CN109797173A - A kind of production method of β-farnesene - Google Patents

Abstract

The present invention relates to field of biotechnology, especially a kind of production method of β-farnesene.This kind of β-farnesene production method includes the following steps: that heterologous inducing expression synthesis β-farnesene, S4 are carried out in the culture medium of the molasses after recombinant host cell is inoculated into containing edulcoration purification by S1 molasses edulcoration purification, the S2 β-building of farnesene recombinant host cell, S3 improves β-farnesene yield by ferment control.A kind of production method of β-farnesene of the invention, it is primary raw material using cheap molasses, β-farnesene is produced using recombination bacillus coli as production strain, shorten fermentation period, reduce production cost, through β-farnesene method detection in a kind of gas chromatography detection fermentation liquid of the invention, after fermentation 72 hours, β-farnesene is at concentrations up to 20.5g/L in fermentation liquid.

Description

A kind of production method of β-farnesene

Technical field

The present invention relates to field of biotechnology, especially a kind of production method of β-farnesene.

Background technique

β-farnesene is a kind of important dilute terpenoid of sesquialter, is had a wide range of applications in many fields, On the one hand, vitamin E production field, β-farnesene can greatly reduce the synthesis step of vitamin E as intermediate, with Prior synthesizing method reduces production cost compared to reaction efficiency and yield is improved, and energy special science and technology in Hubei has utilized β-at present The production of farnesene progress vitamin E；On the other hand, β-farnesene can be used for preparing high heating value, it is dynamical cleaning it is renewable Fuel alleviates global energy crisis, and U.S. Amyris carries out the production of β-farnesene, but its using patent saccharomycete at present Using sucrose as primary raw material, production cost is relatively expensive, and fermentation period is long, and this technology is main former using cheap molasses Material produces β-farnesene using recombination bacillus coli as production strain, shortens fermentation period, reduce production cost.

Summary of the invention

The purpose of the present invention is to provide a kind of production methods of β-farnesene, are main former using cheap molasses Material produces β-farnesene using recombination bacillus coli as production strain, shortens fermentation period, reduce production cost.

The technical scheme adopted by the invention to solve the technical problem is that:

A kind of production method of β-farnesene includes the following steps: S1 molasses edulcoration purification, S2 β-farnesene recombinant host cell Building, S3 recombinant host cell is inoculated into containing edulcoration purification after molasses culture medium in carry out heterologous inducing expression conjunction β-farnesene yield is improved by ferment control at β-farnesene, S4.

Preferably, the step S1 is specifically included:

(1) it weighs: weighing the water that molasses stoste is added 1.5-2.0 times, molasses are diluted to 16-17 Baume degrees, obtain dilute liquid glucose, often Batch molasses stock solution quality has a fluctuation, subject to the data that dilution water is measured according to scene Baume degrees meter；

(2) be acidified: dilute liquid glucose pours into fermentor, and the concentrated sulfuric acid is added and concentrated phosphoric acid adjusts acidity, and pH4.0-4.5 is acidified, After acidification, impurity≤1% such as sandy soil, dross, concentration >=80 ° BX, full sugar >=52%, acidity < 9%；

(3) divulge information: dilute liquid glucose after acidification is boiled in logical steam heating, and heating promotes colloid protein to solidify, and convenient for precipitating centrifugation, adds After heat is boiled, 90 DEG C are kept the temperature, while being passed through into ullage gas 1h, to remove removing and harmful gas NO₂、SO₂Equal pernicious gases and volatility Sour and other volatile materials；

(4) add alkali neutralization: dilute liquid glucose after ventilation process is packed into temporary storage barrel, and milk of lime is added and adjusts pH to 5.0-5.5, milk of lime Matched by calcium oxide plus water, matching while using；

(5) centrifugal clarification: the impurity being centrifuged off in dilute liquid glucose after adding alkali neutralization with supercentrifuge takes clear liquid；

(6) heat sterilization: clear liquid pours into the fermentor cleaned up, and steaming is heated to 90 DEG C, keeps the temperature 0.5h；

(7) pressure maintaining is stored: after sterilizing is good that fermentor is closed, ventilate pressure maintaining 1.0Mpa, and room temperature is kept in spare.

Preferably, the step S2 is specifically included: (1) being obtained by ncbi database inquiry and technique for gene engineering means β-farnesene related gene is synthesized, related gene includes acetoacetyl CoA thiolase (AtoB) base in mevalonate pathway Because of atoB, HMG-CoA synthase (ERG13) gene erg13, HMG-CoA reductase (tHMG1) gene thmg1, mevalonate kinase (ERG12) gene erg12, phosphomevalonate kinase (ERG8) gene erg8, mevalonate pyrophosphate decarboxylase (MVD1) base Because of mvd1, isopentenylpyrophosphate isomerase (IDI) gene idi, farnesyl pyrophosphate synzyme (FPPS) gene ispA, farnesene Synthase (AFS) gene afs；

(2) building multiple recombinant plasmid compatible in same host cell, the multiple recombinant plasmid lead in host cell Cross promoter transcription and host cell translation coexpression synthesis β-farnesene GAP-associated protein GAP；

(3) multiple recombinant plasmid transformed, which is imported into host cell, obtains synthesis β-farnesene recombinant host cell.

Preferably, the step S3 is specifically included: recombinant host cell being activated and accesses 250mL and is cultivated equipped with LB Base (tryptone 10g, yeast extract 5g, NaCl10g, is cultivated, shaking table temperature in the triangular flask of water constant volume to 1000mL) 30-38 DEG C, revolving speed 180-220r/min, culture to OD₆₀₀To 0.8 ± 0.2, with the inoculum concentration access 1000mL second level hair of 3-20% Ferment shaking flask culture, 35-38 DEG C of shaking table temperature, revolving speed 180-250r/min, culture to OD₆₀₀To 1.2 ± 0.2, with connecing for 5%-8% Kind amount access 30L ferment tank culture.

Preferably, the step S4 is specifically included: entire fermentation process ventilation quantity control is in 0.5vvm-1.5vvm, fermentation For the control of tank speed of agitator in 200r/min-400r/min, the control of fermentation liquid dissolved oxygen amount is 7.0 ± 0.4(with 20% in 2%-90%, pH Sulfuric acid and 30% ammonium hydroxide control fermentation liquid pH)；Earlier fermentation (0h-20h) fermentation temperature is 35 DEG C -37 DEG C, ferment middle (21h- 48h) fermentation temperature is 30 DEG C -35 DEG C, and fermentation later period (49h- terminates) is 28 DEG C -35 DEG C；When culture to OD₆₀₀For 8-20, Xiang Fa Final concentration of 0.1-0.5mmol/L isopropylthiogalactoside (IPTG) is accessed in ferment culture medium, is accessed after 2h-10h hours The extractant of fermentating liquid volume 10%-20% is accounted for, the extractant is one of n-decane, n-dodecane, petroleum ether, normal octane Or it is a variety of；Start feed supplement in fermentation process when sugared concentration (° BX) drops to (4 ± 1) %, controls sugared concentration (° BX) in fermentation liquid 4.0 ± 0.2.

Preferably, the molasses in the molasses stoste are the combination of cane molasses or beet molasses or both, combined situation Under, cane molasses, beet molasses weight ratio be 100:25-1000.

