CN109797173B - Production method of beta-farnesene - Google Patents
Production method of beta-farnesene Download PDFInfo
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- CN109797173B CN109797173B CN201910239474.4A CN201910239474A CN109797173B CN 109797173 B CN109797173 B CN 109797173B CN 201910239474 A CN201910239474 A CN 201910239474A CN 109797173 B CN109797173 B CN 109797173B
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Abstract
The invention relates to the technical field of biology, in particular to a production method of beta-farnesene. The production method of the beta-farnesene comprises the following steps: s1 molasses impurity removal and purification, S2 beta-farnesene recombinant host cell construction, S3 inoculation of recombinant host cells to a culture medium containing the molasses impurity removal and purification for heterologous induction expression synthesis of beta-farnesene, and S4 improvement of the yield of the beta-farnesene through fermentation control. According to the production method of beta-farnesene, disclosed by the invention, molasses with low price is used as a main raw material, recombinant escherichia coli is used as a production strain to produce beta-farnesene, the fermentation period is shortened, and the production cost is reduced.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a production method of beta-farnesene.
Background
Beta-farnesene is an important sesquiterpene compound, has wide application prospect in many fields, on one hand, in the field of vitamin E production, the beta-farnesene is taken as an intermediate, so that the synthesis steps of the vitamin E can be greatly reduced, compared with the traditional synthesis method, the reaction efficiency and yield are improved, the production cost is reduced, and the beta-farnesene is utilized to produce the vitamin E in Hubei energy science and technology at present; on the other hand, the beta-farnesene can be used for preparing clean renewable fuels with high heat value and high efficiency, the global energy crisis is relieved, the American Amyris utilizes the patent yeast to produce the beta-farnesene at present, but the American Amyris takes the sucrose as the main raw material, the production cost is relatively expensive, the fermentation period is long, the technology utilizes the molasses with low price as the main raw material, and the recombinant escherichia coli as the production strain to produce the beta-farnesene, the fermentation period is shortened, and the production cost is reduced.
Disclosure of Invention
The invention aims to provide a method for producing beta-farnesene, which uses molasses with low price as a main raw material and recombinant escherichia coli as a production strain to produce the beta-farnesene, shortens the fermentation period and reduces the production cost.
The technical scheme adopted by the invention for solving the technical problem is as follows:
a production method of beta-farnesene comprises the following steps: s1 molasses impurity removal and purification, S2 beta-farnesene recombinant host cell construction, S3 inoculation of recombinant host cells to a culture medium containing the molasses impurity removal and purification for heterologous induction expression synthesis of beta-farnesene, and S4 improvement of the yield of the beta-farnesene through fermentation control.
Preferably, the step S1 specifically includes:
(1) weighing: weighing molasses stock solution, adding 1.5-2.0 times of water, diluting molasses to 16-17 Baume degrees to obtain diluted sugar solution, wherein the mass of each batch of molasses stock solution fluctuates, and the dilution water amount is based on data measured by a Baume meter on site;
(2) acidifying: pouring the dilute sugar solution into a fermentation tank, adding concentrated sulfuric acid and concentrated phosphoric acid to adjust the acidity, adjusting the pH to 4.0-4.5, acidifying, wherein after acidification, the impurities such as sand, scum and the like are less than or equal to 1%, the concentration is more than or equal to 80 degrees BX, the total sugar content is more than or equal to 52%, and the acidity is less than 9%;
(3) ventilating: introducing steam, boiling acidified dilute sugar solution, heating to promote colloidal protein solidification, centrifuging for precipitation, heating to boil, keeping temperature at 90 deg.C, introducing air into tank for 1 hr to remove harmful gas NO 2 、SO 2 And other harmful gases as well as volatile acids and other volatile substances;
(4) adding alkali for neutralization: filling the ventilated dilute sugar solution into a temporary storage barrel, adding lime milk to adjust the pH value to 5.0-5.5, wherein the lime milk is prepared by adding water into calcium oxide and is prepared in situ;
(5) and (3) centrifugal clarification: centrifuging by a high-speed centrifuge to remove impurities in the diluted sugar solution neutralized by adding alkali, and taking clear liquid;
(6) heating and sterilizing: pouring the clear liquid into a clean fermentation tank, introducing steam, heating to 90 ℃, and preserving heat for 0.5 h;
(7) and (4) pressure maintaining storage: sealing the fermentation tank after sterilization, ventilating and maintaining the pressure at 1.0Mpa, and temporarily storing at normal temperature for later use.
Preferably, the step S2 specifically includes: (1) obtaining related genes for synthesizing beta-farnesene by NCBI database query and genetic engineering technical means, wherein the related genes comprise acetoacetyl CoA thiolase (AtoB) gene atoB, HMG-CoA synthase (ERG 13) gene ERG13, HMG-CoA reductase (tHMG 1) gene tHMG1, mevalonate kinase (ERG 12) gene ERG12, phosphomevalonate kinase (ERG 8) gene ERG8, mevalonate pyrophosphate decarboxylase (MVD 1) gene MVD1, isopentene pyrophosphate isomerase (IDI) gene IDI, farnesyl pyrophosphate synthetase FPPS (PS) gene ispA, farnesene synthase (AFS) gene AFS in a mevalonate pathway;
(2) constructing multiple recombinant plasmids compatible in the same host cell, wherein the multiple recombinant plasmids co-express and synthesize beta-farnesene related protein in the host cell through promoter transcription and host cell translation;
(3) and transforming and introducing the multiple recombinant plasmids into host cells to obtain the recombinant host cells for synthesizing the beta-farnesene.
