CN111378587A - Gene engineering bacterium for synthesizing β -farnesene and application thereof - Google Patents

Abstract

The invention discloses a gene engineering bacterium for synthesizing β -farnesene and application thereof, belonging to the technical field of gene engineering, the gene engineering bacterium of the invention knocks down GAL80, ALD6, ALD4, ADH5 and RHR2 genes in saccharomyces cerevisiae, integrates AaFS, LmPK, CkPTA, Dzeute, SpHMGR, GAL4 and HMG1 genes, wherein the promoter and terminator utilized are P respectively_GAL1，P_GAL10，P_GAL4And T_CYC1，T_ADH1The gene engineering bacteria is utilized to carry out fermentation, the yield of β -farnesene can reach 56mg/L, and the gene engineering bacteria is utilized to carry out fermentation, so that β -farnesene can be produced in a green and environment-friendly manner at low cost by combining the biotransformation capability of the gene engineering bacteria and taking corn hydrolysate as a culture mediumThe bacterium and the method for synthesizing β -farnesene are suitable for actual industrial production.

Description

Gene engineering bacterium for synthesizing β -farnesene and application thereof

Technical Field

The invention relates to a gene engineering bacterium for synthesizing β -farnesene and application thereof, belonging to the technical field of gene engineering.

Background

Farnesene (C15H24) with a density of 0.813g/cm³The sesquiterpene is an acyclic sesquiterpene which is produced in plants such as apples and sweet wormwood to resist the invasion of external insects or animals (Nieuwenhuizen et al, 2014; Yang et al, 2011), and the characteristics of low hygroscopicity and high energy density make the sesquiterpene become a research object of high-density fuel.

However, currently, the production of farnesene in microbial cells is not ideal, and the production of farnesene by using microorganisms is always slow and cannot be industrialized, in the existing report, the yield of α -farnesene produced by fermentation of farnesene by using escherichia coli as a bottom plate cell can reach 380mg/L (Zhu et al, 2014), and β -farnesene8.74g/L (inactivation of biodiesei by-product as substrate for high-production of β -farnesene virtual balanced plasmid contained in page).

At present, Chandra et al firstly use Saccharomyces cerevisiae as a production cell to heterologously express β -farnesene synthetase in Artemisia annua to achieve the research of converting 100g of glucose into 23.8g of β -farnesene (Chandra et al, 2011), and later, Tippmann et al also use Saccharomyces cerevisiae and three heterologue terpene synthetases to express α -farnesene, the yield of fed-batch culture reaches 170mg/L (Tippmann et al, 2015), Adam et al finally improve β -farnesene yield to 110g/L by improving the metabolic balance of Saccharomyces cerevisiae, however, the sucrose price is high, and the production cost is limited by fermentation in large scale.

Disclosure of Invention

In order to solve the problem of low-cost production of β -farnesene, the invention provides a genetically engineered bacterium which takes corn hydrolysate as a culture medium and has β -farnesene synthesis capacity and application thereof, and the adopted technical scheme is as follows:

the invention aims to provide a genetically engineered bacterium capable of synthesizing β -farnesene, the starting strain of the genetically engineered bacterium is a yeast, the yeast is knocked out endogenous genes GAL80, ALD6, ALD4, ADH5 and RHR2 of the yeast, β -farnesene synthetase genes AaFS, LmpK, CkPTA, Dzeute, SpHMGR, GAL4 and tHMG1 are integrated, and the required promoter and terminator are respectively P_GAL1，P_GAL10，P_GAL4And T_CYC1，T_ADH1。

The yeast is Saccharomyces cerevisiae (Saccharomyces cerevisiae), and the genotype is as follows: CEN. PK2-1C (MATa; ura 3-52; trp 1-289; leu2-3_ 112; his 3. DELTA.1; MAL 2-8C; SUC 2).

The farnesene synthetase gene AaFS is optimized by codons, the original farnesene synthetase gene AaFS is from Artemisia annua, the GenBank accession number is AAX39387.1, the sequence of the optimized farnesene synthetase gene AaFS is shown in SEQ ID NO.1, the β -farnesene synthetase gene AaFS is integrated at the GAL80 locus of the starting strain yeast, and the promoter and the terminator are P respectively_GAL10And T_CYC1. The LmPK gene is from Leuconostocemesteerietides and NCBI access number YP-819405.1, the LmPK gene is integrated at the ALD6 site of the yeast chromosome of the starting strain, and a promoter and a terminator are P respectively_GAL10And T_CYC1. The CkPTA gene is from Clostridium kluyveri, NCBI access number YP _001394780.1, the CkPTA gene is integrated at the ALD6 site of the yeast chromosome of the starting strain, and a promoter and a terminator are P respectively_GAL1And T_ADH1. The Dzeute gene is from Dickeya zeae, NCBI access number WP-012768716.1, and is integrated in the yeast of the original strainChromosome ALD4 site, promoter and terminator P_GAL1And T_ADH1. The SpHMGR gene is from Silicabacterpomeroyi and NCBI access number YP-164994, the SpHMGR gene is integrated at the ALD4 site of the yeast chromosome of the starting strain, and a promoter and a terminator are P respectively_GAL1And T_ADH1. The GAL4 gene is endogenous gene of the starting strain yeast, GenBank accession number is AQN77051.1, the GAL4 gene is integrated at the chromosome RHR2 site of the starting strain yeast, and the promoter is P (OC)_GAL4The terminator is its own terminator. The tHMG1 gene is endogenous gene of the starting strain yeast, GenBank accession number is CAA86503.1, the tHMG1 gene is integrated at the chromosome RHR2 site of the starting strain yeast, and the promoter is P_GAL1The terminator is its own terminator.

Secondly, the invention also provides application of the gene engineering bacteria for synthesizing β -farnesene in synthesis of β -farnesene, wherein the gene engineering bacteria can produce farnesene by fermentation of corn hydrolysate, and the method comprises the steps of inoculating the gene engineering bacteria to a primary seed culture medium, culturing to obtain a secondary seed, and inoculating the obtained secondary seed to a secondary seed culture medium for fermentation to obtain β -farnesene.

The primary seed culture medium is an YPD culture medium and contains 20g/L of glucose, 10g/L of yeast powder and 20g/L of peptone; the primary seed culture solution, OD₆₀₀Is 0.5.

The preparation method of the secondary seed culture medium comprises the steps of weighing 1500g of corn mash, adding 4500mL of water with the temperature of 60-70 ℃, uniformly stirring, standing for gelatinization for 20-120min, adding α -amylase with the high temperature resistance of 0.9mL and 3 ten thousand U/mL, uniformly stirring, liquefying at the temperature of 85-90 ℃ for 1.5h, rapidly cooling to 60 ℃ after liquefying is finished, adding 3mL and 10 ten thousand U/mL of saccharifying enzyme, uniformly stirring, saccharifying at the temperature of 55-60 ℃ for 20h, filtering with a filter cloth after saccharifying is finished, obtaining a clear solution, enabling the pH to be natural, and sterilizing at the temperature of 105 ℃ for 10min to obtain the corn hydrolysate.