Preferably, the host cell is Escherichia coli, and recombinant host cell is Escherichia coli B21, the recombination place of building Chief cell Escherichia coli B21 can express acetoacetyl CoA thiolase (AtoB), HMG-CoA synthase, HMG-CoA reductase, Mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, isopentenylpyrophosphate isomerase, farnesyl Pyrophosphate synthetase and Farmesene synthase totally 9 kinds of enzymes, wherein the nucleotide sequence of gene atoB is as shown in SEQ ID NO.4, base Because the nucleotide sequence of erg13 is as shown in SEQ ID NO.5, the nucleotide sequence of gene thmg1 as shown in SEQ ID NO.6, The nucleotide sequence of gene erg12 as shown in SEQ ID NO.7, the nucleotide sequence of gene erg8 as shown in SEQ ID NO.8, The nucleotide sequence of gene mvd1 as shown in SEQ ID NO.9, the nucleotide sequence of gene idi as shown in SEQ ID NO.10, The nucleotide sequence of gene ispA is as shown in SEQ ID NO.11, and the nucleotide sequence of gene afs is as shown in SEQ ID NO.12.

Preferably, recombinant host cell Escherichia coli B21 includes tri- kinds of plasmids of pKK223-3, PMCSG9, pMal-c4X, Wherein include tri- kinds of genes of atoB, thmg1, mvd1 on pKK223-3, includes erg13, erg12, erg8, ispA tetra- on PMCSG9 Gene is planted, includes two kinds of genes of idi, afs on pMal-c4X.

Preferably, the plasmid pKK223-3 selects tac promoter (as shown in SEQ ID NO.1), plasmid PMCSG9 choosing Select tac promoter with T7 promoter (as shown in SEQ ID NO.2), plasmid pMal-c4X (as shown in SEQ ID NO.3).

Preferably, in the second order fermentation shaking flask culture and 30L ferment tank incubation, the main composition of culture medium As follows: the molasses after edulcoration purification account for culture medium specific gravity 2%-20%, the combination (combined situation of yeast powder or yeast extract or both Lower yeast powder: yeast extract 100:1-10000), account for culture medium specific gravity 1%-10%, peptone accounts for culture medium specific gravity 1.5%-8.5%, Ammonium citrate accounts for culture medium specific gravity 0.1%-5%, magnesium sulfate or magnesium chloride and accounts for culture medium specific gravity 0.1%-2%, and manganese sulfate accounts for culture medium Specific gravity 0.05%-0.9%, zinc sulfate account for culture medium specific gravity 0.02%-0.8%.

Preferably, the weight of material percentage supplemented during the feed supplement is the molasses 40%-60% after edulcoration purification, sulphur Sour ammonium 20%-50%, peptone 5%-10%, above-mentioned material are spare in 110-120 DEG C of high pressure sterilization 25-40min.

β-farnesene method in a kind of gas chromatography detection fermentation liquid, includes the following steps:

H1, fermentation liquid pre-treatment: taking 10mL or more fermentation liquid, and 9000-12000r/min is centrifuged 3-8min, and oil mutually divides with water phase Layer draws upper oil phase with the organic membrane filtration of 0.44um and obtains filter liquor；

H2, Agilent 7890A gas chromatograph, 19091JHP-5 chromatographic column (column length 30m are used;Internal diameter 0.32mm;Film thickness 250um), β-farnesene series standard solution is configured, the β-Fa Ni that concentration is respectively 5g/L, 10g/L, 15g/L, 20g/L is made Alkene standard solution, sample detection, draws equation of linear regression, and calculates R value respectively, makes R value 99% or more, above-mentioned β-Fa Ni Alkene testing conditions are as follows: post case temperature:, retaining 2min by 65-75 DEG C of initial temperature, rises to 220-280 DEG C with 10 DEG C/min, retains 1min, 220-280 DEG C of injector temperature, sample volume 1uL, product injection split ratio 1:50,260-280 DEG C of temperature of detector (FID), For nitrogen as carrier gas, inlet pressure is 12-18psi, and mode is constant current mode.

The beneficial effects of the present invention are: compared with prior art, a kind of production method of β-farnesene of the invention utilizes Cheap molasses are primary raw material, produce β-farnesene using recombination bacillus coli as production strain, shorten fermentation week Phase reduces production cost, detects through β-farnesene method in a kind of gas chromatography detection fermentation liquid of the invention, fermentation After 72 hours, β-farnesene is at concentrations up to 20.5g/L in fermentation liquid.

Specific embodiment

A kind of production method of the β-farnesene of embodiment 1

A kind of production method of β-farnesene includes the following steps: S1 molasses edulcoration purification, S2 β-farnesene recombinant host cell Building, S3 recombinant host cell is inoculated into containing edulcoration purification after molasses culture medium in carry out heterologous inducing expression conjunction β-farnesene yield is improved by ferment control at β-farnesene, S4.

The step S1 is specifically included:

(1) it weighs: weighing the water that molasses stoste is added 1.5-2.0 times, molasses are diluted to 16-17 Baume degrees, obtain dilute liquid glucose；

(2) be acidified: dilute liquid glucose pours into fermentor, and the concentrated sulfuric acid is added and concentrated phosphoric acid adjusts acidity, and pH4.0-4.5 is acidified, After acidification, impurity≤1% such as sandy soil, dross, concentration >=80 ° BX, full sugar >=52%, acidity < 9%；

(3) divulge information: dilute liquid glucose after acidification is boiled in logical steam heating, and heating promotes colloid protein to solidify, and convenient for precipitating centrifugation, adds After heat is boiled, 90 DEG C are kept the temperature, while being passed through into ullage gas 1h, to remove removing and harmful gas NO₂、SO₂Equal pernicious gases and volatility Sour and other volatile materials；

(4) add alkali neutralization: dilute liquid glucose after ventilation process is packed into temporary storage barrel, and milk of lime is added and adjusts pH to 5.0-5.5, milk of lime Matched by calcium oxide plus water, matching while using；

(5) centrifugal clarification: the impurity being centrifuged off in dilute liquid glucose after adding alkali neutralization with supercentrifuge takes clear liquid；

(6) heat sterilization: clear liquid pours into the fermentor cleaned up, and steaming is heated to 90 DEG C, keeps the temperature 0.5h；

(7) pressure maintaining is stored: after sterilizing is good that fermentor is closed, ventilate pressure maintaining 1.0Mpa, and room temperature is kept in spare.

The step S2 is specifically included:

(1) the acetoacetyl CoA thiolase (AtoB) in MVA approach is found by ncbi database BLAST, HMG-CoA is closed Enzyme, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, iso-amylene are burnt Phosphoric acid isomerase, farnesyl pyrophosphate synzyme and Farmesene synthase totally 9 kinds of enzymes and corresponding cDNA sequence；

(2) building multiple recombinant plasmid compatible in same host cell, it may be assumed that according to the cDNA sequence design primer of known enzyme Corresponding gene order is synthesized, the cDNA sequence to design synthesis synthesizes corresponding gene sequence by pcr clone as template Clone's synthetic gene sequence is connected on corresponding plasmid with DNA recombinant technique, forms multiple recombinant plasmid by column, multiple Recombinant plasmid passes through promoter transcription and host cell translation coexpression synthesis β-farnesene GAP-associated protein GAP in host cell；

(3) multiple recombinant plasmid transformed, which is imported into host cell, obtains synthesis β-farnesene recombinant host cell, it may be assumed that will even The recombinant plasmid connected imports e. coli host cell, and culture amplification recombinant host cell extracts plasmid and carries out sequence verification Target gene has been coupled on corresponding plasmid, and the bacterial strain after verifying is carried out culture preservation.

The step S3 is specifically included: recombinant host cell being activated and accesses 250mL equipped with LB culture medium (pancreas egg White peptone 10g, yeast extract 5g, NaCl10g, it is cultivated, 37 DEG C of shaking table temperature, is turned in the triangular flask of water constant volume to 1000mL) Fast 200r/min, culture to OD₆₀₀To 0.8 ± 0.2, the shaking flask culture of 1000mL second order fermentation, shaking table are accessed with 10% inoculum concentration 37 DEG C of temperature, revolving speed 220r/min, culture to OD₆₀₀To 1.2 ± 0.2, the culture of 30L ferment tank is accessed with 7% inoculum concentration.