Preferably, the step S3 specifically includes: activating the recombinant host cells, inoculating the recombinant host cells into 250mL of triangular flask containing LB medium (10 g of tryptone, 5g of yeast extract and 10g of NaCl10g, and diluting the volume to 1000mL with water) for culture, culturing the cells at the temperature of 30-38 ℃ and the rotation speed of 180-220r/min until OD is achieved 600 Inoculating to 1000mL of secondary fermentation shake flask for culturing at the inoculation amount of 0.8 +/-0.2% with 3-20%, and shaking tableThe temperature is 35-38 ℃, the rotation speed is 180- 600 Inoculating into a 30L fermentation tank for fermentation culture in an inoculation amount of 5-8% to 1.2 +/-0.2.
Preferably, the step S4 specifically includes: the ventilation rate of the whole fermentation process is controlled to be 0.5vvm-1.5vvm, the stirring speed of a fermentation tank is controlled to be 200r/min-400r/min, the dissolved oxygen content of the fermentation liquor is controlled to be 2% -90%, and the pH value is 7.0 +/-0.4 (the pH value of the fermentation liquor is controlled by 20% sulfuric acid and 30% ammonia water); the fermentation temperature in the early fermentation period (0 h-20 h) is 35-37 ℃, the fermentation temperature in the middle fermentation period (21 h-48 h) is 30-35 ℃, and the fermentation temperature in the later fermentation period (49 h-end) is 28-35 ℃; when cultured to OD 600 8-20, inoculating isopropyl thiogalactoside (IPTG) with the final concentration of 0.1-0.5mmol/L into a fermentation medium, inoculating an extracting agent accounting for 10-20% of the volume of the fermentation liquid after 2-10 h, wherein the extracting agent is one or more of n-decane, n-dodecane, petroleum ether and n-octane; feeding is started when the sugar concentration (DEG BX) is reduced to (4 +/-1)% in the fermentation process, and the sugar concentration (DEG BX) in the fermentation liquor is controlled to be 4.0 +/-0.2.
Preferably, the molasses in the molasses stock solution is cane molasses or beet molasses or the combination of the cane molasses and the beet molasses, and the weight ratio of the cane molasses to the beet molasses is 100:25-1000 in the combination case.
Preferably, the host cell is escherichia coli, the recombinant host cell is escherichia coli B21, the constructed recombinant host cell escherichia coli B21 can express 9 enzymes of acetoacetyl CoA thiolase (AtoB), HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthetase and farnesene synthase, wherein the nucleotide sequence of the gene AtoB is shown in SEQ ID No.4, the nucleotide sequence of the gene erg13 is shown in SEQ ID No.5, the nucleotide sequence of the gene thmg1 is shown in SEQ ID No.6, the nucleotide sequence of the gene erg12 is shown in SEQ ID No.7, the nucleotide sequence of the gene erg8 is shown in SEQ ID No.8, the nucleotide sequence of the gene mvd1 is shown in SEQ ID No.9, and the nucleotide sequence of the gene idi is shown in SEQ ID No.10, the nucleotide sequence of the gene ispA is shown as SEQ ID NO.11, and the nucleotide sequence of the gene afs is shown as SEQ ID NO. 12.
Preferably, the recombinant host cell Escherichia coli B21 comprises three plasmids of pKK223-3, PMCSSG 9 and pMal-c4X, wherein the pKK223-3 comprises three genes of atoB, thmg1 and mvd1, the PMCSSG 9 comprises four genes of erg13, erg12, erg8 and ispA, and the pMal-c4X comprises two genes of idi and afs.
Preferably, the plasmid pKK223-3 adopts a tac promoter (shown as SEQ ID NO. 1), the plasmid PMCSG9 adopts a T7 promoter (shown as SEQ ID NO. 2), and the plasmid pMal-c4X adopts a tac promoter (shown as SEQ ID NO. 3).
Preferably, in the processes of the secondary fermentation shake flask culture and the 30L fermentation tank fermentation culture, the main compositions of the culture medium are as follows: the molasses after impurity removal and purification accounts for 2-20% of the weight of the culture medium, yeast powder or yeast extract or the combination of the two (in the combined case, the yeast powder is 100: 1-10000), accounts for 1-10% of the weight of the culture medium, peptone accounts for 1.5-8.5% of the weight of the culture medium, ammonium citrate accounts for 0.1-5% of the weight of the culture medium, magnesium sulfate or magnesium chloride accounts for 0.1-2% of the weight of the culture medium, manganese sulfate accounts for 0.05-0.9% of the weight of the culture medium, and zinc sulfate accounts for 0.02-0.8% of the weight of the culture medium.
Preferably, the materials supplemented in the material supplementing process comprise 40-60 wt% of molasses subjected to impurity removal and purification, 20-50 wt% of ammonium sulfate and 5-10 wt% of peptone, and the materials are sterilized under high pressure at the temperature of 110-120 ℃ for 25-40min for later use.