The primary seed culture solution is inoculated into a secondary seed culture medium, the volume percentage of the inoculated primary seed culture solution accounts for 10% of the secondary seed culture medium, and the shaking fermentation culture is carried out for 5-6 d.

The culture temperature of the genetic engineering bacteria inoculated into the first-level seed culture medium is 30 ℃, the culture mode is shake flask culture, and the rotation speed is 150-250 rpm; the fermentation temperature is 30 ℃, and the oscillation speed is 150-250 rpm.

Wherein the fermentation container is a triangular flask with a baffle plate. When the thallus is from the initial state to the middle stage of the logarithmic growth phase, 10% dodecane is added as an extracting agent. And (3) absorbing an upper dodecane organic phase after fermenting for 5-6 days, filtering by using a filter membrane, taking 1 mu L of dodecane organic phase for gas phase detection, and obtaining the yield of the farnesene produced by the corn hydrolysate according to a farnesene standard curve.

The invention provides a corn hydrolysate culture medium for fermentation production of β -farnesene, which is prepared by weighing 1500g of corn mash, adding 4500mL of water at 60-70 ℃, uniformly stirring, standing for gelatinization for 20-120min, adding α -amylase with high temperature resistance of 0.9mL and 3 ten thousand U/mL, uniformly stirring, liquefying at 85-90 ℃ for 1.5h, rapidly cooling to 60 ℃ after liquefying, adding 3mL and 10 ten thousand U/mL of saccharifying enzyme, uniformly stirring, saccharifying at 55-60 ℃ for 20h, filtering with filter cloth after saccharifying, obtaining a clear solution, sterilizing at 105 ℃ for 10min, adding a sterilized buffer solution before fermentation to adjust the pH value, adding a sterilized trace element mother solution, uniformly mixing, obtaining the corn hydrolysate culture medium for fermentation production of β -farnesene, wherein the buffer solution is 0.5M succinic acid solution, the pH value is adjusted to adjust the pH value of the system to 6.0, the trace element mother solution contains 5.75g of calcium sulfate, the medium is prepared from a water containing 2.32 g of calcium sulfate, and the volume of calcium chloride, and the water containing no copper sulfate, and the volume of the raw water.

The third aspect of the invention provides an application of the corn hydrolysate culture medium for producing β -farnesene through fermentation in the production of β -farnesene through fermentation, specifically, a strain for producing β -farnesene is inoculated to a primary seed culture medium, secondary seeds are obtained after the culture, the obtained secondary seeds are inoculated to the corn hydrolysate culture medium for producing β -farnesene through fermentation to obtain β -farnesene, wherein the primary seed culture medium is a YPD culture medium and contains 20g/L of glucose, 10g/L of yeast powder and 20g/L of peptone, and the OD of the primary seed culture medium is OD₆₀₀0.5, inoculating the primary seed culture solution into a corn hydrolysate culture medium for fermenting and producing β -farnesene, wherein the inoculated volume percentage of the primary seed culture solution accounts for 10 percent of the corn hydrolysate culture medium for fermenting and producing β -farnesene, performing shake fermentation culture 5-6d, inoculating a strain for producing β -farnesene into the primary seed culture medium, wherein the culture temperature is 30 ℃, the culture mode is shake flask culture, the rotation speed is 150-250rpm, the fermentation temperature is 30 ℃, the oscillation speed is 150-250rpm, wherein a fermentation container is a triangular flask with a baffle plate, when the bacteria are from an initial state to a logarithmic growth phase, 10% dodecane is added as an extracting agent, absorbing an upper dodecane organic phase after fermenting 5-8656 d, performing gas phase detection by taking 1 mu L after filtering through a filter membrane, obtaining the yield of the farnesene produced by the corn according to a farnesene standard curve, the strain for producing β -farnesene is a genetically engineered enzyme gene for synthesizing β -farnesene, the starting strain of the yeast is a yeast strain, the yeast strain for knocking off genes of yeast, the genes of synthesizing the yeast, the yeast strain, the genes of the yeast, the yeast strain for terminating RHUTYP 5-35, the genes of the yeast strain, the genes of synthesizing the yeast strain, the genes of the yeast strain, the genes of_GAL1， P_GAL10，P_GAL4And T_CYC1，T_ADH1. The yeast is Saccharomyces cerevisiae (Saccharomyces cerevisiae), and the genotype is as follows: CEN. PK2-1C (MATa; ura 3-52; trp 1-289; leu2-3_ 112; his 3. DELTA.1; MAL 2-8C; SUC 2). The farnesene synthetase gene AaFS is optimized by codon, the original farnesene synthetase gene AaFS is from Artemisiaannua, the GenBank accession number is AAX39387.1, and the optimized farnesene synthetase gene AaFSThe sequence of the alkene synthetase gene AaFS is shown in SEQ ID NO.1, the β -farnesene synthetase gene AaFS is integrated at GAL80 locus of the starting strain yeast chromosome, and the promoter and the terminator are P respectively_GAL10And T_CYC1. The LmpK gene is from Leuconostoc mesenteroides, NCBIaccesion number YP-819405.1, the LmpK gene is integrated at the ALD6 site of the yeast chromosome of the starting strain, and the promoter and the terminator are P respectively_GAL10And T_CYC1. The CkPTA gene is from Clostridium kluyveri, NCBIaccesssion number YP _001394780.1, the CkPTA gene is integrated at the ALD6 site of the yeast chromosome of the starting strain, and a promoter and a terminator are P respectively_GAL1And T_ADH1. The Dzeute gene is from Dickeya zeae, NCBIaccesion number WP-012768716.1, the Dzeute gene is integrated at the ALD4 site of the yeast chromosome of the starting strain, and a promoter and a terminator are P respectively_GAL1And T_ADH1. The SpHMGR gene is from Silicabacterpomeroyi, NCBI access number YP-164994, the SpHMGR gene is integrated at the ALD4 site of the yeast chromosome of the starting strain, and a promoter and a terminator are P respectively_GAL1And T_ADH1. The GAL4 gene is endogenous gene of the starting strain yeast, GenBank accession number is AQN77051.1, the GAL4 gene is integrated at the chromosome RHR2 site of the starting strain yeast, and the promoter is P (OC)_GAL4The terminator is its own terminator. The tHMG1 gene is endogenous gene of the starting strain yeast, GenBank accession number is CAA86503.1, the tHMG1 gene is integrated at the chromosome RHR2 site of the starting strain yeast, and the promoter is P_GAL1The terminator is its own terminator.