The step S4 is specifically included: entire fermentation process ventilation quantity control is in 0.8vvm, the control of fermentor speed of agitator In 300r/min, the control of fermentation liquid dissolved oxygen amount is that 7.0 ± 0.4(, 20% sulfuric acid and 30% ammonium hydroxide control fermentation liquid in 20%, pH PH)；Earlier fermentation (0h-20h) fermentation temperature is 36 DEG C, and ferment middle (21h-48h) fermentation temperature is 32 DEG C, is fermented the later period (49h-72h) is 30 DEG C；When culture to OD₆₀₀It is 16, final concentration of 0.1mmol/L isopropyl sulphur is accessed into fermentation medium For galactoside (IPTG), access accounts for the extractant of fermentating liquid volume 15% after 3h hours, and the extractant is n-dodecane；Hair Start feed supplement when sugared concentration (° BX) drops to (4 ± 1) % during ferment, control in fermentation liquid sugar concentration (° BX) 4.0 ± 0.2。

Molasses in the molasses stoste are the combination of both cane molasses and beet molasses, cane molasses, beet molasses Weight ratio be 2.5:6.

The host cell is Escherichia coli, and recombinant host cell is Escherichia coli B21, and the recombinant host cell of building is big Enterobacteria B21 can express acetoacetyl CoA thiolase (AtoB), HMG-CoA synthase, HMG-CoA reductase, mevalonic acid Kinases, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, isopentenylpyrophosphate isomerase, farnesyl pyrophosphate close At enzyme and Farmesene synthase totally 9 kinds of enzymes；Recombinant host cell Escherichia coli B21 includes pKK223-3, PMCSG9, pMal-c4X Three kinds of plasmids wherein include tri- kinds of genes of atoB, thmg1, mvd1 on pKK223-3, on PMCSG9 comprising erg13, erg12, Tetra- kinds of genes of erg8, ispA include two kinds of genes of idi, afs on pMal-c4X；The plasmid pKK223-3 selects tac starting Son, plasmid PMCSG9 select T7 promoter, plasmid pMal-c4X to select tac promoter.

In the second order fermentation shaking flask culture and 30L ferment tank incubation, the main composition of culture medium is as follows: removing Miscellaneous molasses after purification account for culture medium specific gravity 8.5%, and yeast powder 3.5%, peptone account for culture medium specific gravity 4.8%, ammonium citrate accounts for training Feeding base specific gravity 1.8%, magnesium sulfate account for culture medium specific gravity 1.5%, and manganese sulfate accounts for culture medium specific gravity 0.12%, and zinc sulfate accounts for culture medium ratio Weigh 0.08%.

The weight of material percentage supplemented during the feed supplement is the molasses 50% after edulcoration purification, ammonium sulfate 30%, egg White peptone 20%, above-mentioned material are spare in 110-120 DEG C of high pressure sterilization 25-40min.

A kind of production method of the β-farnesene of embodiment 2

A kind of production method of β-farnesene includes the following steps: S1 molasses edulcoration purification, S2 β-farnesene recombinant host cell Building, S3 recombinant host cell is inoculated into containing edulcoration purification after molasses culture medium in carry out heterologous inducing expression conjunction β-farnesene yield is improved by ferment control at β-farnesene, S4.

The step S1 is specifically included:

(1) it weighs: weighing the water that molasses stoste is added 1.5-2.0 times, molasses are diluted to 16-17 Baume degrees, obtain dilute liquid glucose；

(2) be acidified: dilute liquid glucose pours into fermentor, and the concentrated sulfuric acid is added and concentrated phosphoric acid adjusts acidity, and pH4.0-4.5 is acidified, After acidification, impurity≤1% such as sandy soil, dross, concentration >=80 ° BX, full sugar >=52%, acidity < 9%；

(3) divulge information: dilute liquid glucose after acidification is boiled in logical steam heating, and heating promotes colloid protein to solidify, and convenient for precipitating centrifugation, adds After heat is boiled, 90 DEG C are kept the temperature, while being passed through into ullage gas 1h, to remove removing and harmful gas NO₂、SO₂Equal pernicious gases and volatility Sour and other volatile materials；

(4) add alkali neutralization: dilute liquid glucose after ventilation process is packed into temporary storage barrel, and milk of lime is added and adjusts pH to 5.0-5.5, milk of lime Matched by calcium oxide plus water, matching while using；

(5) centrifugal clarification: the impurity being centrifuged off in dilute liquid glucose after adding alkali neutralization with supercentrifuge takes clear liquid；

(6) heat sterilization: clear liquid pours into the fermentor cleaned up, and steaming is heated to 90 DEG C, keeps the temperature 0.5h；

(7) pressure maintaining is stored: after sterilizing is good that fermentor is closed, ventilate pressure maintaining 1.0Mpa, and room temperature is kept in spare.

The step S2 is specifically included:

(1) the acetoacetyl CoA thiolase (AtoB) in MVA approach is found by ncbi database BLAST, HMG-CoA is closed Enzyme, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, iso-amylene are burnt Phosphoric acid isomerase, farnesyl pyrophosphate synzyme and Farmesene synthase totally 9 kinds of enzymes and corresponding cDNA sequence；

(2) building multiple recombinant plasmid compatible in same host cell, it may be assumed that according to the cDNA sequence design primer of known enzyme Corresponding gene order is synthesized, the cDNA sequence to design synthesis synthesizes corresponding gene sequence by pcr clone as template Clone's synthetic gene sequence is connected on corresponding plasmid with DNA recombinant technique, forms multiple recombinant plasmid by column, multiple Recombinant plasmid passes through promoter transcription and host cell translation coexpression synthesis β-farnesene GAP-associated protein GAP in host cell；

(3) multiple recombinant plasmid transformed, which is imported into host cell, obtains synthesis β-farnesene recombinant host cell, it may be assumed that will even The recombinant plasmid connected imports e. coli host cell, and culture amplification recombinant host cell extracts plasmid and carries out sequence verification Target gene has been coupled on corresponding plasmid, and the bacterial strain after verifying is carried out culture preservation.

The step S3 is specifically included: recombinant host cell being activated and accesses 250mL equipped with LB culture medium (pancreas egg White peptone 10g, yeast extract 5g, NaCl10g, it is cultivated, 35 DEG C of shaking table temperature, is turned in the triangular flask of water constant volume to 1000mL) Fast 180r/min, culture to OD₆₀₀To 0.8 ± 0.2, the shaking flask culture of 1000mL second order fermentation, shaking table temperature are accessed with 5% inoculum concentration 35 DEG C, revolving speed 180r/min of degree, culture to OD₆₀₀To 1.2 ± 0.2, the culture of 30L ferment tank is accessed with 5% inoculum concentration.

The step S4 is specifically included: entire fermentation process ventilation quantity control is in 0.5vvm, the control of fermentor speed of agitator In 200r/min, the control of fermentation liquid dissolved oxygen amount is that 7.0 ± 0.4(, 20% sulfuric acid and 30% ammonium hydroxide control fermentation liquid in 10%, pH PH)；Earlier fermentation (0h-20h) fermentation temperature is 35 DEG C, and ferment middle (21h-48h) fermentation temperature is 30 DEG C, is fermented the later period (49h-72h) is 28 DEG C；When culture to OD₆₀₀It is 10, final concentration of 0.1mmol/L isopropyl sulphur is accessed into fermentation medium For galactoside (IPTG), access accounts for the extractant of fermentating liquid volume 10% after 2h hours, and the extractant is n-decane；Fermentation Start feed supplement when sugared concentration (° BX) drops to (4 ± 1) % in the process, sugared concentration (° BX) is 4.0 ± 0.2 in control fermentation liquid.