A method for detecting beta-farnesene in fermentation liquor by gas chromatography comprises the following steps:
h1, pretreatment of fermentation liquor: taking more than 10mL of fermentation liquor, centrifuging for 3-8min at 9000-;
h2, preparing a beta-farnesene series standard solution by using an Agilent 7890A gas chromatograph, a 19091JHP-5 chromatographic column (the column length is 30m, the inner diameter is 0.32mm, and the film thickness is 250um), preparing the beta-farnesene standard solution with the concentration of 5g/L, 10g/L, 15g/L and 20g/L respectively, carrying out sample injection detection respectively, drawing a linear regression equation, and calculating the R value to ensure that the R value is more than 99%, wherein the beta-farnesene detection conditions are as follows: temperature of the column box: the initial temperature is 65-75 ℃, the initial temperature is kept for 2min, the temperature is increased to 280 ℃ plus 220 ℃ at the speed of 10 ℃/min, the initial temperature is kept for 1min, the injection port temperature is 280 ℃ plus 220 ℃, the injection amount is 1uL, the injection split ratio is 1:50, the temperature of the detector (FID) is 280 ℃ plus 260 ℃, nitrogen is used as carrier gas, the inlet pressure is 12-18psi, and the mode is a constant-flow mode.
The invention has the beneficial effects that: compared with the prior art, the production method of beta-farnesene provided by the invention has the advantages that the molasses with low price is used as a main raw material, the recombinant escherichia coli is used as a production strain to produce the beta-farnesene, the fermentation period is shortened, the production cost is reduced, and the concentration of the beta-farnesene in the fermentation broth is up to 20.5g/L after the fermentation is carried out for 72 hours through the detection of the method for detecting the beta-farnesene in the fermentation broth through the gas chromatography provided by the invention.
Detailed Description
Example 1 production method of beta-farnesene
A production method of beta-farnesene comprises the following steps: s1 molasses impurity removal and purification, S2 beta-farnesene recombinant host cell construction, S3 inoculating the recombinant host cell to a culture medium containing the molasses impurity removal and purification for heterologous induction expression synthesis of the beta-farnesene, and S4 improves the yield of the beta-farnesene through fermentation control.
The step S1 specifically includes:
(1) weighing: weighing molasses stock solution, adding 1.5-2.0 times of water, and diluting molasses to 16-17 Baume degree to obtain diluted sugar solution;
(2) acidifying: pouring the dilute sugar solution into a fermentation tank, adding concentrated sulfuric acid and concentrated phosphoric acid to adjust the acidity, adjusting the pH to 4.0-4.5, acidifying, wherein after acidification, the impurities such as sand, scum and the like are less than or equal to 1%, the concentration is more than or equal to 80 degrees BX, the total sugar content is more than or equal to 52%, and the acidity is less than 9%;
(3) ventilating: introducing steam, boiling acidified dilute sugar solution, heating to promote colloidal protein solidification, centrifuging for precipitation, heating to boil, keeping temperature at 90 deg.C, introducing air into tank for 1 hr to remove harmful gas NO 2 、SO 2 And other harmful gases as well as volatile acids and other volatile substances;
(4) adding alkali for neutralization: filling the diluted sugar solution after ventilation treatment into a temporary storage barrel, adding lime milk to adjust the pH to 5.0-5.5, wherein the lime milk is prepared by adding water into calcium oxide and is prepared at present;
(5) and (3) centrifugal clarification: centrifuging by a high-speed centrifuge to remove impurities in the diluted sugar solution neutralized by adding alkali, and taking clear liquid;
(6) heating and sterilizing: pouring the clear liquid into a clean fermentation tank, introducing steam, heating to 90 ℃, and preserving heat for 0.5 h;
(7) and (4) pressure maintaining storage: after sterilization, the fermentation tank is sealed, ventilated and pressure-maintained at 1.0Mpa, and kept temporarily at normal temperature for standby.
The step S2 specifically includes:
(1) finding 9 enzymes of acetoacetyl-CoA thiolase (AtoB), HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthase and farnesene synthase in MVA pathway and corresponding cDNA sequences by NCBI database BLAST;
(2) constructing multiple recombinant plasmids compatible in the same host cell, namely: designing a primer according to a cDNA sequence of a known enzyme to synthesize a corresponding gene sequence, cloning and synthesizing the corresponding gene sequence by using the designed and synthesized cDNA sequence as a template through a PCR (polymerase chain reaction) technology, connecting the cloned and synthesized gene sequence to a corresponding plasmid by using a DNA (deoxyribonucleic acid) recombination technology to form a multiple recombinant plasmid, and co-expressing the multiple recombinant plasmid in a host cell through promoter transcription and host cell translation to synthesize beta-farnesene related protein;
(3) transforming and introducing the multiple recombinant plasmids into host cells to obtain recombinant host cells for synthesizing the beta-farnesene, namely: introducing the connected recombinant plasmid into an escherichia coli host cell, culturing and amplifying the recombinant host cell, extracting the plasmid, performing sequencing verification to verify that the target gene is connected to the corresponding plasmid, and culturing and preserving the verified strain.
The step S3 specifically includes: activating the recombinant host cell, inoculating into 250mL triangular flask containing LB medium (tryptone 10g, yeast extract 5g, NaCl10g, and water to reach volume of 1000 mL), culturing at 37 deg.C with rotation speed of 200r/min in shaking table until OD 600 To 0.8. + -. 0.2, by 1Inoculating 0% of the inoculum size into 1000mL of secondary fermentation shake flask for culture, wherein the shaking table temperature is 37 ℃, the rotating speed is 220r/min, and the culture is carried out until OD is reached 600 When the inoculation amount is 1.2 +/-0.2, inoculating the mixture into a 30L fermentation tank for fermentation culture at the inoculation amount of 7 percent.