The invention has the beneficial effects that:

1. the genetically engineered bacteria can be used as a culture medium in corn hydrolysate, a carbon source, a nitrogen source and an inducer do not need to be additionally added, the fermentation cost is saved, and high-density fuel β -farnesene can be produced specifically and efficiently.

2. The gene engineering bacteria constructed according to the invention can obtain the product β -farnesene with high purity under proper conditions and no other impurities.

3. The corn hydrolysate culture medium for producing β -farnesene by fermentation does not need to add a carbon source, a nitrogen source and an inducer additionally, saves the fermentation cost, can produce β -farnesene which is a high-density fuel in a green and environment-friendly manner, and utilizes the same bacterial strain to produce β -farnesene by adding trace elements with specific types and concentrations and adjusting a specific pH value, so that the yield reaches 89.1mg/L and is increased by about 35%.

4. The β -farnesene production process does not relate to high-temperature and high-pressure operation, is economic and green, fundamentally solves the defect that the β -farnesene can only be separated and extracted from plants or essential oil at present, and provides a method for synthesizing β -farnesene by utilizing a sustainable method for relieving the problem of insufficient raw materials in the high-density fuel industry.

Drawings

FIG. 1 is a β -farnesene biosynthetic metabolic pathway;

FIG. 2 is a gas phase detection diagram of β -farnesene synthesized by engineering bacteria;

FIG. 3 is a gas phase-mass spectrometry detection chart of cis- β -farnesene synthesized by engineering bacteria;

FIG. 4 is a gas phase-mass spectrometric detection diagram of the engineering bacteria for synthesizing (E) - β -farnesene;

FIG. 5 is a graph of the effect of corn hydrolysate at various pH values on β -farnesene production;

FIG. 6 is a graph showing the effect of addition of a microelement mother liquor and nutrient salts on the yield of β -farnesene.

Detailed Description

The present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.

Example 1

The genetically engineered bacterium capable of synthesizing β -farnesene has starting strain of saccharomycete, which has endogenous genes GAL80, ALD6, ALD4, ADH5 and RHR2 knocked out and β -farnesene synthetase genes AaFS, LmPK, CkPTA and Dzeut integratedE. SpHMGR, GAL4 and tHMG1 genes, the required promoter and terminator are P respectively_GAL1，P_GAL10，P_GAL4And T_CYC1，T_ADH1。

The yeast is Saccharomyces cerevisiae (Saccharomyces cerevisiae), and the genotype is as follows: CEN. PK2-1C (MATa; ura 3-52; trp 1-289; leu2-3_ 112; his 3. DELTA.1; MAL 2-8C; SUC 2). The detailed construction process is as follows:

constructing a yeast engineering strain containing a farnesene synthetase gene AaFS.

(1) The farnesene synthetase gene AaFS is inserted into GAL80 site of yeast genome. The farnesene synthetase gene is derived from Artemisia annua L (Artemisia annua L.) with the sequence registration number of GenBank: AY835398.1, and the gene AaFS is subjected to codon optimization, and the sequence is shown as SEQ 1. AaFS is cleaved from the synthesized plasmid by BamHI and Sal I, and pESC-HIS plasmid is ligated to obtain recombinant plasmid pESC-HIS-AaFS. Obtaining P by PCR_GAL1-AaFS-T_CYC1Fragment, P_GAL1Is GAL1 promoter, T_CYC1Is CYC1 terminator. These were ligated into the pUC19 plasmid using the restriction enzymes Nde I and Pst I. Then sequentially connecting the upstream (GAL80US)511bp (the sequence is shown as SEQ 2) of GAL80 and the downstream (GAL80DS)501bp (the sequence is shown as SEQ 3) of GAL80 into P by Nde I and Pst I_GAL1-AaFS- T_CYC1To obtain a recombinant plasmid pUC19-GAL80US-P_GAL1-AaFS-T_CYC1-GAL80 DS. The donor fragment was obtained by Nde I and Pst I cleavage: GAL80US-P_GAL1-AaFS-T_CYC1GAL80DS, the primers used are shown in Table 1.

(2) An expression fragment of gRNA was constructed using sequence TAAGGCTGCTGCTGAACGT as a target sequence. A19 bp target sequence is introduced when a primer is designed, a linearized pML104 is used as a template, each 500bp DNA sequence at two ends of the primer is amplified by PCR, two upstream and downstream fragments both contain 19bp GAL80 target sequences, the two fragments are fused together by overlapping PCR to obtain a complete gRNA expression fragment, and the 19bp target sequence is positioned in the center of the gRNA expression fragment. The primers used are shown in Table 1. Carrying out double enzyme digestion on a plasmid pML104 by using restriction enzymes Swa I and Bcl I to obtain a linearized plasmid; and (3) simultaneously transferring the donor DNA fragment, the linearized pML104 plasmid and the gRNA expression fragment into a yeast competent cell CEN. PK2-1C by a chemical conversion method to obtain the yeast genetic engineering bacteria FS 1.

Constructing a yeast engineering strain containing a phosphoketolase gene LmpK and a phosphotransacetylase gene CkPTA.

(1) On the basis of the FS1 strain, gene integration was carried out by means of homologous recombination. Phosphotransacetylase gene CkPTA and phosphoketolase gene LmPK are inserted into ALD6 site of yeast genome. The phosphotransacetylase gene CkPTA is derived from Clostridium kluyveri (Clostridium kluyveri), and the sequence registration number is GenBank: CP 018335.1; the phosphoketolase gene LMPK is derived from Leuconostoc mesenteroides (Leuconostoc mesenteroides) and has the sequence registration number of GenBank: AY 804190.1. The CkPTA gene is connected into pESC-URA plasmid through EcoRI and SacI to obtain recombinant plasmid pESC-URA-CkPTA. The LmPK gene is connected into a pESC-URA-CkPTA plasmid through Bam HI and Sal I to obtain a recombinant plasmid pESC-URA-LmPK-CkPTA. Obtaining P by PCR_GAL1-LmPK-T_CYC1_P_GAL10-CkPTA-T_ADH1The fragments, primers used are shown in Table 2.