Molasses in the molasses stoste are the combination of both cane molasses and beet molasses, cane molasses, beet molasses Weight ratio be 2.5:6.

The host cell is Escherichia coli, and recombinant host cell is Escherichia coli B21, and the recombinant host cell of building is big Enterobacteria B21 can express acetoacetyl CoA thiolase (AtoB), HMG-CoA synthase, HMG-CoA reductase, mevalonic acid Kinases, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, isopentenylpyrophosphate isomerase, farnesyl pyrophosphate close At enzyme and Farmesene synthase totally 9 kinds of enzymes；Recombinant host cell Escherichia coli B21 includes pKK223-3, PMCSG9, pMal-c4X Three kinds of plasmids wherein include tri- kinds of genes of atoB, thmg1, mvd1 on pKK223-3, on PMCSG9 comprising erg13, erg12, Tetra- kinds of genes of erg8, ispA include two kinds of genes of idi, afs on pMal-c4X；The plasmid pKK223-3 selects tac starting Son, plasmid PMCSG9 select T7 promoter, plasmid pMal-c4X to select tac promoter.

In the second order fermentation shaking flask culture and 30L ferment tank incubation, the main composition of culture medium is as follows: removing Miscellaneous molasses after purification account for culture medium specific gravity 5%, and yeast powder 2%, peptone account for culture medium specific gravity 2%, lemon acid ammonium accounts for culture medium specific gravity 1%, magnesium sulfate accounts for culture medium specific gravity 0.5%, and manganese sulfate accounts for culture medium specific gravity 0.08%, and zinc sulfate accounts for culture medium specific gravity 0.1%.

The weight of material percentage supplemented during the feed supplement is the molasses 55% after edulcoration purification, ammonium sulfate 40%, egg White peptone 5%, above-mentioned material are spare in 110-120 DEG C of high pressure sterilization 25-40min.

A kind of production method of the β-farnesene of embodiment 3

A kind of production method of β-farnesene includes the following steps: S1 molasses edulcoration purification, S2 β-farnesene recombinant host cell Building, S3 recombinant host cell is inoculated into containing edulcoration purification after molasses culture medium in carry out heterologous inducing expression conjunction β-farnesene yield is improved by ferment control at β-farnesene, S4.

The step S1 is specifically included:

(1) it weighs: weighing the water that molasses stoste is added 1.5-2.0 times, molasses are diluted to 16-17 Baume degrees, obtain dilute liquid glucose；

(2) be acidified: dilute liquid glucose pours into fermentor, and the concentrated sulfuric acid is added and concentrated phosphoric acid adjusts acidity, and pH4.0-4.5 is acidified, After acidification, impurity≤1% such as sandy soil, dross, concentration >=80 ° BX, full sugar >=52%, acidity < 9%；

(3) divulge information: dilute liquid glucose after acidification is boiled in logical steam heating, and heating promotes colloid protein to solidify, and convenient for precipitating centrifugation, adds After heat is boiled, 90 DEG C are kept the temperature, while being passed through into ullage gas 1h, to remove removing and harmful gas NO₂、SO₂Equal pernicious gases and volatility Sour and other volatile materials；

(4) add alkali neutralization: dilute liquid glucose after ventilation process is packed into temporary storage barrel, and milk of lime is added and adjusts pH to 5.0-5.5, milk of lime Matched by calcium oxide plus water, matching while using；

(5) centrifugal clarification: the impurity being centrifuged off in dilute liquid glucose after adding alkali neutralization with supercentrifuge takes clear liquid；

(6) heat sterilization: clear liquid pours into the fermentor cleaned up, and steaming is heated to 90 DEG C, keeps the temperature 0.5h；

(7) pressure maintaining is stored: after sterilizing is good that fermentor is closed, ventilate pressure maintaining 1.0Mpa, and room temperature is kept in spare.

The step S2 is specifically included:

(1) the acetoacetyl CoA thiolase (AtoB) in MVA approach is found by ncbi database BLAST, HMG-CoA is closed Enzyme, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, iso-amylene are burnt Phosphoric acid isomerase, farnesyl pyrophosphate synzyme and Farmesene synthase totally 9 kinds of enzymes and corresponding cDNA sequence；

(2) building multiple recombinant plasmid compatible in same host cell, it may be assumed that according to the cDNA sequence design primer of known enzyme Corresponding gene order is synthesized, the cDNA sequence to design synthesis synthesizes corresponding gene sequence by pcr clone as template Clone's synthetic gene sequence is connected on corresponding plasmid with DNA recombinant technique, forms multiple recombinant plasmid by column, multiple Recombinant plasmid passes through promoter transcription and host cell translation coexpression synthesis β-farnesene GAP-associated protein GAP in host cell；

(3) multiple recombinant plasmid transformed, which is imported into host cell, obtains synthesis β-farnesene recombinant host cell, it may be assumed that will even The recombinant plasmid connected imports e. coli host cell, and culture amplification recombinant host cell extracts plasmid and carries out sequence verification Target gene has been coupled on corresponding plasmid, and the bacterial strain after verifying is carried out culture preservation.

The step S3 is specifically included: recombinant host cell being activated and accesses 250mL equipped with LB culture medium (pancreas egg White peptone 10g, yeast extract 5g, NaCl10g, it is cultivated, 38 DEG C of shaking table temperature, is turned in the triangular flask of water constant volume to 1000mL) Fast 220r/min, culture to OD₆₀₀To 0.8 ± 0.2, the shaking flask culture of 1000mL second order fermentation, shaking table are accessed with 20% inoculum concentration 38 DEG C of temperature, revolving speed 250r/min, culture to OD₆₀₀To 1.2 ± 0.2, the culture of 30L ferment tank is accessed with 8% inoculum concentration.

The step S4 is specifically included: entire fermentation process ventilation quantity control is in 1.2vvm, the control of fermentor speed of agitator In 400r/min, the control of fermentation liquid dissolved oxygen amount is that 7.0 ± 0.4(, 20% sulfuric acid and 30% ammonium hydroxide control fermentation liquid in 50%, pH PH)；Earlier fermentation (0h-20h) fermentation temperature is 37 DEG C, and ferment middle (21h-48h) fermentation temperature is 35 DEG C, is fermented the later period (49h-72h) is 30 DEG C；When culture to OD₆₀₀It is 20, final concentration of 0.3mmol/L isopropyl sulphur is accessed into fermentation medium For galactoside (IPTG), access accounts for the extractant of fermentating liquid volume 20% after 3h hours, and the extractant is n-dodecane；Hair Start feed supplement when sugared concentration (° BX) drops to (4 ± 1) % during ferment, control in fermentation liquid sugar concentration (° BX) 4.0 ± 0.2。

Molasses in the molasses stoste are the combination of both cane molasses and beet molasses, cane molasses, beet molasses Weight ratio be 2.5:6.

The host cell is Escherichia coli, and recombinant host cell is Escherichia coli B21, and the recombinant host cell of building is big Enterobacteria B21 can express acetoacetyl CoA thiolase (AtoB), HMG-CoA synthase, HMG-CoA reductase, mevalonic acid Kinases, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, isopentenylpyrophosphate isomerase, farnesyl pyrophosphate close At enzyme and Farmesene synthase totally 9 kinds of enzymes；Recombinant host cell Escherichia coli B21 includes pKK223-3, PMCSG9, pMal-c4X Three kinds of plasmids wherein include tri- kinds of genes of atoB, thmg1, mvd1 on pKK223-3, on PMCSG9 comprising erg13, erg12, Tetra- kinds of genes of erg8, ispA include two kinds of genes of idi, afs on pMal-c4X；The plasmid pKK223-3 selects tac starting Son, plasmid PMCSG9 select T7 promoter, plasmid pMal-c4X to select tac promoter.

