CN110964678B - Genetically engineered bacterium for synthesizing farnesene and construction method and application thereof - Google Patents

Abstract

A genetically engineered bacterium for synthesizing farnesene and a construction method and application thereof belong to the technical field of microorganisms. In order to solve the problems of low catalytic efficiency of a downstream pathway of heterologous MVA, toxicity of an intermediate metabolite such as IPP/DMAPP to a host and further improvement of farnesene yield in the farnesene synthesis process by escherichia coli, the invention provides a gene engineering bacterium for synthesizing farnesene, wherein the gene engineering bacterium overexpresses mvaE, mvaS, ERG8, ERG12, ERG19, a beta-farnesene synthetase FS gene, idi and ispA, and the copy number of the beta-farnesene synthetase AaFS gene, idi and ispA gene on a plasmid is 2. The genetic engineering bacteria can obviously improve the synthesis yield of farnesene and is beneficial to promoting the industrial process of synthesizing farnesene by a biological method.

Description

Genetically engineered bacterium for synthesizing farnesene and construction method and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a genetically engineered bacterium for synthesizing farnesene as well as a construction method and application thereof.

Background

Farnesene (farnesene), also called farnesene and sesquicitronellaene, is a chain sesquiterpene compound with molecular formula C ₁₅ H ₂₄ There are 6 different configurations, cis/trans, -alpha, -beta, etc., and commercial farnesene is usually a mixture of various isomers. Farnesene has floral, green and balsamic aromas and is present in a variety of plant essential oils. The farnesene can be used as an additive to be applied to the industries of daily chemicals, medicines, foods and the like; can also be used as biological pheromone for preventing and treating pests such as aphid and the like; furthermore, farnesene is also an important intermediate for the synthesis of vitamin E; and can be used as novel biofuelThe aerospace field.

The synthetic pathways for natural farnesene are mainly the mevalonate pathway (MVA) and the 2-methyl-D-erythritol-4-phosphate pathway (2-methyl-D-erythrorito-4-phosphate, MEP) present in plants, as well as the terpenes synthetic pathway downstream of both. The farnesene extracted from the plant has low yield, high cost and limitation of raw materials, and the components are complex, so that the product with single configuration and high purity cannot be obtained. Therefore, from the last 60 s, methods for synthesizing farnesene from nerolidol, farnesol, myrcene, geranyl bromide, and the like, respectively, have been developed. The chemical synthesis method improves the yield of farnesene, can obtain a product with single configuration and higher purity, but also has the problems of special instrument and equipment, complex operation flow, high energy consumption, easy pollution generation and the like.

In recent years, farnesene can be synthesized by using engineering microorganisms through metabolic engineering, but the problems of unbalanced metabolic flow of a heterologous pathway, toxicity of intermediate metabolites to host cells and the like still exist.

Disclosure of Invention

In order to solve the problems that the downstream pathway of heterologous MVA has low catalytic efficiency, intermediate metabolites such as IPP/DMAPP have toxicity to a host and the yield of farnesene is further improved in the process of synthesizing farnesene by using escherichia coli, the invention adopts the following technical scheme:

the invention improves the catalytic efficiency of the MVA downstream pathway and reduces the accumulation of toxic intermediates by increasing the copy number of key genes idi and ispA in a host. The plasmid pACYC-mvaE-mvaS-ispA-AaFS is constructed by utilizing an enzyme digestion-connection method, and two plasmids pET28a-AaFS-ispA-idi are constructed by utilizing a Gibbson assembly method. The above two plasmids and the third plasmid pTrc-low were transformed into E.coli BL21 (DE 3) and subjected to shake flask fermentation and 5L fermenter fed-batch fermentation, respectively. A sample containing farnesene is obtained through IPTG induced expression and n-dodecane in-situ extraction, and the sample is quantitatively analyzed through gas chromatography and a farnesene standard substance curve.

Based on the technical scheme, the invention provides a genetically engineered bacterium for synthesizing farnesene, which is a recombinant bacterium for over-expressing acetyl Co-A acyltransferase/HMG-CoA reductase mvaE gene, HMG-CoA synthetase mvaS gene, mevalonate-5-phosphokinase ERG8, mevalonate kinase ERG12, mevalonate-5-diphosphate decarboxylase ERG19, beta-farnesene synthetase AaFS gene, isopentenyl diphosphate isomerase idi gene and farnesyl diphosphate synthetase ispA gene, wherein the copy number of the beta-farnesene synthetase AaFS gene, idi gene and ispA gene is2, and the starting strain is escherichia coli.