The step S4 specifically includes: the ventilation rate of the whole fermentation process is controlled at 0.8vvm, the stirring speed of the fermentation tank is controlled at 300r/min, the dissolved oxygen content of the fermentation liquor is controlled at 20%, and the pH value is 7.0 +/-0.4 (the pH value of the fermentation liquor is controlled by 20% sulfuric acid and 30% ammonia water); the fermentation temperature in the early fermentation period (0 h-20 h) is 36 ℃, the fermentation temperature in the middle fermentation period (21 h-48 h) is 32 ℃, and the fermentation temperature in the later fermentation period (49 h-72 h) is 30 ℃; when cultured to OD 600 16, inoculating isopropyl thiogalactoside (IPTG) with the final concentration of 0.1mmol/L into a fermentation medium, and inoculating an extractant accounting for 15% of the volume of the fermentation liquid after 3 hours, wherein the extractant is n-dodecane; feeding is started when the sugar concentration (BX) is reduced to (4 +/-1)% in the fermentation process, and the sugar concentration (BX) in the fermentation liquor is controlled to be 4.0 +/-0.2.
The molasses in the molasses stock solution is the combination of cane molasses and beet molasses, and the weight ratio of the cane molasses to the beet molasses is 2.5: 6.
The host cell is escherichia coli, the recombinant host cell is escherichia coli B21, the constructed recombinant host cell escherichia coli B21 can express 9 enzymes including acetoacetyl CoA thiolase (AtoB), HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthase and farnesene synthase; the recombinant host cell Escherichia coli B21 comprises three plasmids of pKK223-3, PMCSG9 and pMal-c4X, wherein the pKK223-3 comprises three genes of atoB, thmg1 and mvd1, the PMCSG9 comprises four genes of erg13, erg12, erg8 and ispA, and the pMal-c4X comprises two genes of idi and afs; the plasmid pKK223-3 adopts a tac promoter, the plasmid PMCSG9 adopts a T7 promoter, and the plasmid pMal-c4X adopts a tac promoter.
In the processes of secondary fermentation shake flask culture and 30L fermentation tank fermentation culture, the culture medium mainly comprises the following components: the molasses after impurity removal and purification accounts for 8.5 percent of the proportion of the culture medium, the yeast powder 3.5 percent, the peptone accounts for 4.8 percent of the proportion of the culture medium, the ammonium citrate accounts for 1.8 percent of the proportion of the culture medium, the magnesium sulfate accounts for 1.5 percent of the proportion of the culture medium, the manganese sulfate accounts for 0.12 percent of the proportion of the culture medium, and the zinc sulfate accounts for 0.08 percent of the proportion of the culture medium.
The materials supplemented in the material supplementing process comprise 50% of molasses, 30% of ammonium sulfate and 20% of peptone after impurity removal and purification, and the materials are sterilized under high pressure at the temperature of 110-120 ℃ for 25-40min for later use.
Example 2 production method of beta-farnesene
A production method of beta-farnesene comprises the following steps: s1 molasses impurity removal and purification, S2 beta-farnesene recombinant host cell construction, S3 inoculation of recombinant host cells to a culture medium containing the molasses impurity removal and purification for heterologous induction expression synthesis of beta-farnesene, and S4 improvement of the yield of the beta-farnesene through fermentation control.
The step S1 specifically includes:
(1) weighing: weighing molasses stock solution, adding 1.5-2.0 times of water, and diluting molasses to 16-17 Baume degree to obtain diluted sugar solution;
(2) acidifying: pouring the diluted sugar solution into a fermentation tank, adding concentrated sulfuric acid and concentrated phosphoric acid to adjust the acidity, wherein the pH value is 4.0-4.5, and acidifying, wherein after acidification, impurities such as sandy soil, floating slag and the like are less than or equal to 1%, the concentration is more than or equal to 80 degrees BX, the total sugar content is more than or equal to 52%, and the acidity is less than 9%;
(3) ventilating: introducing steam, boiling acidified dilute sugar solution, heating to promote colloidal protein solidification, centrifuging for precipitation, heating to boil, keeping temperature at 90 deg.C, introducing air into tank for 1 hr to remove harmful gas NO 2 、SO 2 And other harmful gases as well as volatile acids and other volatile substances;
(4) adding alkali for neutralization: filling the diluted sugar solution after ventilation treatment into a temporary storage barrel, adding lime milk to adjust the pH to 5.0-5.5, wherein the lime milk is prepared by adding water into calcium oxide and is prepared at present;
(5) and (3) centrifugal clarification: centrifuging by a high-speed centrifuge to remove impurities in the diluted sugar solution neutralized by adding alkali, and taking clear liquid;
(6) heating and sterilizing: pouring the clear liquid into a clean fermentation tank, introducing steam, heating to 90 ℃, and preserving heat for 0.5 h;
(7) and (4) pressure maintaining storage: sealing the fermentation tank after sterilization, ventilating and maintaining the pressure at 1.0Mpa, and temporarily storing at normal temperature for later use.