(2) The upstream and downstream fragments ALD6US (539bp, whose sequence is shown in SEQ 4) and ALD6DS (557bp, whose sequence is shown in SEQ 5) of the ALD6 gene were obtained by PCR, and the primers used were shown in Table 2. HIS expression fragments (the sequence is shown in SEQ 6) are obtained by PCR with pESC-HIS plasmid as a template, and the primers are shown in Table 2. The pUC19 plasmid was linearized by restriction enzymes Sma I and Pst I, followed by a rapid cloning kit by a multi-fragment one-step method (Hieff)

Plus Multi OneStep Cloning Kit, 10912ES10) will P_GAL1-LmPK-T_CYC1_P_GAL10-CkPTA-T_ADH1ALD6US, ALD6DS and HIS were ligated to pUC19 to obtain recombinant plasmid pUC19-ALD6US-P_GAL1-LmPK-T_CYC1_ P_GAL10-CkPTA-T_ADH1HIS-ALD6 DS. The donor fragment was obtained by cleavage of the recombinant plasmid by Sma I and Sph I: ALD6US-P_GAL1-LmPK-T_CYC1_P_GAL10-CkPTA-T_ADH1HIS-ALD6 DS. Will be provided withThe donor fragment was transferred into yeast strain FS1 by chemical transformation to give yeast strain FS 2.

Constructing a yeast engineering strain containing acetaldehyde dehydrogenase gene Dzeute.

(1) On the basis of the FS2 strain, gene integration was carried out by means of homologous recombination. The acetaldehyde dehydrogenase gene Dzeute and the farnesene synthetase gene AaFS are inserted into the ALD4 site of the yeast genome. The acetaldehyde dehydrogenase gene Dzeute is derived from rice basic rot fungus (Dickeya zeae), and has a sequence registration number of GenBank: CP 006929.1.

The Dzeute gene is linked into pESC-HIS-AaFS plasmid through Not I and Pac I to obtain recombinant plasmid pESC-HIS-AaFS-Dzeute. Obtaining P by PCR_GAL1-AaFS-T_CYC1_P_GAL10-DzeutE-T_ADH1Fragments, primers used are shown in Table 3.

(2) The upstream and downstream fragments ALD4US (569bp, whose sequence is shown in SEQ 7) and ALD4DS (865bp, whose sequence is shown in SEQ 8) of the ALD4 gene were obtained by PCR, and the primers used are shown in Table 3. URA expression fragments (the sequence is shown in SEQ 9) are obtained by PCR with pESC-URA plasmid as a template, and the used primers are shown in Table 3. Will P_GAL1-AaFS-T_CYC1_ P_GAL10-DzeutE-T_ADH1The yeast strain FS3 was obtained by transferring ALD4US, ALD4DS, URA4 fragments into yeast strain FS2 by chemical conversion.

Constructing a yeast engineering strain containing hydroxymethyl glutaryl coenzyme A reductase gene RpHGR.

(1) On the basis of the FS3 strain, gene integration was carried out by means of homologous recombination. The hydroxymethyl glutaryl coenzyme A reductase gene RpHGR and the farnesene synthetase gene AaFS are inserted into the ADH5 site of the yeast genome. The hydroxymethyl glutaryl coenzyme A reductase gene RpHGR is derived from Ruegeriapomeroyi and has the sequence registration number of NCBI ReferenceSequence: NC-006569.1. The RpHMGR disclosed by the invention is an optimized sequence, and the sequence of the RpHMGR is shown as SEQ10

The RpHMGR gene is obtained through PCR, and the pESC-HIS-AaFS plasmid is connected through Spe I and Sac I to obtain a recombinant plasmid pESC-HIS-AaFS-RpHMGR. Obtaining P by PCR_GAL1-AaFS-T_CYC1_P_GAL10-RpHMGR-T_ADH1Fragments of, or ofThe primers used are shown in Table 4.

(2) The upstream and downstream fragments ADH5US (493bp, sequence shown in SEQ 11) and ADH5DS (543bp, sequence shown in SEQ 12) of the ADH5 gene were obtained by PCR, and the primers used are shown in Table 4. LEU expression fragments (the sequence is shown in SEQ 13) are obtained by PCR using pESC-LEU plasmid as a template, and the primers are shown in Table 4. Will P_GAL1-AaFS-T_CYC1_ P_GAL10-RpHMGR-T_ADH1The 4 fragments ADH5US, ADH5DS and LEU were transformed into yeast strain FS3 by chemical transformation to obtain yeast strain FS 4.

Construction of engineered yeast strains overexpressing GAL4 gene and tHMG1 gene.

(1) On the basis of the FS4 strain, gene integration was carried out by means of homologous recombination. The GAL4 gene is derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) with the Sequence accession number NCBI Reference Sequence: NC-001148.4. The GAL4 gene contains a downstream sequence of 251bp after the stop codon. The GAL4 gene promoter is an optimized GAL4 promoter, and the sequence of the GAL4 gene promoter is shown in SEQ 14. the tHMG1 gene is derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) with the Sequence accession number NCBI Reference Sequence NC-001145.3. The tHMG1 gene is a truncation body with 529 amino acids of the N end deleted and contains a 225bp sequence behind a stop codon, the sequence is shown as SEQ15, and the promoter is P_GAL1The promoter has the sequence shown in SEQ 16. GAL4 gene and tHMG1 are inserted into the RHR2 site of the yeast genome.

(2) GAL4 expression fragment (including promoter, coding gene and terminator sequence) was divided into A, B, C fragments, which were amplified by PCR, and the primers used were shown in Table 5. the tHMG1 expression fragment (including promoter, coding gene and terminator sequences) was divided into D, E fragments, which were amplified by PCR, and the primers used were shown in Table 5. The sequence of the selection marker gene KanMX with loxP sites is shown in SEQ17, and the selection marker gene KanMX with plasmid pUC6 as a template is obtained by PCR. The fragment RHR2US (580bp, shown in SEQ 18) and RHR2DS (587bp, shown in SEQ 19) are located above and below the RHR2 gene and obtained by PCR, and the primers are shown in Table 5.

(3) The fragments RHR2US and A, C and D, E, KanMX and RHR2-DS are respectively fused into one fragment by means of Overlap extension PCR, the fused RHR2US + A and B are fused into one fragment, and the four fragments are simultaneously transferred into a yeast strain FS4 by means of a chemical conversion method to obtain a yeast strain FS5 which is the genetic engineering bacterium for synthesizing β -farnesene.

TABLE 1 primers

TABLE 2 primers

TABLE 3 primers

TABLE 4 primers

TABLE 5 primers

Example 2 application of genetically engineered bacteria for synthesizing β -farnesene in synthesis of β -farnesene

1. Second-stage seed culture medium

Weighing 1500g of corn mash, adding 4500mL of 65-70 ℃ water, uniformly stirring, standing for gelatinization for 20min, adding 0.9mL of high temperature resistant α -amylase and 3 ten thousand U/mL of high temperature resistant α -amylase, uniformly stirring, liquefying at 85-90 ℃ for 1.5h, rapidly cooling to 60 ℃ after liquefaction, adding 3mL of saccharifying enzyme and 10 ten thousand U/mL of saccharifying enzyme, uniformly stirring, saccharifying at 55-60 ℃ for 20h, filtering with filter cloth after saccharification, obtaining a clear solution, naturally adjusting pH, and sterilizing at 105 ℃ for 10min to obtain the corn hydrolysate.

