obtained.It is 60.3g/L by determining 3-hydroxy-2-butanone yield.
The activation method of the ECTMP7 engineering bacterias bacterium solution：ECTMP7 engineered strains are scoring to containing quality volume respectively
Than for 1.8% agar and on LB flat boards containing 100 μ g/mL ampicillins, 37 DEG C of culture 15h；Under sterile conditions,
With a single bacterium colony on toothpick picking LB flat boards, the LB fluid nutrient mediums containing 100 μ g/mL ampicillins are then seeded into
In, 37 DEG C of shaking table concussion and cultivate 10h；Described LB culture medium prescriptions are：10g/L peptones, 5g/L dusty yeasts, 10g/L chlorinations
Sodium.
3rd, precursor substance 3-hydroxy-2-butanone and NH4 +Reaction synthesis Tetramethylpyrazine
Application Example 7
Diammonium hydrogen phosphate concentration converts the influence for producing Tetramethylpyrazine to 3-hydroxy-2-butanone：
Zymotic fluid of the Application Example 3 containing 3-hydroxy-2-butanone is taken, 10min is centrifuged with 8000rpm, precipitation is removed, takes supernatant
Determine 3-hydroxy-2-butanone concentration be 67.2g/L, take respectively 100mL add 3 beakers in, be then respectively adding 3-hydroxy-2-butanone 1,2.0,
The diammonium hydrogen phosphate of 2.5 times of molar concentrations, after dissolving, adjusts pH to 7.5, respectively takes 20mL to add in 100mL triangular flasks, 65
DEG C, react 20h, 3-hydroxy-2-butanone and NH in shaking bath under 150rpm4 +Reaction synthesis Tetramethylpyrazine.By determining various concentrations phosphorus
The yield of the corresponding Tetramethylpyrazine of the sour ammonium of hydrogen two is 12.9g/L, 17.9g/L, 18.3g/L respectively.
Application Example 8
Add hydrogen peroxide and the influence for producing Tetramethylpyrazine is converted to 3-hydroxy-2-butanone：
Zymotic fluid of the Application Example 5 containing 3-hydroxy-2-butanone is taken, 10min is centrifuged with 8000rpm, precipitation is removed, takes supernatant
The concentration for determining 3-hydroxy-2-butanone is 68.5g/L, takes 400mL to add in beaker, then adds the phosphoric acid of 2.0 times of molar concentrations of 3-hydroxy-2-butanone
The ammonium of hydrogen two, after dissolving, adjusts pH to 7.5, takes six parts of 20mL solution to add in 100mL triangular flasks respectively, in 90 DEG C, 100rpm
React, be separately added into after reaction 3h as 3-hydroxy-2-butanone molar concentration 0,0.1,0.5,1.0,1.5,2.0 times of mistake in lower shaking bath
Hydrogen oxide, then react 3h, 3-hydroxy-2-butanone and NH4 +Reaction synthesis Tetramethylpyrazine.By determining corresponding Tetramethylpyrazine yield
For 18.7,20.8,22.6,25.3,23.7,21.9g/L.
SEQUENCE LISTING
<110>Zhongnuo Bioengineering Co., Ltd., Nanning, Guangxi Academy Of Sciences
<120>Produce construction method and its application of (R) -3-hydroxy-2-butanone engineering strain
<130> 3849
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 3849
<212> DNA
<213>It is artificial synthesized
<400> 1
taaggaggat atacatatga actcggagaa acagtcgcgc caatgggcgc atggcgccga 60