The step S2 specifically includes:
(1) finding 9 enzymes of acetoacetyl CoA thiolase (AtoB), HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthetase and farnesene synthase in MVA pathway and corresponding cDNA sequences by NCBI database BLAST;
(2) constructing multiple recombinant plasmids compatible in the same host cell, namely: designing a primer according to a cDNA sequence of a known enzyme to synthesize a corresponding gene sequence, cloning and synthesizing the corresponding gene sequence by using the designed and synthesized cDNA sequence as a template through a PCR (polymerase chain reaction) technology, connecting the cloned and synthesized gene sequence to a corresponding plasmid by using a DNA (deoxyribonucleic acid) recombination technology to form a multiple recombinant plasmid, and co-expressing the multiple recombinant plasmid in a host cell through transcription of a promoter and translation of the host cell to synthesize a related protein of beta-farnesene;
(3) transforming and introducing the multiple recombinant plasmids into host cells to obtain recombinant host cells for synthesizing the beta-farnesene, namely: and introducing the connected recombinant plasmid into an escherichia coli host cell, culturing and amplifying the recombinant host cell, extracting the plasmid, performing sequencing verification to verify that the target gene is connected to the corresponding plasmid, and culturing and preserving the verified strain.
The step S3 specifically includes: activating the recombinant host cell, inoculating into 250mL triangular flask containing LB medium (tryptone 10g, yeast extract 5g, NaCl10g, and water to reach volume of 1000 mL), culturing at 35 deg.C with rotation speed of 180r/min until OD 600 Inoculating to 1000mL of secondary fermentation shake flask for culturing at the inoculation amount of 5% to 0.8 + -0.2 at the shaking table temperature of 35 deg.C and rotation speed of 180r/min to OD 600 When the inoculation amount is 1.2 +/-0.2, inoculating the mixture into a 30L fermentation tank for fermentation culture at the inoculation amount of 5 percent.
The step S4 specifically includes: the ventilation of the whole fermentation process is controlled at 0.5vvm, the stirring speed of the fermentation tank is controlled at 200r/min, the dissolved oxygen content of the fermentation liquor is controlled at 10%, and the pH value is 7.0 +/-L0.4 (pH of fermentation broth controlled with 20% sulfuric acid and 30% ammonia water); the fermentation temperature in the early fermentation period (0 h-20 h) is 35 ℃, the fermentation temperature in the middle fermentation period (21 h-48 h) is 30 ℃, and the fermentation temperature in the later fermentation period (49 h-72 h) is 28 ℃; when cultured to OD 600 10, inoculating isopropyl thiogalactoside (IPTG) with the final concentration of 0.1mmol/L into a fermentation medium, and inoculating an extracting agent accounting for 10% of the volume of the fermentation liquid after 2 hours, wherein the extracting agent is n-decane; feeding is started when the sugar concentration (BX) is reduced to (4 +/-1)% in the fermentation process, and the sugar concentration (BX) in the fermentation liquor is controlled to be 4.0 +/-0.2.
The molasses in the molasses stock solution is the combination of cane molasses and beet molasses, and the weight ratio of the cane molasses to the beet molasses is 2.5: 6.
The host cell is escherichia coli, the recombinant host cell is escherichia coli B21, the constructed recombinant host cell escherichia coli B21 can express 9 enzymes including acetoacetyl CoA thiolase (AtoB), HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthase and farnesene synthase; the recombinant host cell Escherichia coli B21 comprises three plasmids of pKK223-3, PMCSG9 and pMal-c4X, wherein the pKK223-3 comprises three genes of atoB, thmg1 and mvd1, the PMCSG9 comprises four genes of erg13, erg12, erg8 and ispA, and the pMal-c4X comprises two genes of idi and afs; the plasmid pKK223-3 adopts a tac promoter, the plasmid PMCSG9 adopts a T7 promoter, and the plasmid pMal-c4X adopts a tac promoter.
In the processes of secondary fermentation shake flask culture and 30L fermentation tank fermentation culture, the culture medium mainly comprises the following components: the molasses after impurity removal and purification accounts for 5% of the weight of the culture medium, the yeast powder 2%, the peptone 2%, the ammonium citrate 1%, the magnesium sulfate 0.5%, the manganese sulfate 0.08% and the zinc sulfate 0.1%.
The materials supplemented in the material supplementing process comprise 55% of molasses, 40% of ammonium sulfate and 5% of peptone after impurity removal and purification, and the materials are sterilized under high pressure at the temperature of 110-120 ℃ for 25-40min for later use.
Example 3 production method of beta-farnesene
A production method of beta-farnesene comprises the following steps: s1 molasses impurity removal and purification, S2 beta-farnesene recombinant host cell construction, S3 inoculation of recombinant host cells to a culture medium containing the molasses impurity removal and purification for heterologous induction expression synthesis of beta-farnesene, and S4 improvement of the yield of the beta-farnesene through fermentation control.