2. The primary seed culture medium is YPD culture medium containing 20g/L glucose, 10g/L yeast powder and 20g/L peptone;

3. and (3) fermenting thalli:

the gene engineering bacteria of β -farnesene synthesis prepared in example 1 are inoculated into the primary seed culture medium and cultured in a shake flask to OD₆₀₀Obtaining a first-stage seed culture solution for 0.5, adding 50mL of a second-stage seed culture medium into a 250mL baffle triangular flask, inoculating the first-stage seed culture solution into the second-stage seed culture medium in a volume ratio of 10%, performing shaking fermentation culture, and fermenting to OD₆₀₀Adding about 2 of dodecane serving as an extractant, continuously fermenting for 4 days, sampling, measuring the growth condition of thalli, and carrying out gas phase detection on an organic phase. .

The culture temperature of the genetic engineering bacteria inoculated into the first-level seed culture medium is 30 ℃, the culture mode is shake flask culture, and the rotating speed is 150 rpm; and (3) fermenting, wherein the fermentation temperature is 30 ℃, and the oscillation speed is 250 rpm.

Dodecane was added in an amount of 10 vol%.

Example 3

1. Second-stage seed culture medium

Weighing 1500g of corn mash, adding 4500mL of 65-70 ℃ water, uniformly stirring, standing for gelatinization for 40min, adding 0.9mL of high temperature resistant α -amylase and 3 ten thousand U/mL of high temperature resistant α -amylase, uniformly stirring, liquefying at 85-90 ℃ for 1.5h, rapidly cooling to 60 ℃ after liquefaction, adding 3mL of saccharifying enzyme and 10 ten thousand U/mL of saccharifying enzyme, uniformly stirring, saccharifying at 55-60 ℃ for 20h, filtering with filter cloth after saccharification, obtaining a clear solution, naturally adjusting the pH, and sterilizing at 105 ℃ for 10min to obtain the corn hydrolysate.

2. The primary seed culture medium is YPD culture medium containing 20g/L glucose, 10g/L yeast powder and 20g/L peptone;

3. and (3) fermenting thalli:

mixing all the materialsThe gene engineering bacterium of β -farnesene synthesis prepared in example 1 was inoculated into the above-mentioned primary seed culture medium, and cultured in a shake flask to OD₆₀₀Obtaining a first-stage seed culture solution for 0.5, adding 50mL of a second-stage seed culture medium into a 250mL baffle triangular flask, inoculating the first-stage seed culture solution into the second-stage seed culture medium in a volume ratio of 10%, performing shaking fermentation culture, and fermenting to OD₆₀₀Adding about 2 of dodecane serving as an extractant, continuously fermenting for 4 days, sampling, measuring the growth condition of thalli, and carrying out gas phase detection on an organic phase. .

The culture temperature of the genetic engineering bacteria inoculated into the first-level seed culture medium is 30 ℃, the culture mode is shake flask culture, and the rotating speed is 250 rpm; and (3) fermenting, wherein the fermentation temperature is 30 ℃, and the oscillation speed is 250 rpm.

Dodecane was added in an amount of 10 vol%.

Example 4

1. Second-stage seed culture medium

Weighing 1500g of corn mash, adding 4500mL of water with the temperature of 60-65 ℃, uniformly stirring, standing for gelatinization for 1h, adding 0.9mL of high-temperature resistant α -amylase with the temperature of 3 ten thousand U/mL, uniformly stirring, liquefying at 85-90 ℃ for 1.5h, rapidly cooling to 60 ℃ after liquefaction, adding 3mL of saccharifying enzyme with the temperature of 10 ten thousand U/mL, uniformly stirring, saccharifying at 55-60 ℃ for 20h, filtering with filter cloth after saccharification is completed, obtaining a clear solution, naturally adjusting the pH, and sterilizing at 105 ℃ for 10min to obtain the corn hydrolysate.

2. The primary seed culture medium is YPD culture medium containing 20g/L glucose, 10g/L yeast powder and 20g/L peptone;

3. and (3) fermenting thalli:

the gene engineering bacteria of β -farnesene synthesis prepared in example 1 are inoculated into the primary seed culture medium and cultured in a shake flask to OD₆₀₀Obtaining a first-stage seed culture solution for 0.5, adding 50mL of a second-stage seed culture medium into a 250mL baffle triangular flask, inoculating the first-stage seed culture solution into the second-stage seed culture medium in a volume ratio of 10%, performing shaking fermentation culture, and fermenting to OD₆₀₀Adding about 2 of dodecane serving as an extractant, continuously fermenting for 4 days, sampling, measuring the growth condition of thalli, and carrying out gas phase detection on an organic phase. .

The culture temperature of the genetic engineering bacteria inoculated into the first-level seed culture medium is 30 ℃, the culture mode is shake flask culture, and the rotating speed is 200 rpm; and (3) fermenting, wherein the fermentation temperature is 30 ℃, and the oscillation speed is 250 rpm.

Dodecane was added in an amount of 10 vol%.

Example 5

1. Second-stage seed culture medium

Weighing 1500g of corn mash, adding 4500mL of water with the temperature of 60-65 ℃, uniformly stirring, standing for gelatinization for 2h, adding 0.9mL of high-temperature resistant α -amylase with the temperature of 3 ten thousand U/mL, uniformly stirring, liquefying at 85-90 ℃ for 1.5h, rapidly cooling to 60 ℃ after liquefaction, adding 3mL of saccharifying enzyme with the temperature of 10 ten thousand U/mL, uniformly stirring, saccharifying at 55-60 ℃ for 20h, filtering with filter cloth after saccharification is completed, obtaining a clear solution, naturally adjusting the pH, and sterilizing at 105 ℃ for 10min to obtain the corn hydrolysate.

2. The primary seed culture medium is YPD culture medium containing 20g/L glucose, 10g/L yeast powder and 20g/L peptone;

3. and (3) fermenting thalli:

the gene engineering bacteria of β -farnesene synthesis prepared in example 1 are inoculated into the primary seed culture medium and cultured in a shake flask to OD₆₀₀Obtaining a first-stage seed culture solution for 0.5, adding 50mL of a second-stage seed culture medium into a 250mL baffle triangular flask, inoculating the first-stage seed culture solution into the second-stage seed culture medium in a volume ratio of 10%, performing shaking fermentation culture, and fermenting to OD₆₀₀Adding about 2 of dodecane serving as an extractant, continuously fermenting for 4 days, sampling, measuring the growth condition of thalli, and carrying out gas phase detection on an organic phase. .