In the second order fermentation shaking flask culture and 30L ferment tank incubation, the main composition of culture medium is as follows: removing Miscellaneous molasses after purification account for culture medium specific gravity 12%, and yeast powder 8%, peptone account for culture medium specific gravity 8.5%, ammonium citrate accounts for culture Base specific gravity 4%, magnesium sulfate account for culture medium specific gravity 1.5%, and manganese sulfate accounts for culture medium specific gravity 0.4%, and zinc sulfate accounts for culture medium specific gravity 0.4%。

The weight of material percentage supplemented during the feed supplement is the molasses 60% after edulcoration purification, ammonium sulfate 30%, egg White peptone 10%, above-mentioned material are spare in 110-120 DEG C of high pressure sterilization 25-40min.

A kind of gas chromatography of embodiment 4 detects β-farnesene method in fermentation liquid

β-farnesene method in a kind of gas chromatography detection fermentation liquid, includes the following steps:

H1, fermentation liquid pre-treatment: taking 10mL or more fermentation liquid, and 10000r/min is centrifuged 5min, and oil is mutually layered with water phase, in absorption The oily phase of layer obtains filter liquor with the organic membrane filtration of 0.44um；

H2, Agilent 7890A gas chromatograph, 19091JHP-5 chromatographic column (column length 30m are used;Internal diameter 0.32mm;Film thickness 250um), β-farnesene series standard solution is configured, the β-Fa Ni that concentration is respectively 5g/L, 10g/L, 15g/L, 20g/L is made Alkene standard solution, sample detection, draws equation of linear regression, and calculates R value respectively, makes R value 99% or more, above-mentioned β-Fa Ni Alkene testing conditions are as follows: post case temperature:, retaining 2min by 70 DEG C of initial temperature, rises to 250 DEG C with 10 DEG C/min, retains 1min, sample introduction 250 DEG C of temperature, sample volume 1uL of mouth, product inject split ratio 1:50, and 260 DEG C of temperature of detector (FID), nitrogen is as carrier gas, entrance Pressure is 15psi, and mode is constant current mode.

Using β-farnesene method in the kind gas chromatography detection fermentation liquid in the present embodiment to raw in embodiment 1-3 β-farnesene of production is detected, and embodiment 1-3 ferments 72h, it detects β-farnesene concentration in fermentation liquid and successively reaches 20.5g/L, 20.3g/L and 20.2g/L.

Above-mentioned specific embodiment is only specific case of the invention, and scope of patent protection of the invention includes but is not limited to The product form and style of above-mentioned specific embodiment, any claims of the present invention and any technical field of meeting The appropriate changes or modifications that those of ordinary skill does it, all shall fall within the protection scope of the present invention.

Sequence table

<110>Shandong Hongda Biology S&T Co., Ltd., Shandong Gunther Biotechnology Co., Ltd

<120>a kind of production method of β-farnesene

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 28

<212> DNA

<213>artificial sequence ()

<400> 1

gacaattaat catcggctcg tataatgt 28

<210> 2

<211> 17

<212> DNA

<213>artificial sequence ()

<400> 2

taatacgact cactata 17

<210> 3

<211> 29

<212> DNA

<213>artificial sequence ()

<400> 3

tgacaattaa tcatcggctc gtataatgt 29

<210> 4

<211> 1185

<212> DNA

<213>artificial sequence ()

<400> 4

atgaaaaatt gtgtcatcgt cagtgcggta cgtactgcta tcggtagttt taacggttca 60

ctcgcttcca ccagcgccat cgacctgggg gcgacagtaa ttaaagccgc cattgaacgt 120

gcaaaaatcg attcactaca cgttgatgaa gtgattatgg gtaacgtgtt gcaagccgga 180

ctggggcaaa atccggcacg tcaggcgctg ctaaaaagcg gactagctga aacggtgtgc 240

ggattcacgg tcaacaaagt gtgcggttca ggtctgaaaa gcgtggcgct tgctgcacag 300

gcgattcagg caggtcaggc acagagcatt gtggcggggg gtatggaaaa tatgagttta 360

gcgccctact tactcgatgc aaaagcacgc tctggttatc gtcttggaga cggacaggtt 420

tatgacgtaa tcctgcgcga tggcctgatg tgcgccaccc atggttatca tatggggatt 480

accgccgaaa acgtggctaa agagtacgga attacccgtg aaatgcagga tgaactggcg 540

ctacattcac agcgtaaagc ggcagccgca attgagtccg gtgcttttac agccgaaatc 600

gtcccggtaa atgttgtcac ccggaagaaa accttcgtct tcagtcaaga cgaattcccg 660

aaagcggatt ctacggctga agcgttaggc gcattgcgcc cggccttcga taaagcagga 720

acagtcaccg ccgggaacgc gtcaggtatt aacgacggtg ctgccgctct ggtgattatg 780

gaagaatctg cggcgctggc agcaggcctg aatcccctgg cacgcattaa aagttatgcc 840

agcggtggcg tgccccccgc attgatgggt atggggccag tacctgctac gcaaaaagcg 900

ttacaactgg cggggctgca actggcggat attgatctca ttgaggctaa tgaagcattt 960

gctgcacagt tccttgccgt tgggaaaacc ctgggctttg atcctgagaa agtgaatgtc 1020

aacggcgggg ccatcgcgct cggacatcct atcggtgcca gtggtgctcg tattctggtc 1080

acactattac atgcaatgca ggcacgcgat aaaacgctgg ggctggcaac actatgcatt 1140

ggtggcggcc agggaattgc gatggtgatt gagcggttga attaa 1185

<210> 5

<211> 1476

<212> DNA

<213>artificial sequence ()

<400> 5

ttattttttt acatcgtaag atcttctaaa tttgtcatcg atgttggtca agtagtaaac 60

accactttgc aaatgctcaa tggaaccttg aggtttgaag ttcttcttca aatgggcatt 120

ttctctcaat tcgatggcag cttcgtaatc ctttggagtt tcggtgattc tcttggctaa 180

tttgttagta atatctaatt ccttgataat atgttggacg tcaccaacaa ttttgcaaga 240

atatagagat gcagctaaac cggaaccgta agaaaataaa ccaacacgct tgccttgtaa 300

gtcgtcagat ccaacatagt ttaatagaga tgcaaaggcg gcataaacag atgcggtgta 360

catgttacct gtgtttgttg gaacaatcaa agattgggca actctctctt tgtggaatgg 420

cttagcaaca ttaacaaaag ttttttcaat gttcttatcg gttaaagatt cgtcataatc 480

gcgagtagct aattcggcgt caacttctgg gaacaattga ggattggctc tgaaatcgtt 540

atatagtaat ctaccgtatg attttgtgac caatttacag gttggaacat ggaaaacgtt 600

gtagtcgaaa tatttcaaaa cgttcaaagc atccgaacca gcgggatcgc taaccaaccc 660

tttagaaata gccttcttgg aataactctt gtaaacttga tcaagagcct tgacgtaaca 720

agttaatgaa aaatgaccat cgacgtaagg atattcgctg gtgaaatctg gcttgtaaaa 780

atcgtaggcg tgttccatgt aagaagctct tacagagtca aatacaattg gagcatcagg 840

accgatccac atagcaacag taccggcacc accggttggt cttgcggcac ccttatcgta 900

gatggcaata tcaccgcaaa ctacaatggc gtctctacca tcccatgcgt tagattcaat 960

ccagttcaaa gagttgaaca acgcgttggt accaccgtaa caggcattaa gcgtgtcaat 1020

accttcgacg tcagtgtttt caccaaacaa ttgcatcaag acagacttga cagacttgga 1080

cttgtcaatc agagtttcag taccgacttc taatctacca attttgttag tgtcgatatt 1140

gtaactcttg atcaacttag acaaaacagt tagggacatc gagtagatat cttctctgtc 1200

attgacaaaa gacatgttgg tttggcccag accaattgtg tatttacctt gagaaacgcc 1260

atcaaatttc tctagctcag attggttgac acattgagtt gggatgtaaa tttggatacc 1320

tttaataccg acattttgag gtctggtttt ttgttcagcg gtcttttgtt tttttagttc 1380

agtcatttgc aagtttgtat tgtgtaattg ttgttgcttt tgcggcctaa gtcttccctt 1440

aataccacac caacaaagtt tagttgagag tttcat 1476

<210> 6

<211> 381

<212> DNA

<213>artificial sequence ()