The step S1 specifically includes:
(1) weighing: weighing molasses stock solution, adding 1.5-2.0 times of water, and diluting molasses to 16-17 Baume degree to obtain diluted sugar solution;
(2) acidifying: pouring the diluted sugar solution into a fermentation tank, adding concentrated sulfuric acid and concentrated phosphoric acid to adjust the acidity, wherein the pH value is 4.0-4.5, and acidifying, wherein after acidification, impurities such as sandy soil, floating slag and the like are less than or equal to 1%, the concentration is more than or equal to 80 degrees BX, the total sugar content is more than or equal to 52%, and the acidity is less than 9%;
(3) ventilating: introducing steam, boiling acidified dilute sugar solution, heating to promote colloidal protein solidification, centrifuging for precipitation, heating to boil, keeping temperature at 90 deg.C, introducing air into tank for 1 hr to remove harmful gas NO 2 、SO 2 And the like, as well as volatile acids and other volatile substances;
(4) adding alkali for neutralization: filling the ventilated dilute sugar solution into a temporary storage barrel, adding lime milk to adjust the pH value to 5.0-5.5, wherein the lime milk is prepared by adding water into calcium oxide and is prepared in situ;
(5) and (3) centrifugal clarification: centrifuging by a high-speed centrifuge to remove impurities in the diluted sugar solution neutralized by adding alkali, and taking clear liquid;
(6) heating and sterilizing: pouring the clear liquid into a clean fermentation tank, introducing steam, heating to 90 ℃, and preserving heat for 0.5 h;
(7) and (4) pressure maintaining storage: sealing the fermentation tank after sterilization, ventilating and maintaining the pressure at 1.0Mpa, and temporarily storing at normal temperature for later use.
The step S2 specifically includes:
(1) finding 9 enzymes of acetoacetyl CoA thiolase (AtoB), HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthetase and farnesene synthase in MVA pathway and corresponding cDNA sequences by NCBI database BLAST;
(2) constructing multiple recombinant plasmids compatible in the same host cell, namely: designing a primer according to a cDNA sequence of a known enzyme to synthesize a corresponding gene sequence, cloning and synthesizing the corresponding gene sequence by using the designed and synthesized cDNA sequence as a template through a PCR (polymerase chain reaction) technology, connecting the cloned and synthesized gene sequence to a corresponding plasmid by using a DNA (deoxyribonucleic acid) recombination technology to form a multiple recombinant plasmid, and co-expressing the multiple recombinant plasmid in a host cell through promoter transcription and host cell translation to synthesize beta-farnesene related protein;
(3) transforming and introducing the multiple recombinant plasmids into host cells to obtain the recombinant host cells for synthesizing the beta-farnesene, namely: introducing the connected recombinant plasmid into an escherichia coli host cell, culturing and amplifying the recombinant host cell, extracting the plasmid, performing sequencing verification to verify that the target gene is connected to the corresponding plasmid, and culturing and preserving the verified strain.
The step S3 specifically includes: activating the recombinant host cells, inoculating the cells into 250mL triangular flask containing LB medium (tryptone 10g, yeast extract 5g, NaCl10g, water to volume 1000 mL) for culturing at 38 ℃ and 220r/min until OD is reached 600 Inoculating to 1000mL of secondary fermentation shake flask for culturing at 0.8 + -0.2 with an inoculum size of 20%, and culturing at a shaker temperature of 38 deg.C and a rotation speed of 250r/min to OD 600 When the inoculation amount is 1.2 +/-0.2 percent, inoculating the mixture into a 30L fermentation tank for fermentation culture by using the inoculation amount of 8 percent.
The step S4 specifically includes: the ventilation rate of the whole fermentation process is controlled at 1.2vvm, the stirring speed of the fermentation tank is controlled at 400r/min, the dissolved oxygen content of the fermentation liquor is controlled at 50%, and the pH value is 7.0 +/-0.4 (the pH value of the fermentation liquor is controlled by 20% sulfuric acid and 30% ammonia water); the fermentation temperature in the early fermentation period (0 h-20 h) is 37 ℃, the fermentation temperature in the middle fermentation period (21 h-48 h) is 35 ℃, and the fermentation temperature in the later fermentation period (49 h-72 h) is 30 ℃; when cultured to OD 600 20, inoculating isopropyl thiogalactoside (IPTG) with the final concentration of 0.3mmol/L into a fermentation medium, and inoculating an extractant accounting for 20 percent of the volume of the fermentation liquid after 3 hours, wherein the extractant is n-dodecane(ii) a Feeding is started when the sugar concentration (BX) is reduced to (4 +/-1)% in the fermentation process, and the sugar concentration (BX) in the fermentation liquor is controlled to be 4.0 +/-0.2.
The molasses in the molasses stock solution is the combination of cane molasses and beet molasses, and the weight ratio of the cane molasses to the beet molasses is 2.5: 6.
The host cell is escherichia coli, the recombinant host cell is escherichia coli B21, the constructed recombinant host cell escherichia coli B21 can express 9 enzymes including acetoacetyl CoA thiolase (AtoB), HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthase and farnesene synthase; the recombinant host cell Escherichia coli B21 comprises three plasmids of pKK223-3, PMCSG9 and pMal-c4X, wherein the pKK223-3 comprises three genes of atoB, thmg1 and mvd1, the PMCSG9 comprises four genes of erg13, erg12, erg8 and ispA, and the pMal-c4X comprises two genes of idi and afs; the plasmid pKK223-3 adopts a tac promoter, the plasmid PMCSSG 9 adopts a T7 promoter, and the plasmid pMal-c4X adopts a tac promoter.
In the processes of secondary fermentation shake flask culture and 30L fermentation tank fermentation culture, the culture medium mainly comprises the following components: the molasses after impurity removal and purification accounts for 12% of the proportion of the culture medium, the yeast powder 8%, the peptone 8.5%, the ammonium citrate 4%, the magnesium sulfate 1.5%, the manganese sulfate 0.4% and the zinc sulfate 0.4% of the proportion of the culture medium.
The materials supplemented in the material supplementing process comprise 60% of molasses, 30% of ammonium sulfate and 10% of peptone after impurity removal and purification, and the materials are sterilized under high pressure at the temperature of 110-.