Further limiting, the beta-farnesene synthetase AaFS gene is derived from artemisia apiacea, is obtained after optimization according to the codon preference of escherichia coli, and has a nucleotide sequence shown as SEQ ID No. 1; the ispA gene is derived from escherichia coli Top10, and the nucleotide sequence of the ispA gene is shown as SEQ ID No. 2; the idi gene is derived from Escherichia coli BL21 (DE 3), and the nucleotide sequence of the idi gene is shown in SEQ ID No. 3.

Further defined, the escherichia coli is BL21 (DE 3).

The invention also provides a construction method of the gene engineering bacteria for synthesizing farnesene, which comprises the following steps:

1) Construction of plasmid pACYC-mvaE-mvaS-ispA-AaFS: after the gene sequence of the beta-farnesene synthetase AaFS is optimized according to the codon preference of escherichia coli, constructing a pACYC-mvaE-mvaS-ispA carrier in a digestion-connection mode to obtain a plasmid pACYC-mvaE-mvaS-ispA-AaFS;

2) Construction of plasmid pET28 a-AaFS-ispA-idi:

respectively amplifying a pET28a vector sequence, an AaFS gene, an ispA gene and an idi gene sequence, and constructing and obtaining a pET28a-AaFS-ispA-idi plasmid by a Gibson Assembly method; the pET28a vector sequence is shown as SEQ ID No. 4;

3) And (3) plasmid transformation: and (3) transforming the plasmids constructed in the steps 1) and 2) and the pTrc-low plasmid into an escherichia coli host cell together to obtain the gene engineering bacteria for synthesizing farnesene.

Further limiting, after the beta-farnesene synthetase AaFS gene is optimized in the step 1), adding restriction enzyme BglII and XhoI enzyme cutting sites at two ends of the gene respectively, and synthesizingThen cloning to a pUC57-simple vector, carrying out double digestion on the obtained plasmid pUC57-AaFS by restriction enzymes BglII and XhoI, and recovering an AaFS gene digestion product; double digestion of pACYC-mvaE-mvaS-ispA with restriction enzymes BglII and XhoI to recover 8260bp segment, and T treatment of the recovered segment ₄ And connecting the DNA ligase to obtain a plasmid pACYC-mvaE-mvaS-ispA-AaFS.

Further limited, the upstream primer sequence for pET28a vector sequence amplification in the step 2) is shown as SEQ ID No. 5, and the downstream primer sequence is shown as SEQ ID No. 6; the sequence of the upstream primer for AaFS gene amplification is shown as SEQ ID No. 7, and the sequence of the downstream primer is shown as SEQ ID No. 8; the upstream primer sequence for ispA gene amplification is shown as SEQ ID No. 9, and the downstream primer sequence is shown as SEQ ID No. 10; the sequence of the upstream primer for the amplification of the idi gene is shown as SEQ ID No. 11, and the sequence of the downstream primer is shown as SEQ ID No. 12.

The invention also provides application of the genetic engineering bacteria in synthesizing farnesene.

Further limiting, the application is that after the genetically engineered bacteria are cultured by a primary seed culture medium, the obtained seed liquid is inoculated into a shake flask fermentation culture medium for fermentation to obtain farnesene; or after the genetically engineered bacteria are sequentially cultured by a first-stage seed culture medium and a second-stage seed culture medium, inoculating the obtained second-stage seed liquid into a fermentation culture medium of a fermentation tank for fermentation to obtain farnesene.

Further limited, the primary seed culture medium is an LB culture medium, and the components of the primary seed culture medium are as follows: 10g/L NaCl, 10g/L peptone, 5g/L yeast extract, and the balance water.

Further defined, the secondary seed medium comprises: 20g/L glucose, 9.8g/L K ₂ HPO ₄ 5g/L beef extract, 0.3g/L ferric ammonium citrate, 2.1g/L citric acid monohydrate, 0.06g/L MgSO ₄ 1mL/L of trace element solution containing (NH) ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O 0.37g/L、ZnSO ₄ ·7H ₂ O 0.29g/L、H ₃ BO ₃ 2.47 g/L、CuSO ₄ ·5H ₂ O0.25 g/L and MnCl ₂ ·4H ₂ O1.58 g/L; the fermentation medium is added with betaine with the final concentration of 1g/L on the basis of the secondary seed culture medium, and the final concentration of the trace element solution is 1.5mL/L.