catggttgtg ggccaactgg aagcccaggg cgtgaagcag gtgtttggca tcccgggcgc 120
gaaaattgac aaggtgtttg actcgctgct ggatagcagc attgagatca ttcctgtgcg 180
ccatgaggcg aacgccgcct ttatggccgc cgcggttggt cgtctgaccg gtaaggccgg 240
cgtggcctta gtgaccagcg gtcctggttg ttctaatctg attaccggca ttgcgaccgc 300
gaactcggag ggcgatcctg tggtggcctt aggcggtgcg gttaagcgcg cggataaagc 360
caaactggtg caccagagca tggataccgt ggcgatgttt agcccggtga cgaagtacgc 420
cgttgaagtt agctcgccgg acgcgattgc ggaggtggtt agcaacgcgt ttcgtgccgc 480
ggaacatggc cgtcctggtg gcgcgttcgt ttcgctgccg caggacattg ttgatcagcc 540
tgcgaccggt gcgatcttac cggccagcgg tcctgccctg atgggtccgg cccctgaaag 600
cgcgatcaat gatgttgcga aattaattga caatgcgaaa aacccggtga ttctgctggg 660
cttaatggcc tcgcagcctg ccaatagcgc ggcgttacgc aaactgctgg agaagagccg 720
tattccggtg acttctactt accaagccgc cggcgcggtg aatcaggaac actttacccg 780
ctttgccggt cgcgtgggtc tgttcaacaa tcaagcgggt gaccgcctgt tacacctggc 840
ggatctgatc atttgcattg gctacagccc ggttgaatac gaaccgagca tgtggaacag 900
cggcgatgcc acgctggtgc atatcgatgt tctgcctgcg tacgaggaac gcaattatgt 960
gccggatatc gagctggtgg gtgacattgc cgcgaccctg aatttactgg cctcgcgtat 1020
tgaccacaag ctggaactga gccaacgcgc gtcggagatt ctggtggacc gccaacatca 1080
gcgcgattta ttagaccgcc gtggtgcgtc gctgaaccag tttgcgctgc atcctctgcg 1140
cattgttcgc gcgatgcagg atatcgtgaa caacgatgtg accctgaccg tggacatggg 1200
cagcttccat atctggatcg cgcgttatct gtacagcttc cgcgcccgtc aggtgatgat 1260
cagcaacggc cagcagacta tgggtgttgc cttaccgtgg gcgatcggcg cgtggctggt 1320
taacccgggt cgtaaggttg tgagcgtgtc gggcgatggc ggtttcttac agagcagcat 1380
ggagctggaa acggccgtgc gcctgaacgc caacgtgtta catattattt gggtggataa 1440
cggctataac atggtggcca tccaagagga gaagaagtat cagcgcctga gcggtgttgc 1500
gtttggcccg gtggatttca aggcctatgc ggatgccttt ggcgcccgtg gcttcgccgt 1560
ggaaagcgcc gatgcgttag aaagcaccct gcgcgcggct atggatgtta atggtccggc 1620
ggttgttgcg attccggtgg attacagcga taacccgctg ctgatgggcc agctgcatct 1680
gtcgcagatt ctgtaataag gaggatatac atatgatgca cagctcggcg tgcgattgtg 1740
aagcgtcgct gtgcgaaacc ctgcgcggct ttagcgccaa acatccggac agcgttatct 1800
accagacctc gctgatgagc gcgctgctgt cgggcgttta cgaaggcgac accacgatcg 1860
ccgatctgct ggctcatggt gatttcggtc tgggcacctt caacgaactg gacggcgaaa 1920
tgattgcctt ttcttctcag gtgtaccagt tacgcgccga cggcagcgcg cgcgcggcca 1980
aaccggaaca gaaaacgccg ttcgccgtga tgacgtggtt ccaaccgcag tatcgcaaaa 2040
cgtttgatgc cccggtttcg cgtcagcaga ttcatgacgt gatcgatcag cagattccga 2100
gcgacaacct gttctgcgcg ctgcgcattg acggcaattt tcgccacgcc cacacccgca 2160
cggttcctcg ccaaacgccg ccgtaccgtg ccatgaccga tgtgctggac gaccagccgg 2220
tgtttcgttt taaccaacgc gaaggcgtgc tggttggctt ccgcacgccg cagcacatgc 2280
agggcattaa tgttgccggc taccatgagc actttattac cgacgatcgc cagggcggcg 2340
gtcatttact ggattatcag ctggagagcg gtgtgctgac cttcggcgag attcacaaac 2400
tgatgatcga cctgccggcc gacagcgcct tcttacaagc gaatctgcac ccgagcaacc 2460
tggacgccgc gattcgttcg gtggaaaatt aataaggagg atatacatat gaaaatcgtt 2520
gttatcggta ccaaccacgc tggtatcgct accgctaaca ccctgctgga acagtacccg 2580
ggtcacgaaa tcgttatgat cgaccgtaac tctaacatgt cttacctggg ttgcggtacc 2640
gctatctggg ttggtcgtca gatcgaaaaa ccggacgaac tgttctacgc taaagctgaa 2700