Example 4 method for detecting beta-farnesene in fermentation broth by gas chromatography
A method for detecting beta-farnesene in fermentation liquor by gas chromatography comprises the following steps:
h1, pretreatment of fermentation liquor: centrifuging above 10mL fermentation liquid at 10000r/min for 5min, layering an oil phase and a water phase, absorbing an upper oil phase, and filtering with a 0.44um organic filter membrane to obtain a filtrate;
h2, preparing a beta-farnesene series standard solution by using an Agilent 7890A gas chromatograph, a 19091JHP-5 chromatographic column (the column length is 30m, the inner diameter is 0.32mm, and the film thickness is 250um), preparing the beta-farnesene standard solution with the concentration of 5g/L, 10g/L, 15g/L and 20g/L respectively, carrying out sample injection detection respectively, drawing a linear regression equation, and calculating the R value to ensure that the R value is more than 99%, wherein the beta-farnesene detection conditions are as follows: temperature of the column box: the initial temperature is 70 ℃, the temperature is kept for 2min, the temperature is increased to 250 ℃ at the speed of 10 ℃/min, the temperature is kept for 1min, the temperature of a sample inlet is 250 ℃, the sample injection amount is 1uL, the product injection split ratio is 1:50, the temperature of a detector (FID) is 260 ℃, nitrogen is used as a carrier gas, the inlet pressure is 15psi, and the mode is a constant flow mode.
The method for detecting beta-farnesene in the fermentation liquor by using the seed gas chromatography in the embodiment is adopted to detect the beta-farnesene produced in the embodiments 1-3, the embodiments 1-3 are fermented for 72 hours, and the concentrations of the beta-farnesene in the fermentation liquor are detected to sequentially reach 20.5g/L, 20.3g/L and 20.2 g/L.
The above embodiments are only specific examples of the present invention, and the protection scope of the present invention includes but is not limited to the product forms and styles of the above embodiments, and any suitable changes or modifications made by those skilled in the art according to the claims of the present invention shall fall within the protection scope of the present invention.
Sequence listing
<110> Shandong Hongda Biotech Co., Ltd
<120> production method of beta-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 (5)
1. A method for producing beta-farnesene is characterized by comprising the following steps: the method comprises the following steps: s1 molasses impurity removal and purification, S2 beta-farnesene recombinant host cell construction, S3 inoculating the recombinant host cell to a culture medium containing the molasses impurity removal and purification for heterologous induction expression synthesis of beta-farnesene, and S4 improvement of the yield of the beta-farnesene through fermentation control;
the step S1 specifically includes:
(1) weighing: weighing molasses stock solution, adding 1.5-2.0 times of water, and diluting molasses to 16-17 Baume degree to obtain diluted sugar solution;
(2) acidifying: pouring the diluted sugar solution into a fermentation tank, adding concentrated sulfuric acid and concentrated phosphoric acid to adjust the acidity, and acidifying at a pH of 4.0-4.5;
(3) ventilating: introducing steam, boiling acidified dilute sugar solution, heating to promote colloidal protein solidification, centrifuging for precipitation, heating to boil, keeping temperature at 90 deg.C, introducing air into tank for 1 hr to remove harmful gas NO 2 、SO 2 And a volatile substance;
(4) adding alkali for neutralization: filling the ventilated dilute sugar solution into a temporary storage barrel, and adding lime milk to adjust the pH value to 5.0-5.5;
(5) and (3) centrifugal clarification: centrifuging by a high-speed centrifuge to remove impurities in the diluted sugar solution neutralized by adding alkali, and taking clear liquid;
(6) heating and sterilizing: pouring the clear liquid into a clean fermentation tank, introducing steam, heating to 90 ℃, and preserving heat for 0.5 h;
(7) and (4) pressure maintaining storage: sealing the fermentation tank after sterilization, ventilating and maintaining the pressure at 1.0Mpa, and temporarily storing at normal temperature for later use;
the step S2 specifically includes: (1) obtaining related genes for synthesizing beta-farnesene by NCBI database query and genetic engineering technical means, wherein the related genes comprise acetoacetyl CoA thiolase gene atoB, HMG-CoA synthase gene erg13, HMG-CoA reductase gene thmg1, mevalonate kinase gene erg12, phosphomevalonate kinase gene erg8, mevalonate pyrophosphate decarboxylase gene mvd1, isopentenyl pyrophosphate isomerase gene idi, farnesyl pyrophosphate synthase gene ispA and farnesene synthase gene afs in a mevalonate pathway;
(2) constructing multiple recombinant plasmids compatible in the same host cell, wherein the multiple recombinant plasmids co-express and synthesize beta-farnesene related protein in the host cell through promoter transcription and host cell translation;
(3) transforming and introducing the multiple recombinant plasmids into host cells to obtain recombinant host cells for synthesizing beta-farnesene;
the step S3 specifically includes: activating the recombinant host cells and inoculating the cells into 250mL of triangular flask filled with LB culture medium for cultureCulturing at 30-38 deg.C and 180-220r/min until OD 600 Inoculating to 1000mL of secondary fermentation shake flask for culturing at the inoculation amount of 3-20% to 0.8 +/-0.2 at the shaking table temperature of 35-38 ℃ and the rotation speed of 180- 600 Inoculating the mixture into a 30L fermentation tank for fermentation culture in an inoculation amount of 5-8% to 1.2 +/-0.2;