The gene engineering bacterium for synthesizing farnesene is used in the construction process as follows:

plasmid pACYC-mvaE-mvaS-ispA, the original empty vector used in the construction is pACYCDuet-1, and the plasmid pACYC-mvaE-mvaS-ispA is described in Zhang H, liu Q, cao Y, feng X, zheng Y, zou H, liu H, yang J, xian M.2014.Microbiological production of sabinene-a new depends-based precursor of advanced biological.Microbiological cells Factories 13.

Plasmid pTrc-low, vector map as shown in fig. 2 c, original empty vector used in construction is pTrcHIS2b, and the plasmid pTrc-low is described in Zhang H, liu Q, cao Y, feng X, zheng Y, zou H, liu H, yang J, xian M2014. Microbiological production of sabene-a new terpen-based plasmid prediction of advanced bio-Cell factors 13, which contains one copy number of idi gene.

Advantageous effects

1. The invention increases the heterologous copy numbers of key genes idi, ispA and AaFS in the farnesene synthesis pathway to 2. Compared with BL21 (DE 3) engineering bacteria introduced with heterologous MVA ways respectively containing one gene, the yield of farnesene synthesized by the engineering bacteria containing three plasmids provided by the invention reaches 2.01g/L in a shake flask level, and the yield is improved by nearly 300 times; the fermentation tank level reaches 12.73g/L, which is the highest yield of farnesene synthesized by the currently known Escherichia coli.

2. The method disclosed by the invention has the characteristics of short growth and fermentation period, low culture cost, simple genetic operation and the like, the yield of the farnesene synthesized by escherichia coli is further improved, the produced farnesene has the advantages of higher yield and purity, no toxicity and harmlessness, and compared with plant extraction and chemical synthesis, the method is a more economic, environment-friendly and sustainable production mode and is more beneficial to promoting the industrial process of synthesizing the farnesene by a biological method.

Drawings

FIG. 1 is a scheme for the synthesis of farnesene.

In FIG. 2, a is a plasmid map of pACYC-mvaE-mvaS-ispA-AaFS; b is a plasmid map of pET28a-AaFS-ispA-idi, and c is a vector map of pTrc-low.

FIG. 3 shows the yield of farnesene by horizontal shake flask fermentation, a is the shake flask fermentation result of genetically engineered bacteria containing two plasmids, the abscissa is the fermentation time (h) and the ordinate (left) is OD ₆₀₀ The ordinate (right) of the absorbance value of (1) is the farnesene yield (mg/L); b is the shake flask fermentation result containing the three-plasmid genetic engineering bacteria, the abscissa is the fermentation time (h), and the ordinate (left) is OD ₆₀₀ The absorbance of (D) is plotted on the ordinate (right) as farnesene production (g/L).

FIG. 4 shows the horizontal fermentation yield of farnesene in a fermenter, the abscissa shows the fermentation time (h) and the ordinate shows the farnesene yield (g/L).

Detailed Description

The plasmids pACYCDuet-1, pTrcHIS2b, pET28a, BL21 (DE 3) in E.coli, competent cells, primers and reagents, etc., used in the examples were all commercially available or obtained by conventional means well known to those skilled in the art.

Wherein:

restriction enzyme BglII was purchased from Thermo Scientific, cat #: FD0083.

Restriction enzyme XhoI was purchased from Thermo Scientific, cat #: FD0694.

T ₄ Ligase was purchased from NEB, cat no: M0202S.

The primers were synthesized from: qingdao Zhixi Biotechnology Limited.

Coli Top10, purchased from Huada Gene.

Cm ^r Represents chloramphenicol resistance; kan ^r Represents kanamycin resistance; amp ^r Represents ampicillin. The strain provided by the invention is Escherichia coli (Escherichia coli) BL21 (DE 3), the contained plasmids are pACYC-mvaE-mvaS-ispA-AaFS, pTrc-low and pET28a-AaFS-ispA-idi, and the plasmids are respectively constructed by using a method of enzyme digestion-ligation or Gibson assembly (Gibson assembly).