<400> 6

atgtcagact gcggagcgcg ccccaaaaaa cccatgagcg ccttcatgtt gtggatgaat 60

tccaccgggc ggaagcacat aaaagcggag catcccgatt ttagtgtcca agaagtgtct 120

gtgaagggcg gagagatgtg gcgagccatg gccgatgagg acaagatcgt gtggcaggag 180

tcggccacca cggcaatggc cgagtacaag gagaagttga agcagtggaa tttccccaag 240

gagcaccgct tttcggacac gcaatgtatt tgttcctcaa atactaacca atgccccacc 300

ctttttgtgt acgacaccat ggatgactcg atgactccga tctgcaggaa gtgcttatca 360

aagaccaggt gccttcacta a 381

<210> 7

<211> 1332

<212> DNA

<213>artificial sequence ()

<400> 7

atgtcattac cgttcttaac ttctgcaccg ggaaaggtta ttatttttgg tgaacactct 60

gctgtgtaca acaagcctgc cgtcgctgct agtgtgtctg cgttgagaac ctacctgcta 120

ataagcgagt catctgcacc agatactatt gaattggact tcccggacat tagctttaat 180

cataagtggt ccatcaatga tttcaatgcc atcaccgagg atcaagtaaa ctctcaaaaa 240

ttggccaagg ctcaacaagc caccgatggc ttgtctcagg aactcgttag tcttttggat 300

ccgttgttag ctcaactatc cgaatccttc cactaccatg cagcgttttg tttcctgtat 360

atgtttgttt gcctatgccc ccatgccaag aatattaagt tttctttaaa gtctacttta 420

cccatcggtg ctgggttggg ctcaagcgcc tctatttctg tatcactggc cttagctatg 480

gcctacttgg gggggttaat aggatctaat gacttggaaa agctgtcaga aaacgataag 540

catatagtga atcaatgggc cttcataggt gaaaagtgta ttcacggtac cccttcagga 600

atagataacg ctgtggccac ttatggtaat gccctgctat ttgaaaaaga ctcacataat 660

ggaacaataa acacaaacaa ttttaagttc ttagatgatt tcccagccat tccaatgatc 720

ctaacctata ctagaattcc aaggtctaca aaagatcttg ttgctcgcgt tcgtgtgttg 780

gtcaccgaga aatttcctga agttatgaag ccaattctag atgccatggg tgaatgtgcc 840

ctacaaggct tagagatcat gactaagtta agtaaatgta aaggcaccga tgacgaggct 900

gtagaaacta ataatgaact gtatgaacaa ctattggaat tgataagaat aaatcatgga 960

ctgcttgtct caatcggtgt ttctcatcct ggattagaac ttattaaaaa tctgagcgat 1020

gatttgagaa ttggctccac aaaacttacc ggtgctggtg gcggcggttg ctctttgact 1080

ttgttacgaa gagacattac tcaagagcaa attgacagtt tcaaaaagaa attgcaagat 1140

gattttagtt acgagacatt tgaaacagac ttgggtggga ctggctgctg tttgttaagc 1200

gcaaaaaatt tgaataaaga tcctaaaatc aaatccctag tattccaatt atttgaaaat 1260

aaaactacca caaagcaaca aattgacgat ctattattgc caggaaacac aaatttacca 1320

tggacttcat aa 1332

<210> 8

<211> 1356

<212> DNA

<213>artificial sequence ()

<400> 8

atgtcagagt tgagagcctt cagtgcccca gggaaagcgt tactagctgg tggatattta 60

gttttagata caaaatatga agcatttgta gtcggattat cggcaagaat gcatgctgta 120

gcccatcctt acggttcatt gcaagggtct gataagtttg aagtgcgtgt gaaaagtaaa 180

caatttaaag atggggagtg gctgtaccat ataagtccta aaagtggctt cattcctgtt 240

tcgataggcg gatctaagaa ccctttcatt gaaaaagtta tcgctaacgt atttagctac 300

tttaaaccta acatggacga ctactgcaat agaaacttgt tcgttattga tattttctct 360

gatgatgcct accattctca ggaggatagc gttaccgaac atcgtggcaa cagaagattg 420

agttttcatt cgcacagaat tgaagaagtt cccaaaacag ggctgggctc ctcggcaggt 480

ttagtcacag ttttaactac agctttggcc tccttttttg tatcggacct ggaaaataat 540

gtagacaaat atagagaagt tattcataat ttagcacaag ttgctcattg tcaagctcag 600

ggtaaaattg gaagcgggtt tgatgtagcg gcggcagcat atggatctat cagatataga 660

agattcccac ccgcattaat ctctaatttg ccagatattg gaagtgctac ttacggcagt 720

aaactggcgc atttggttga tgaagaagac tggaatatta cgattaaaag taaccattta 780

ccttcgggat taactttatg gatgggcgat attaagaatg gttcagaaac agtaaaactg 840

gtccagaagg taaaaaattg gtatgattcg catatgccag aaagcttgaa aatatataca 900

gaactcgatc atgcaaattc tagatttatg gatggactat ctaaactaga tcgcttacac 960

gagactcatg acgattacag cgatcagata tttgagtctc ttgagaggaa tgactgtacc 1020

tgtcaaaagt atcctgaaat cacagaagtt agagatgcag ttgccacaat tagacgttcc 1080

tttagaaaaa taactaaaga atctggtgcc gatatcgaac ctcccgtaca aactagctta 1140

ttggatgatt gccagacctt aaaaggagtt cttacttgct taatacctgg tgctggtggt 1200

tatgacgcca ttgcagtgat tactaagcaa gatgttgatc ttagggctca aaccgctaat 1260

gacaaaagat tttctaaggt tcaatggctg gatgtaactc aggctgactg gggtgttagg 1320

aaagaaaaag atccggaaac ttatcttgat aaataa 1356

<210> 9

<211> 1191

<212> DNA

<213>artificial sequence ()