gacttcgaag ctaaaggtgt taaaatcctg accgaaaccg aagtttctga aatcgacttc 2760
gctaacaaaa aagtttacgc taaaaccaaa tctgacgacg aaatcatcga agcttacgac 2820
aaactggttc tggctaccgg ttctcgtccg atcatcccga acctgccggg taaagacctg 2880
aaaggtatcc acttcctgaa actgttccag gaaggtcagg ctatcgacgc tgaattcgct 2940
aaagaaaaag ttaaacgtat cgctgttatc ggtgctggtt acatcggtac cgaaatcgct 3000
gaagctgcta aacgtcgtgg taaagaagtt ctgctgttcg acgctgaaaa cacctctctg 3060
gcttcttact acgacgaaga attcgctaaa ggtatggacg aaaacctggc tcagcacggt 3120
atcgaactgc acttcggtga actggctaaa gaattcaaag ctaacgaaga aggttacgtt 3180
tctcagatcg ttaccaacaa agctacctac gacgttgacc tggttatcaa ctgcatcggt 3240
ttcaccgcta actctgctct ggcttctgac aaactggcta ccttcaaaaa cggtgctatc 3300
aaagttgaca aacaccagca gtcttctgac ccggacgttt acgctgttgg tgacgttgct 3360
accatctact ctaacgctct gcaggacttc acctacatcg ctctggcttc taacgctgtt 3420
cgttctggta tcgttgctgg tcacaacatc ggtggtaaag aactggaatc tgttggtgtt 3480
cagggttcta acggtatctc tatcttcggt tacaacatga cctctaccgg tctgtctgtt 3540
aaagctgcta aaaaactggg tctggaagtt tctttctctg acttcgaaga caaacagaaa 3600
gcttggttcc tgcacgaaaa caacgactct gttaaaatcc gtatcgttta cgaaaccaaa 3660
tctcgtcgta tcatcggtgc tcagctggct tctaaatctg aaatcatcgc tggtaacatc 3720
aacatgttct ctctggctat ccaggaaaaa aaaaccatcg acgaactggc tctgctggac 3780
ctgttcttcc tgccgcactt caactctccg tacaactaca tgaccgttgc tgctctgaac 3840
gctaaataa 3849
Claims (10)
1. a kind of method that utilization cheap raw material produces Tetramethylpyrazine, it is characterised in that：Comprise the following steps：
（1）By the nucleotides sequence of α-acetolactate synthase gene, alpha -acetolactate decarboxylase gene and nadh oxidase gene
Row carry out codon optimization；
（2）By α-acetolactate synthase gene through codon optimization, alpha -acetolactate decarboxylase gene and nadh oxidase
Gene carries out splicing and obtains the gene cluster for including three genes；
（3）Gene cluster is inserted in expression vector, polycistronic expression plasmid is obtained；
（4）Polycistronic expression plasmid is imported into Host StrainsE. coli, obtain the engineering strain of production 3-hydroxy-2-butanone；
（5）Tapioca starch, nitrogen source raw material are hydrolyzed, fermentation medium is obtained, by the genetic engineering bacterium of the production 3-hydroxy-2-butanone of activation
Strain is seeded to fermentation medium and carries out fermented and cultured, obtains the zymotic fluid containing 3-hydroxy-2-butanone；
（6）Take above-mentioned zymotic fluid to carry out centrifugal treating, take supernatant, add diammonium hydrogen phosphate, in a heated condition, 3-hydroxy-2-butanone
With NH4 +Reaction synthesis Tetramethylpyrazine.
2. the method for Tetramethylpyrazine is produced using cheap raw material according to claim 1, it is characterised in that：α-the acetyl
The codon optimization of lactic acid synthase gene, alpha -acetolactate decarboxylase gene and nadh oxidase gene is comprised the concrete steps that
The nucleotide sequence TAAGGAGGATATACA containing ribosome bind site and intervening sequence is added before each gene.