The farnesene synthesis pathway constructed by the invention is shown in figure 1, and consists of plasmids pACYC-mvaE-mvaS-ispA-AaFS, pET28a-AaFS-ispA-idi, pTrc-low and MEP pathway of escherichia coli. Among them, acetyl Co-A acyltransferase/HMG-CoA reductase mvaE gene is one copy, which catalyzes two-step reaction.

Example 1. Construction method of genetically engineered bacterium for synthesizing farnesene.

1) Construction of plasmid pACYC-mvaE-mvaS-ispA-AaFS: after the gene sequence of beta-farnesene synthetase AaFS from southernwood is optimized by the codon preference of escherichia coli, restriction enzymes BglII and XhoI enzyme cutting sites are respectively added at two ends, and the sequence synthesis is carried out by Huada gene, and the synthesized sequence is shown as SEQ ID No. 1. The AaFS gene with BglII and XhoI cleavage sites after synthesis was cloned into a pUC57-simple vector, and the cloning method was performed with reference to the vector instructions to obtain a plasmid designated as pUC57-AaFS. The construction of plasmid pACYC-mvaE-mvaS-ispA-AaFS (shown as a in the vector map in figure 2) adopts an enzyme digestion-connection method. Firstly, carrying out double enzyme digestion on plasmids pACYC-mvaE-mvaS-ispA and pUC57-AaFS by using restriction enzymes BglII and XhoI respectively, wherein the enzyme digestion system is as follows:

carrying out agarose gel electrophoresis and target band gel cutting recovery on products after enzyme digestion, wherein pACYC-mvaE-mvaS-ispA is subjected to BglII and XhoI double enzyme digestion to recover 8260bp fragments as a vector, pUC57-AaFS is subjected to BglII and XhoI double enzyme digestion to recover 1725bp fragments as inserted fragments, and the recovered products are subjected to ligation reaction:

ligation product 10. Mu.L of heat shock transformed DH 5. Alpha. Competent cells and plated with LB Cm ^r Plates were incubated overnight at 37 ℃. Observing the colony condition on the plate the next day, picking out single bacterium, dropping into liquid culture medium, culturing at 37 deg.C to relatively high concentration, and performingColony PCR identification or extraction plasmid enzyme digestion identification, and sending to sequencing.

2) Construction of plasmid pET28 a-AaFS-ispA-idi:

construction of plasmid pET28a-AaFS-ispA-idi (shown as b in the attached drawing 2) firstly, pET28a vector and three gene fragments of AaFS, ispA and idi are amplified by adopting a Gibson Assembly method, and respectively taking pET28a plasmid, pUC57-AaFS plasmid, pACYC-mvaE-mvaS-ispA plasmid and Escherichia coli BL21 (DE 3) bacterial liquid as templates:

the upstream primer sequence for pET28a vector sequence amplification is shown as SEQ ID No. 5, and the downstream primer sequence is shown as SEQ ID No. 6; the sequence of an upstream primer for AaFS gene amplification is shown as SEQ ID No. 7, and the sequence of a downstream primer is shown as SEQ ID No. 8; the upstream primer sequence for ispA gene amplification is shown as SEQ ID No. 9, and the downstream primer sequence is shown as SEQ ID No. 10; the sequence of the upstream primer for the amplification of the idi gene is shown as SEQ ID No. 11, and the sequence of the downstream primer is shown as SEQ ID No. 12.

Performing agarose gel electrophoresis and target band gel cutting recovery on the PCR product, determining the concentration of the gel recovery product, performing Gibson analysis by using a NEBuilder kit, calculating the proportion of fragments and the amount of each component according to the instruction, performing ligation reaction at 50 ℃ for 60min, diluting the product with sterile water with the same volume, taking 5 mu L of heat shock transformed DH5 alpha competent cells, and coating LB Kan competent cells ^r Plates were incubated overnight at 37 ℃. Observing the colony condition on the plate the next day, selecting single bacteria, dropping the single bacteria into a liquid culture medium, culturing the single bacteria to be concentrated at 37 ℃, carrying out colony PCR identification or extraction plasmid enzyme digestion identification, and sending to sequencing.

The ispA nucleotide sequence is shown as SEQ ID No. 2; the idi nucleotide sequence is shown as SEQ ID No. 3, and the pET28a vector sequence is shown as SEQ ID No. 4.