The culture temperature of the genetic engineering bacteria inoculated into the first-level seed culture medium is 30 ℃, the culture mode is shake flask culture, and the rotating speed is 150 rpm; and (3) fermenting at the temperature of 30 ℃ and at the oscillation speed of 200 rpm.

Dodecane was added in an amount of 10 vol%.

Example 6

1. Corn hydrolysate culture medium for preparing β -farnesene by fermentation

Weighing 1500g of corn mash, adding 4500mL of 65-70 ℃ water, uniformly stirring, standing for gelatinization for 20min, adding 0.9mL of 3 ten thousand U/mL of high-temperature-resistant α -amylase, uniformly stirring, liquefying at 85-90 ℃ for 1.5h, rapidly cooling to 60 ℃, adding 3mL of 10 ten thousand U/mL of saccharifying enzyme, uniformly stirring, saccharifying at 55-60 ℃ for 20h, filtering by using filter cloth after completing saccharification to obtain a clear solution, wherein the pH is natural, sterilizing at 105 ℃ for 10min, adding a sterilized buffer solution to adjust the pH value, adding a sterilized microelement mother solution, and uniformly mixing to obtain the corn hydrolysate for fermentation production of β -farnesene, wherein the buffer solution is 0.5M succinic acid aqueous solution, the pH value is adjusted to be 6.0, the microelement mother solution contains 5.75g/L of zinc sulfate heptahydrate, 0.32g/L of tetrahydrate of manganese chloride tetrahydrate, 0.32g/L of anhydrous medium, 0.47g/L of cobalt sulfate, and the dosage of a ferrous sulfate mother solution is equal to 48g of a medium, and the dosage of a manganese sulfate mother solution is equal to 48.8 g of a medium prepared by a fermentation method, wherein the dosage of a volume ratio of a raw corn hydrolysate for each corn hydrolysate is not equal to 2.5 g of a calcium sulfate medium, and a volume of a calcium sulfate medium.

2. The primary seed culture medium is YPD culture medium containing 20g/L glucose, 10g/L yeast powder and 20g/L peptone;

3. and (3) fermenting thalli:

the gene engineering bacteria of β -farnesene synthesis prepared in example 1 are inoculated into the primary seed culture medium and cultured in a shake flask to OD₆₀₀Obtaining first-class seed culture solution for 0.5, adding 50mL of corn hydrolysate culture medium for fermentation production of β -farnesene into a 250mL baffle triangular flask, inoculating the first-class seed culture solution into the corn hydrolysate culture medium for fermentation production of β -farnesene according to the volume ratio of 10%, performing shaking fermentation culture, and fermenting to OD₆₀₀Adding about 2 of dodecane serving as an extractant, continuously fermenting for 4 days, sampling, measuring the growth condition of thalli, and carrying out gas phase detection on an organic phase. .

The culture temperature of the genetic engineering bacteria inoculated into the first-level seed culture medium is 30 ℃, the culture mode is shake flask culture, and the rotating speed is 250 rpm; and (3) fermenting at the temperature of 30 ℃ and at the oscillation speed of 200 rpm.

Dodecane was added in an amount of 10 vol%.

Example 7

1. Corn hydrolysate culture medium for preparing β -farnesene by fermentation

Weighing 1500g of corn mash, adding 4500mL of 65-70 ℃ water, uniformly stirring, standing for gelatinization for 40min, adding 0.9mL of 3 ten thousand U/mL of high-temperature-resistant α -amylase, uniformly stirring, liquefying at 85-90 ℃ for 1.5h, rapidly cooling to 60 ℃, adding 3mL of 10 ten thousand U/mL of saccharifying enzyme, uniformly stirring, saccharifying at 55-60 ℃ for 20h, filtering by using filter cloth after completing saccharification to obtain a clear solution, wherein the pH is natural, sterilizing at 105 ℃ for 10min, adding a sterilized buffer solution to adjust the pH value, adding a sterilized microelement mother solution, and uniformly mixing to obtain the corn hydrolysate for fermentation production of β -farnesene, wherein the buffer solution is 0.5M succinic acid aqueous solution, the pH value is adjusted to be 6.0, the microelement mother solution contains 5.75g/L of zinc sulfate heptahydrate, 0.32g/L of tetrahydrate of manganese chloride tetrahydrate, 0.32g/L of anhydrous medium, 0.47g/L of cobalt sulfate, and the dosage of a ferrous sulfate mother solution is equal to 48g of a medium, and the dosage of a manganese sulfate mother solution is equal to 48.8 g of a medium prepared by a fermentation method, wherein the dosage of a volume ratio of a raw corn hydrolysate for each corn hydrolysate is not equal to 2.5 g of a calcium sulfate medium, and a volume of a calcium sulfate medium.

2. The primary seed culture medium is YPD culture medium containing 20g/L glucose, 10g/L yeast powder and 20g/L peptone;

3. and (3) fermenting thalli:

the gene engineering bacteria of β -farnesene synthesis prepared in example 1 are inoculated into the primary seed culture medium and cultured in a shake flask to OD₆₀₀Obtaining first-class seed culture solution for 0.5, adding 50mL of corn hydrolysate culture medium for fermentation production of β -farnesene into a 250mL baffle triangular flask, inoculating the first-class seed culture solution into the corn hydrolysate culture medium for fermentation production of β -farnesene according to the volume ratio of 10%, performing shaking fermentation culture, and fermenting to OD₆₀₀Adding about 2% of dodecane as extractant, continuously fermenting for 4 days, sampling, measuring thallus growth condition, and performing gas phase detection on organic phaseAnd (6) measuring.

The culture temperature of the genetic engineering bacteria inoculated into the first-level seed culture medium is 30 ℃, the culture mode is shake flask culture, and the rotating speed is 200 rpm; and (3) fermenting at the temperature of 30 ℃ and at the oscillation speed of 200 rpm.

Dodecane was added in an amount of 10 vol%.

Example 8

1. Corn hydrolysate culture medium for preparing β -farnesene by fermentation

Weighing 1500g of corn mash, adding 4500mL of water with the temperature of 60-65 ℃, uniformly stirring, standing for gelatinization for 1h, adding α -amylase with the high temperature resistance of 0.9mL and 3 thousand U/mL, uniformly stirring, liquefying at 85-90 ℃ for 1.5h, rapidly cooling to 60 ℃, adding glucoamylase with the temperature of 3mL and 10 thousand U/mL, uniformly stirring, saccharifying at 55-60 ℃ for 20h, filtering by using filter cloth after completing saccharification to obtain a clear solution, wherein the pH is natural, sterilizing at 105 ℃ for 10min, adding a sterilized buffer solution to adjust the pH value, adding a sterilized microelement mother solution, and uniformly mixing to obtain the corn hydrolysate for fermentation production of β -farnesene, wherein the buffer solution is 0.5M succinic acid aqueous solution, the pH value is adjusted to be 6.0, the microelement mother solution contains 5.75g/L of zinc sulfate heptahydrate, 0.32g/L of tetrahydrate of manganese chloride, 0.32g/L of anhydrous medium, 0.47g of cobalt sulfate, and the volume of the raw corn hydrolysate is not equal to the volume of a medium, and the volume of a formula of a ferrous sulfate hydrolysate for producing corn hydrolysate is equal to 48g of calcium sulfate medium, and the volume of a calcium sulfate medium, wherein the corn hydrolysate is equal to 2g of the formula of the corn hydrolysate.