<400> 9

atgaccgttt acacagcatc cgttaccgca cccgtcaaca tcgcaaccct taagtattgg 60

gggaaaaggg acacgaagtt gaatctgccc accaattcgt ccatatcagt gactttatcg 120

caagatgacc tcagaacgtt aacctctgcg gctactgcac ctgagtttga acgcgacact 180

ttgtggttaa atggagaacc acacagcatc gacaatgaaa gaactcaaaa ttgtctgcgc 240

gacctacgcc aattaagaaa ggaaatggaa tcgaaggacg cctcattgcc cacattatct 300

caatggaaac ttcacattgt ctccgaaaat aactttccta cagcagctgg tttagcttcc 360

tccgctgctg gctttgctgc attggtctct gcaattgcta agttatacca attaccacag 420

tcaacttcag aaatatctag aatagcaaga aaggggtctg gttcagcttg tagatcgttg 480

tttggcggat acgtggcctg ggaaatggga aaagctgaag atggtcatga ttccatggca 540

gtacaaatcg cagacagctc taactggcct cagatgaaag cttgtgtcct agttgtcagc 600

gatattaaaa aggatgtgag ttccactcag ggtatgcaat tgaccgtggc aacctccgaa 660

ctatttaaag aaagaattga acatgtcgta ccaaagagat ttgaagtcat gcgtaaagcc 720

attgttgaaa aagatttcgc cacctttgca aaggaaacaa tgatggattc caactctttc 780

catgccacat gtttggactc tttccctcca atattctaca tgaacgacac ttccaagcgt 840

atcatcagtt ggtgccacac cattaatcag ttttacggag aaacaatcgt tgcatacacg 900

tttgatgcag gtccaaatgc tgtgttgtac tacttagctg aaaatgagtc gaaactcttt 960

gcatttatct ataaattgtt tggctctgtt cctggatggg acaagaaatt tactgctgag 1020

cagcttgagg ctttcaacca tcaatttgaa tcttctaact ttactgcacg tgaattggat 1080

cttgagttgc aaaagggtgt tgccagagtg attttaactc aagtcggttc aggcccacaa 1140

gaaacaaatg aatctttgat tgacgcaaag actggtctac caaagaaata a 1191

<210> 10

<211> 549

<212> DNA

<213>artificial sequence ()

<400> 10

atgcaaacgg aacacgtcat tttattgaat gcacagggag ttcccacggg tacgctggaa 60

aagtatgccg cacacacggc agacaccctc ttacatctcg cgttttccag ttggctgttt 120

aatgccaaag ggcaattatt agttacccgc cgcgccctta gcaaaaaagc atggcctggc 180

gtgtggacta actcggtttg tgggcaccca caactgggag aaagcaacga agaggcggtg 240

atccgccgtt gccgttatga gcttggcgta gaaattacgc ctcctgaatc tatctatcct 300

gactttcgct accgcgccac cgatccgaat ggcattgtgg aaaatgaagt gtgtccggta 360

tttgccgcac gcacgaccag tgcgttacag atcaacgatg atgaagtgat ggattatcaa 420

tggtgtgatt tagcagcggt tttacgcggt attgatgcta cgccgtgggc gttcagtccg 480

tggatggtga tgcaggcgac aaatcgcgaa gccagaaaac gattatctgc atttacccaa 540

cttaaataa 549

<210> 11

<211> 543

<212> DNA

<213>artificial sequence ()

<400> 11

atgggggaca ccgtttgcaa tgggacaccc tgcaaaaagg cgtcgctctc ggactctcag 60

ctgtttgacg ccaagttcga ggagctggtg acagagctga ccgagaggga cctccaggat 120

cctgcgctgg cggacgcctt gaaaaggttg agagaggttt tggattacaa tgttcctgga 180

ggcaaaaaga acagaggttt gtctgtgatt ggctccctga gggagcttct tccgccgtcc 240

cagctcagtc aggatgctgt gcagaaagct ctgacggtcg gctggtgcat agagatgctt 300

caagcatttt tcctcatggc ggatgacatc atggatgcat ctgtgacccg gcgaggtcaa 360

ccctgctggt acaagaggaa tggaataggt ctggatgcaa taaatgactc cttcttgtgg 420

aggcatcaat atatagactc cttcgaaggc aatgcagggg tgagccgtat tacgtccatt 480

tactggagct ttttaatgag tccaccttcc agactgaact tggtcaggcc ctggacctca 540

tga 543

<210> 12

<211> 1731

<212> DNA

<213>artificial sequence ()

<400> 12

atggaattca gagttcactt gcaagctgat aatgagcaga aaatttttca aaaccagatg 60

aaacccgaac ctgaagcctc ttacttgatt aatcaaagac ggtctgcaaa ttacaagcca 120

aatatttgga agaacgattt cctagatcaa tctcttatca gcaaatacga tggagatgag 180

tatcggaagc tgtctgagaa gttaatagaa gaagttaaga tttatatatc tgctgaaaca 240

atggatttag tagctaagtt ggagctcatt gacagcgtcc gaaaactagg cctcgcgaac 300

ctcttcgaaa aggaaatcaa ggaagcccta gacagcattg cagctatcga aagcgacaat 360

ctcggcacaa gagacgatct ctatggtact gcattacact tcaagatcct caggcagcat 420

ggctataaag tttcacaaga tatatttggt agattcatgg atgaaaaggg cacattagag 480

aaccgccatt tcgcacattt gaaaggaatg ctggaacttt tcgaggcctc aaacctgggt 540

ttcgaaggtg aagatatttt agatgaggcg aaagcttcct tgacgctagc tctcagagat 600

agtggtcata tttgttatcc ggacagtaac ctttccaggg acgtaattca ttccctggag 660

cttccatcac accgcagagt gcagtggttt gatgtcaaat ggcaaatcaa cgcctatgaa 720

aaagacatct gtcgcgtcaa cgccacgtta ctcgaattag caaagcttaa tttcaacatg 780

gttcaggccc acctccaaaa agacttaagg gaagcatcca agtggtgggc aaatctgggc 840

atcgcagaca acttgaaatt tgcaagagat agactggttg aatgtttcgc atgtgctgtg 900

ggagtagctt tcgagcctga atactcatct tttagaatat gtcttaccaa agtcatcaac 960

ttagtactga tcatagacga cgtctatgat atttatggct cagaggaaga gctaaagcac 1020

ttcaccaatg ctgttgatag gtgggattct agggaaactg agcagcttcc agagtgtatg 1080

aagatgtgtt tccaagtact ctacaacact acttgtgaaa ttgctcatga aattgagaag 1140

gacaatggtt ggaaccaagt attacctcaa ttgaccaaag tgtgggcaga tttttgtaaa 1200

gcattattgg tggaggcaga gtggtataat aagagccata taccaaccct tgaagagtac 1260

ctaagaaatg gatgcgattc atcatcagtt tcaatacttt tggttcactc atttttctct 1320

ataactcatg agggaaccaa agagatggct gattttcttc acaagaatga agatcttttg 1380

tataatctct ctctcattgt tcgcctcaac aatgatttgg gaacttctgc ggctgaacaa 1440

gagagagggg attctccttc atcaatcgta tgttacatga gagaagtgaa tgcctctgaa 1500

gaaatagcta ggaagaacat taagggcatg atagacaatg catggaagaa agtaaatgga 1560

aaatgcttca caacaaacca agtgcctttt ctgtcatcat tcatgaacaa tgccacaaac 1620

atggcacgtg tggcgcacag cctttacaaa gacggagatg ggtttggtga ccaagagaaa 1680

gggcctcgga cccacatcct atctttacta ttccaacctc ttgtaaacta g 1731

Claims (10)

1. a kind of production method of β-farnesene, characterized by the following steps: S1 molasses edulcoration purification, S2 β-Fa Ni The building of alkene recombinant host cell, S3 recombinant host cell is inoculated into containing edulcoration purification after molasses culture medium in carry out Heterologous inducing expression synthesis β-farnesene, S4 improve β-farnesene yield by ferment control.