3. the method that utilization cheap raw material according to claim 1 or claim 2 produces Tetramethylpyrazine, it is characterised in that：The place
Main bacteriumE. coliIt is the mutant strain of polygene deletion, is obtained by being superimposed the key gene of synthesising by-product in knock-out bacterial strain
；The accessory substance includes 2,3- butanediols, succinic acid, lactic acid and acetic acid；The key gene of the synthesising by-product includesgldA、frdABCD、ldhAWithpta。
4. the method for Tetramethylpyrazine is produced using cheap raw material according to claim 3, it is characterised in that：Described α-second
The source strain of acyl lactic acid synthase gene and alpha -acetolactate decarboxylase gene is selected good strains in the field for seed certainlyEnterobacter cloacae、Klebsiella pneumoniae、Enterobacter aerogenes、Klebsiella oxytoca、Serratia marcescensWithBacillus licheniformis。
5. the method for Tetramethylpyrazine is produced using cheap raw material according to claim 3, it is characterised in that：Described NADH
The source strain of oxidase gene is selected good strains in the field for seed certainlyLactobacillus brevis、Lactococcus lactis、Streptococcus pyogenes、Clostridium aminovalericumWithBacillus subtilis。
6. the method that Tetramethylpyrazine is produced according to any utilization cheap raw materials of claim 1-5, it is characterised in that：It is described
Step（5）Concrete operation step be：
S1：Tapioca starch, nitrogen source raw material are taken, water, α-amylase and calcium chloride is added, liquefy 0.5-5 h at 80-100 DEG C, stands
Cooling；When temperature is reduced to below 55 DEG C, carbohydrase and protease are added, be saccharified 5-20 h at 50-60 DEG C, adjusts pH
6.5-7.5, centrifuges or is collected by filtration 15 min that sterilized after supernatant, dilution supernatant under the conditions of 115 DEG C, produce fermented and cultured
Base；Based on 1000 mL water, the consumption of each component is：Tapioca starch 100-200 g, nitrogen source raw material 40-80 g, α-amylase 0.5-2
ML, calcium chloride 0.01-0.5 g, the g of carbohydrase 0.2-1 mL, proteinase-10 .05 ~ 0.5；The tapioca starch is to remove the peel cassava to shine
It is prepared after dry through crushing 50-200 mesh sieves, the nitrogen source raw material is one kind in cottonseed flour, bean cake powder, beancake powder
Or multiple combinations；
S2：Aseptically, 50-100 μ g/mL ampicillins are added in the fermentation medium, then move to fermentation tank
In, the inoculum concentration by the engineering bacteria bacterium solution of activated production 3-hydroxy-2-butanone using volume ratio as 1-10% is inoculated into fermentation medium,
The fermented and cultured 20-72 h under the conditions of 35-42 DEG C of temperature, speed of agitator 200-800 rpm, throughput 0.5-1.5 vvm, work as inspection
When the concentration for surveying 3-hydroxy-2-butanone in zymotic fluid is not further added by, fermentation ends obtain the zymotic fluid containing 3-hydroxy-2-butanone.
7. the method for Tetramethylpyrazine is produced using cheap raw material according to claim 6, it is characterised in that：The activation
The preparation method of the engineering bacteria bacterium solution of 3-hydroxy-2-butanone is produced, is comprised the following steps：
Production 3-hydroxy-2-butanone engineering strain is scoring to containing mass volume ratio for 1.8% agar and containing 100 μ g/mL ammonia benzyls
On the LB flat boards of penicillin, 10-15 h are cultivated at 37 DEG C；Under sterile conditions, a single bacterium colony on picking LB flat boards, so
It is inoculated into afterwards in the LB liquid seed culture mediums containing 100 μ g/mL ampicillins, 37 DEG C of shaking table concussion and cultivate 10-15 h；
The formula of the LB culture mediums is：10 g/L peptones, 5 g/L dusty yeasts, 10 g/L sodium chloride.
8. the method for Tetramethylpyrazine is produced using cheap raw material according to claim 6, it is characterised in that：Step S2's
The concentration of glucose in zymotic fluid is detected in fermentation process, when concentration of glucose is down to 10-30 g/L, cassava reduced sugar is added
Mother liquor makes concentration of glucose in zymotic fluid be 40-80 g/L；The concentration of glucose of the cassava reduced sugar mother liquor is 500-800
g/L。
9. the method that Tetramethylpyrazine is produced according to any utilization cheap raw material of claim 1,2,4,5,7,8, its feature
It is：The step（6）Concrete operation step be：By the zymotic fluid centrifugation containing 3-hydroxy-2-butanone, precipitation is removed, takes supernatant to survey
Determine the content of 3-hydroxy-2-butanone, then add the diammonium hydrogen phosphate of 1-2.5 times of molar concentration of 3-hydroxy-2-butanone, after dissolving, pH is to 7.5 for regulation,
6-20 h are reacted in 65-95 DEG C of shaking bath, Tetramethylpyrazine is produced.
10. the method for Tetramethylpyrazine is produced using cheap raw material according to claim 9, it is characterised in that：Shaking bath
The hydrogen peroxide of 0.1-2 times of molar concentration of 3-hydroxy-2-butanone is added in course of reaction.
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