2. The primary seed culture medium is YPD culture medium containing 20g/L glucose, 10g/L yeast powder and 20g/L peptone;

3. and (3) fermenting thalli:

the gene engineering bacteria of β -farnesene synthesis prepared in example 1 are inoculated into the primary seed culture medium and cultured in a shake flask to OD₆₀₀Obtaining first-grade seed culture solution for 0.5, adding 50mL of corn hydrolysate culture medium for producing β -farnesene by fermentation into a baffle triangular flask with 250mL of baffle,inoculating the first-stage seed culture solution into a corn hydrolysate culture medium for producing β -farnesene by fermentation at a volume ratio of 10%, performing shaking fermentation culture, and fermenting to OD₆₀₀Adding about 2 of dodecane serving as an extractant, continuously fermenting for 4 days, sampling, measuring the growth condition of thalli, and carrying out gas phase detection on an organic phase.

The culture temperature of the genetic engineering bacteria inoculated into the first-level seed culture medium is 30 ℃, the culture mode is shake flask culture, and the rotating speed is 200 rpm; and (3) fermenting at the temperature of 30 ℃ and at the oscillation speed of 150 rpm.

Dodecane was added in an amount of 10 vol%.

Example 9

1. Corn hydrolysate culture medium for preparing β -farnesene by fermentation

Weighing 1500g of corn mash, adding 4500mL of water with the temperature of 60-65 ℃, uniformly stirring, standing for gelatinization for 2h, adding α -amylase with the high temperature resistance of 0.9mL and 3 thousand U/mL, uniformly stirring, liquefying at 85-90 ℃ for 1.5h, rapidly cooling to 60 ℃, adding glucoamylase with the temperature of 3mL and 10 thousand U/mL, uniformly stirring, saccharifying at 55-60 ℃ for 20h, filtering by using filter cloth after completing saccharification to obtain a clear solution, wherein the pH is natural, sterilizing at 105 ℃ for 10min, adding a sterilized buffer solution to adjust the pH value, adding a sterilized microelement mother solution, and uniformly mixing to obtain the corn hydrolysate for fermentation production of β -farnesene, wherein the buffer solution is 0.5M succinic acid aqueous solution, the pH value is adjusted to be 6.0, the microelement mother solution contains 5.75g/L of zinc sulfate heptahydrate, 0.32g/L of tetrahydrate of manganese chloride, 0.32g/L of anhydrous medium, 0.47g of cobalt sulfate, and the volume of the raw corn hydrolysate is not equal to the volume of a medium, and the volume of a formula of a ferrous sulfate hydrolysate for producing corn hydrolysate is equal to 48g of calcium sulfate medium, and the volume of a calcium sulfate medium, wherein the corn hydrolysate is equal to 2g of the calcium sulfate medium.

2. The primary seed culture medium is YPD culture medium containing 20g/L glucose, 10g/L yeast powder and 20g/L peptone;

3. and (3) fermenting thalli:

the gene engineering bacteria of β -farnesene synthesis prepared in example 1 are inoculated into the primary seed culture medium and cultured in a shake flask to OD₆₀₀Obtaining first-class seed culture solution for 0.5, adding 50mL of corn hydrolysate culture medium for fermentation production of β -farnesene into a 250mL baffle triangular flask, inoculating the first-class seed culture solution into the corn hydrolysate culture medium for fermentation production of β -farnesene according to the volume ratio of 10%, performing shaking fermentation culture, and fermenting to OD₆₀₀Adding about 2 of dodecane serving as an extractant, continuously fermenting for 4 days, sampling, measuring the growth condition of thalli, and carrying out gas phase detection on an organic phase.

The culture temperature of the genetic engineering bacteria inoculated into the first-level seed culture medium is 30 ℃, the culture mode is shake flask culture, and the rotating speed is 200 rpm; and (3) fermenting at the temperature of 30 ℃ and at the oscillation speed of 150 rpm.

Dodecane was added in an amount of 10 vol%.

β -farnesene product detection

The fermentation product β -farnesene was analyzed and measured by Gas Chromatography (GC) or gas chromatography-mass spectrometer (GC-MS). The GC detection system was a SP-6890 type gas chromatograph (Shandong Lu nan Rainbow chemical instruments Co., Ltd.), a capillary column Agilent HP-INNOWAX (30m × 0.25.25 mm × 0.25.25 μm). The temperature program was 75 ℃ for 0.5min at the initial column temperature, 5min at a 10 ℃/min ramp rate to 100 ℃, 240 ℃ for 5min at the detector temperature, 220 ℃ at the vaporizer temperature, the β -farnesene product of example 6 was identified most efficiently, and the yield of β -farnesene was 89.1mg/L according to the gas detection result, and the yield of β -farnesene of example 2 was 65.9mg/L, and the yields of β -farnesene of examples 3, 4, 5, 7, 8, and 9 were 59.3mg/L, 59.9mg/L, 88.9 mg/L, 88 mg/L, and 87.8 mg/L, respectively.

Comparative example 1

The secondary seed medium of example 2 was adjusted to pH 3, 4, 5 and 6, respectively, and the same experiment as in example 2 was carried out under the same conditions for the remaining steps, and the yield was examined, and the results are shown in FIG. 6, where it can be seen that the yield was the highest at pH 6 and did not follow an increasing or decreasing law, which was not expected in advance.

Comparative example 2

The microelement mother liquor in the embodiment 6 is replaced by nutrient salt solution, the nutrient salt solution contains 15g/L urea, 8g/L potassium dihydrogen phosphate and 6.15g/L magnesium sulfate, the addition volume percentage is 2%, other steps or conditions are also carried out in the same way as the embodiment 6, the yield is detected, and the result is shown in a figure, so that the yield is 33% higher than that of common nutrient salt by adding the microelement mother liquor provided by the invention, and the increase of the yield is unpredictable in advance.