the step S4 specifically includes: the ventilation rate of the whole fermentation process is controlled to be 0.5vvm-1.5vvm, the stirring rotating speed of a fermentation tank is controlled to be 200r/min-400r/min, the dissolved oxygen of the fermentation liquor is controlled to be 2-90%, and the pH value is 7.0 +/-0.4; the fermentation temperature in the early fermentation stage is 35-37 ℃, the fermentation temperature in the middle fermentation stage is 30-35 ℃, and the fermentation temperature in the later fermentation stage is 28-35 ℃; when cultured to OD 600 8-20, inoculating isopropyl thiogalactoside with the final concentration of 0.1-0.5mmol/L into a fermentation medium, and inoculating an extracting agent accounting for 10-20% of the volume of the fermentation liquid after 2-10 h, wherein the extracting agent is one or more of n-decane, n-dodecane, petroleum ether and n-octane; feeding when the sugar concentration is reduced to (4 +/-1)% in the fermentation process, and controlling the sugar concentration in the fermentation liquor to be 4.0 +/-0.2;
the host cell is escherichia coli, the recombinant host cell is escherichia coli B21, and the constructed recombinant host cell escherichia coli B21 can express 9 enzymes of acetoacetyl CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthase and farnesene synthase; escherichia coli B21, which comprises three plasmids of pKK223-3, PMCSG9 and pMal-c4X, wherein the pKK223-3 comprises three genes of atoB, thmg1 and mvd1, the PMCSG9 comprises four genes of erg13, erg12, erg8 and ispA, and the pMal-c4X comprises two genes of idi and afs; the plasmid pKK223-3 adopts a tac promoter, the plasmid PMCSG9 adopts a T7 promoter, and the plasmid pMal-c4X adopts a tac promoter;
the LB culture medium is 10g of tryptone, 5g of yeast extract and 10g of NaCl, and the volume is fixed to 1000mL by water;
the pH was controlled with 20% sulfuric acid and 30% ammonia;
the early stage of fermentation is 0-20 h; the middle fermentation period is 21-48 h; the late stage of fermentation is 49 h-end.
2. The method for producing beta-farnesene according to claim 1, wherein the method comprises the following steps: the molasses in the molasses stock solution is cane molasses or beet molasses or the combination of the cane molasses and the beet molasses, and the weight ratio of the cane molasses to the beet molasses is 100:25-1000 under the combined condition.
3. The method for producing beta-farnesene according to claim 1, wherein: in the processes of secondary fermentation shake flask culture and 30L fermentation tank fermentation culture, the culture medium mainly comprises the following components: the molasses after impurity removal and purification accounts for 2-20% of the weight of the culture medium, the yeast powder or yeast extract or the combination of the two accounts for 1-10% of the weight of the culture medium, the peptone accounts for 1.5-8.5% of the weight of the culture medium, the ammonium citrate accounts for 0.1-5% of the weight of the culture medium, the magnesium sulfate or magnesium chloride accounts for 0.1-2% of the weight of the culture medium, the manganese sulfate accounts for 0.05-0.9% of the weight of the culture medium, and the zinc sulfate accounts for 0.02-0.8% of the weight of the culture medium.
4. The method for producing beta-farnesene according to claim 1, wherein the method comprises the following steps: the supplementary materials in the material supplementing process comprise 40-60 wt% of molasses subjected to impurity removal and purification, 20-50 wt% of ammonium sulfate and 5-10 wt% of peptone, and the materials are sterilized under high pressure at the temperature of 110-120 ℃ for 25-40min for later use.
5. A method for detecting beta-farnesene in the fermentation broth according to any one of claims 1 to 4 by gas chromatography, which is characterized in that: the method comprises the following steps:
h1, pretreatment of fermentation liquor: taking more than 10mL of fermentation liquor, centrifuging for 3-8min at 9000-;
h2, using Agilent 7890A gas chromatograph, 19091JHP-5 chromatographic column, wherein the 19091JHP-5 chromatographic column has a column length of 30m, an inner diameter of 0.32mm and a film thickness of 250 um; preparing beta-farnesene series standard solutions, preparing the beta-farnesene standard solutions with the concentrations of 5g/L, 10g/L, 15g/L and 20g/L respectively, carrying out sample injection detection respectively, drawing a linear regression equation, and calculating an R value to ensure that the R value is more than 99 percent, wherein the beta-farnesene detection conditions are as follows: temperature of the column box: the initial temperature is 65-75 ℃, the temperature is kept for 2min, the temperature is increased to 280 ℃ plus 220 ℃ at the speed of 10 ℃/min, the temperature is kept for 1min, the temperature of a sample inlet is 280 ℃ plus 220 ℃, the sample injection amount is 1uL, the product injection split ratio is 1:50, the temperature of a detector is 280 ℃ plus 260 ℃, nitrogen is used as carrier gas, the inlet pressure is 12-18psi, and the mode is a constant flow mode.
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Denomination of invention: one kind b- Production method of farnesene Effective date of registration: 20230821 Granted publication date: 20220920 Pledgee: Shandong Yishui Rural Commercial Bank Co.,Ltd. Pledgor: SHANDONG KUNDA BIOTECHNOLOGY CO.,LTD.|SHANDONG HONGDA BIOTECHNOLOGY Co.,Ltd. Registration number: Y2023980052981 |