2. a kind of production method of β-farnesene according to claim 1, it is characterised in that: the step S1 is specifically wrapped It includes:

(1) it weighs: weighing the water that molasses stoste is added 1.5-2.0 times, molasses are diluted to 16-17 Baume degrees, obtain dilute liquid glucose；

(2) be acidified: dilute liquid glucose pours into fermentor, and the concentrated sulfuric acid is added and concentrated phosphoric acid adjusts acidity, pH4.0-4.5 is acidified；

(3) divulge information: dilute liquid glucose after acidification is boiled in logical steam heating, and heating promotes colloid protein to solidify, and convenient for precipitating centrifugation, adds After heat is boiled, 90 DEG C are kept the temperature, while being passed through into ullage gas 1h, to remove removing and harmful gas NO₂、SO₂And volatile materials；

(4) add alkali neutralization: dilute liquid glucose after ventilation process is packed into temporary storage barrel, and milk of lime is added and adjusts pH to 5.0-5.5；

(5) centrifugal clarification: the impurity being centrifuged off in dilute liquid glucose after adding alkali neutralization with supercentrifuge takes clear liquid；

(6) heat sterilization: clear liquid pours into the fermentor cleaned up, and steaming is heated to 90 DEG C, keeps the temperature 0.5h；

(7) pressure maintaining is stored: after sterilizing is good that fermentor is closed, ventilate pressure maintaining 1.0Mpa, and room temperature is kept in spare.

3. a kind of production method of β-farnesene according to claim 1, it is characterised in that: the step S2 is specifically wrapped It includes: (1) synthesis β-farnesene related gene, related gene being obtained by ncbi database inquiry and technique for gene engineering means Including acetoacetyl CoA thiolase (AtoB) gene atoB, HMG-CoA synthase (ERG13) gene in mevalonate pathway Erg13, HMG-CoA reductase (tHMG1) gene thmg1, mevalonate kinase (ERG12) gene erg12, phosphomelovanate Kinases (ERG8) gene erg8, mevalonate pyrophosphate decarboxylase (MVD1) gene mvd1, isopentenylpyrophosphate isomerase (IDI) Gene idi, farnesyl pyrophosphate synzyme (FPPS) gene ispA, Farmesene synthase (AFS) gene afs；

(2) building multiple recombinant plasmid compatible in same host cell, the multiple recombinant plasmid lead in host cell Cross promoter transcription and host cell translation coexpression synthesis β-farnesene GAP-associated protein GAP；

(3) multiple recombinant plasmid transformed, which is imported into host cell, obtains synthesis β-farnesene recombinant host cell.

4. a kind of production method of β-farnesene according to claim 1, it is characterised in that: the step S3 is specifically wrapped Include: recombinant host cell activated and access 250mL equipped with LB culture medium (tryptone 10g, yeast extract 5g, NaCl10g, it is cultivated, 30-38 DEG C of shaking table temperature, revolving speed 180-220r/min, is cultivated in the triangular flask of water constant volume to 1000mL) To OD₆₀₀To 0.8 ± 0.2, the shaking flask culture of 1000mL second order fermentation is accessed with the inoculum concentration of 3-20%, 35-38 DEG C of shaking table temperature turns Fast 180-250r/min, culture to OD₆₀₀To 1.2 ± 0.2, the culture of 30L ferment tank is accessed with the inoculum concentration of 5%-8%.

5. according to a kind of production method of β-farnesene described in claim 1, it is characterised in that: the step S4 is specifically included: In 0.5vvm-1.5vvm, fermentor speed of agitator is controlled in 200r/min-400r/min for entire fermentation process ventilation quantity control, The control of fermentation liquid dissolved oxygen amount is that 7.0 ± 0.4(, 20% sulfuric acid and 30% ammonium hydroxide control fermentation liquid pH in 2%-90%, pH)；Before fermentation Phase (0h-20h) fermentation temperature is 35 DEG C -37 DEG C, and ferment middle (21h-48h) fermentation temperature is 30 DEG C -35 DEG C, is fermented the later period (49h- terminates) is 28 DEG C -35 DEG C；When culture to OD₆₀₀For 8-20, final concentration of 0.1- is accessed into fermentation medium 0.5mmol/L isopropylthiogalactoside (IPTG), access accounts for the extraction of fermentating liquid volume 10%-20% after 2h-10h hours Agent, the extractant are one of n-decane, n-dodecane, petroleum ether, normal octane or a variety of；When sugared concentration in fermentation process (° BX) starts feed supplement when dropping to (4 ± 1) %, sugared concentration (° BX) is 4.0 ± 0.2 in control fermentation liquid.

6. according to a kind of production method of β-farnesene as claimed in claim 2, it is characterised in that: the molasses in the molasses stoste For the combination of cane molasses or beet molasses or both, under combined situation, cane molasses, beet molasses weight ratio be 100: 25-1000。

7. according to a kind of production method of β-farnesene as claimed in claim 3, it is characterised in that: the host cell is large intestine bar Bacterium, recombinant host cell are Escherichia coli B21, and the recombinant host cell Escherichia coli B21 of building can express acetoacetyl CoA thiolase (AtoB), HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, first hydroxyl Valeric acid pyrophosphoric acid decarboxylase, isopentenylpyrophosphate isomerase, farnesyl pyrophosphate synzyme and Farmesene synthase totally 9 kinds of enzymes；Greatly Enterobacteria B21, include tri- kinds of plasmids of pKK223-3, PMCSG9, pMal-c4X, wherein on pKK223-3 comprising atoB, thmg1, Tri- kinds of genes of mvd1 include tetra- kinds of genes of erg13, erg12, erg8, ispA on PMCSG9, include idi, afs on pMal-c4X Two kinds of genes；The plasmid pKK223-3 selects tac promoter, plasmid PMCSG9 to select T7 promoter, plasmid pMal-c4X choosing With tac promoter.

8. according to a kind of production method of β-farnesene as claimed in claim 4, it is characterised in that: the second order fermentation shaking flask culture In 30L ferment tank incubation, the main composition of culture medium is as follows: the molasses after edulcoration purification account for culture medium specific gravity 2%-20%, yeast powder or yeast extract or both combination (yeast powder under combined situation, yeast extract weight ratio be 100:1- 10000) culture medium specific gravity 1%-10%, is accounted for, peptone accounts for culture medium specific gravity 1.5%-8.5%, ammonium citrate accounts for culture medium specific gravity 0.1%-5%, magnesium sulfate or magnesium chloride account for culture medium specific gravity 0.1%-2%, and manganese sulfate accounts for culture medium specific gravity 0.05%-0.9%, zinc sulfate Account for culture medium specific gravity 0.02%-0.8%.

9. according to a kind of production method of β-farnesene described in claim 5, it is characterised in that: supplemented during the feed supplement Weight of material percentage be edulcoration purification after molasses 40%-60%, ammonium sulfate 20%-50%, peptone 5%-10%, above-mentioned material in 110-120 DEG C of high pressure sterilization 25-40min is spare.

10. a kind of β-farnesene method in gas chromatography detection fermentation liquid, characterized by the following steps:

H1, fermentation liquid pre-treatment: taking 10mL or more fermentation liquid, and 9000-12000r/min is centrifuged 3-8min, and oil mutually divides with water phase Layer draws upper oil phase with the organic membrane filtration of 0.44um and obtains filter liquor；

H2, Agilent 7890A gas chromatograph, 19091JHP-5 chromatographic column (column length 30m are used;Internal diameter 0.32mm;Film thickness 250um), β-farnesene series standard solution is configured, the β-Fa Ni that concentration is respectively 5g/L, 10g/L, 15g/L, 20g/L is made Alkene standard solution, sample detection, draws equation of linear regression, and calculates R value respectively, makes R value 99% or more, above-mentioned β-Fa Ni Alkene testing conditions are as follows: post case temperature:, retaining 2min by 65-75 DEG C of initial temperature, rises to 220-280 DEG C with 10 DEG C/min, retains 1min, 220-280 DEG C of injector temperature, sample volume 1uL, product injection split ratio 1:50,260-280 DEG C of temperature of detector (FID), For nitrogen as carrier gas, inlet pressure is 12-18psi, and mode is constant current mode.