The β -farnesene production process does not relate to high-temperature and high-pressure operation, is economical and green, fundamentally solves the defect that the conventional β -farnesene can only be separated and extracted from plants or essential oil, provides a method for synthesizing β -farnesene by utilizing a sustainable method, and is used for relieving the problem of insufficient raw materials in the high-density fuel industry.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> institute of bioenergy and Process in Qingdao, China academy of sciences

<120> genetic engineering bacterium for synthesizing β -farnesene and application thereof

<130>

<160>1

<170>PatentIn version 3.5

<210>1

<211>1725

<212>DNA

<213> optimization sequence

<220>

<221>DNA

<222>(1)..(1725)

<400>1

atgtctactt tgccaatctc ttctgtttct ttctcttctt ctacttctcc attggttgtt 60

gacgacaagg tttctactaa gccagacgtt atcagacaca ctatgaactt caacgcttct 120

atctggggtg accaattctt gacttacgac gaaccagaag acttggttat gaagaagcaa 180

ttggttgaag aattgaagga agaagttaag aaggaattga tcactatcaa gggttctaac 240

gaaccaatgc aacacgttaa gttgatcgaa ttgatcgacg ctgttcaaag attgggtatc 300

gcttaccact tcgaagaaga aatcgaagaa gctttgcaac acatccacgt tacttacggt 360

gaacaatggg ttgacaagga aaacttgcaa tctatctctt tgtggttcag attgttgaga 420

caacaaggtt tcaacgtttc ttctggtgtt ttcaaggact tcatggacga aaagggtaag 480

ttcaaggaat ctttgtgtaa cgacgctcaa ggtatcttgg ctttgtacga agctgctttc 540

atgagagttg aagacgaaac tatcttggac aacgctttgg aattcactaa ggttcacttg 600

gacatcatcg ctaaggaccc atcttgtgac tcttctttga gaactcaaat ccaccaagct 660

ttgaagcaac cattgagaag aagattggct agaatcgaag ctttgcacta catgccaatc 720

taccaacaag aaacttctca cgacgaagtt ttgttgaagt tggctaagtt ggacttctct 780

gttttgcaat ctatgcacaa gaaggaattg tctcacatct gtaagtggtg gaaggacttg 840

gacttgcaaa acaagttgcc atacgttaga gacagagttg ttgaaggtta cttctggatc 900

ttgtctatct actacgaacc acaacacgct agaactagaa tgttcttgat gaagacttgt 960

atgtggttgg ttgttttgga cgacactttc gacaactacg gtacttacga agaattggaa 1020

atcttcactc aagctgttga aagatggtct atctcttgtt tggacatgtt gccagaatac 1080

atgaagttga tctaccaaga attggttaac ttgcacgttg aaatggaaga atctttggaa 1140

aaggaaggta agacttacca aatccactac gttaaggaaa tggctaagga attggttaga 1200

aactacttgg ttgaagctag atggttgaag gaaggttaca tgccaacttt ggaagaatac 1260

atgtctgttt ctatggttac tggtacttac ggtttgatga tcgctagatc ttacgttggt 1320

agaggtgaca tcgttactga agacactttc aagtgggttt cttcttaccc accaatcatc 1380

aaggcttctt gtgttatcgt tagattgatg gacgacatcg tttctcacaa ggaagaacaa 1440

gaaagaggtc acgttgcttc ttctatcgaa tgttactcta aggaatctgg tgcttctgaa 1500

gaagaagctt gtgaatacat ctctagaaag gttgaagacg cttggaaggt tatcaacaga 1560

gaatctttga gaccaactgc tgttccattc ccattgttga tgccagctat caacttggct 1620

agaatgtgtg aagttttgta ctctgttaac gacggtttca ctcacgctga aggtgacatg 1680

aagtcttaca tgaagtcttt cttcgttcac ccaatggttg tttaa 1725

Claims (5)

1. A gene engineering bacterium for synthesizing β -farnesene is characterized in that an initial strain of the gene engineering bacterium is a yeast, endogenous genes GAL80, ALD6, ALD4, ADH5 and RHR2 of the yeast are knocked out, β -farnesene synthetase genes AaFS, LmPK, CkPTA, Dzeute, SpHMGR, GAL4 and tHMG1 are integrated, and a required promoter and a required terminator are P respectively_GAL1，P_GAL10，P_GAL4And T_CYC1，T_ADH1。

2. The genetically engineered bacterium of claim 1, wherein the yeast is Saccharomyces cerevisiae (Saccharomyces cerevisiae) with a genotype of CEN. PK2-1C (MATa; ura 3-52; trp 1-289; leu2-3_ 112; his3 Δ 1; MAL 2-8C; SUC 2).

3. The genetically engineered bacterium of claim 1, wherein the farnesene synthase gene AaFS has a codeThe optimized farnesene synthase gene AaFS is from Artemisia annua, the GenBank accession number is AAX39387.1, the sequence of the optimized farnesene synthase gene AaFS is shown in SEQ ID NO.1, the β -farnesene synthase gene AaFS is integrated at the GAL80 site of the yeast chromosome of the starting strain, and the promoter and the terminator are respectively P_GAL10And T_CYC1(ii) a The LmPK gene is from Leuconostoc mesenteroides and NCBI accession number YP-819405.1, the LmPK gene is integrated at the ALD6 site of the yeast chromosome of the starting strain, and a promoter and a terminator are P respectively_GAL10And T_CYC1(ii) a The CkPTA gene is from Clostridium kluyveri, NCBI accession number YP _001394780.1, the CkPTA gene is integrated at the ALD6 site of the yeast chromosome of the starting strain, and a promoter and a terminator are P respectively_GAL1And T_ADH1(ii) a The Dzeute gene is from Dickeya zeae, NCBI accession number WP-012768716.1, the Dzeute gene is integrated at the ALD4 site of the yeast chromosome of the starting strain, and a promoter and a terminator are P respectively_GAL1And T_ADH1(ii) a The SpHMGR gene is from Silicabacterpomeroyi, NCBIaccesssion number YP-164994, the SpHMGR gene is integrated at the ALD4 site of the yeast chromosome of the starting strain, and a promoter and a terminator are P respectively_GAL1And T_ADH1(ii) a The GAL4 gene is endogenous gene of the starting strain yeast, GenBank accession number is AQN77051.1, the GAL4 gene is integrated at the chromosome RHR2 site of the starting strain yeast, and the promoter is P (OC)_GAL4The terminator is the self terminator; the tHMG1 gene is endogenous gene of the starting strain yeast, GenBank accession number is CAA86503.1, the tHMG1 gene is integrated at the chromosome RHR2 site of the starting strain yeast, and the promoter is P_GAL1The terminator is its own terminator.

4. Use of the genetically engineered bacterium of any one of claims 1 to 3 for synthesizing β -farnesene in synthesizing β -farnesene.

5. The use of claim 4, wherein the genetically engineered bacterium that synthesizes β -farnesene is activated and fermented to obtain β -farnesene in a fermentation broth.

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