3) And (3) plasmid transformation:

e.coli BL21 (DE 3) competent cells are transformed by heat shock of plasmids pACYC-mvaE-mvaS-ispA-AaFS, pTrc-low and pET28a-AaFS-ispA-idi (three plasmids) with correct sequencing, corresponding three-antibody (Cm, amp and Kan) LB medium plates are coated, wherein the final concentration of Cm in LB medium is 34mg/L, the final concentration of Amp in LB medium is 100mg/L, and the final concentration of Kan in LB medium is 50mg/L, and the plasmids are cultured at 37 ℃ until a single colony grows out, so that the gene engineering bacteria for synthesizing farnesene are obtained.

The embodiment obtains recombinant bacteria of genetically engineered bacteria over-expression acetyl Co-A acyltransferase/HMG-CoA reductase mvaE gene, HMG-CoA synthetase mvaS gene, mevalonate-5-phosphokinase ERG8 gene, mevalonate kinase ERG12 gene, mevalonate-5-diphosphonate decarboxylase ERG19 gene, beta-farnesene synthetase AaFS gene, isopentenyl diphosphate isomerase idi gene and farnesyl diphosphate synthetase ispA gene, wherein the copy number of the beta-farnesene synthetase AaFS gene, the idi gene and the ispA gene is 2.

Comparative example 1. Example 1 was repeated, except that in this comparative example, in step 3), E.coli BL21 (DE 3) competent cells were transformed by heat shock using correctly sequenced plasmids pACYC-mvaE-mvaS-ispA-AaFS and pTrc-low (two plasmids in total) to LB medium plates coated with the corresponding diabodies (Cm and Amp) at a final concentration of 34mg/L in LB medium and 100mg/L in LB medium, and cultured at 37 ℃ until single colonies grew out. The ispA, aaFS and idi genes in the genetic engineering bacteria obtained by the comparative example are respectively 1 copy.

Example 2. Application of the genetically engineered bacterium constructed in example 1 in the synthesis of farnesene.

In the embodiment, the farnesene quantitative determination is performed by using gas chromatography, the chromatographic column is an Agilent DB-5MS (30 m × 0.25mm × 0.25 μm) capillary column, and the column temperature-raising program is as follows: the initial temperature of 60 ℃ is kept for 0.75min, the temperature is increased to 180 ℃ at the speed of 10 ℃/min, and then the temperature is reduced to the initial temperature. And (4) quantifying by using a beta-farnesene standard substance as a standard curve.

In this embodiment, the primary seed culture medium is an LB culture medium, and comprises the following components: 10g/L NaCl, 10g/L peptone, 5g/L yeast extract, and the balance water.

The secondary seed culture medium comprises the following components: 20g/L glucose, 9.8g/L K ₂ HPO ₄ 5g/L beef extract, 0.3g/L ferric ammonium citrate, 2.1g/L citric acid monohydrate, 0.06g/L MgSO ₄ 1mL/L of a trace element solution containing (NH) ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O 0.37g/L、ZnSO ₄ ·7H ₂ O 0.29g/L、H ₃ BO ₃ 2.47g/L、CuSO ₄ ·5H ₂ O0.25 g/L and MnCl ₂ ·4H ₂ O1.58 g/L, wherein the concentration is the final concentration of each component in the trace element solution; the fermentation medium is added with betaine with the final concentration of 1g/L on the basis of the secondary seed culture medium, and the final concentration of the microelement solution is 1.5mL/L.

1. Taking a shake flask fermentation method as an example, the application of the genetically engineered bacterium constructed in example 1 in the synthesis of farnesene is described.

Selecting the single colony of the genetically engineered bacteria obtained in the example 1 to 5mL of LB culture medium containing corresponding resistance (Cm/Amp/Kan), carrying out shake culture at 37 ℃ for 8-12 h to obtain a primary seed solution, fermenting by using a saline bottle with the volume of 600mL, transferring 500 mu L of the primary seed solution to 50mL of fermentation culture medium containing corresponding resistance (Cm, amp and Kan), and carrying out culture at 37 ℃ to OD ₆₀₀ About 0.6-0.8, adding IPTG with final concentration of 0.1mM and 10mL of n-dodecane, shake culturing at 30 deg.C, sampling at 24h, 48h and 72h, respectively, and measuring OD with spectrophotometer ₆₀₀ The fermentation product is beta-farnesene identified by gas chromatography-mass spectrometry, and the farnesene yield is determined by gas chromatography, and three repetitions are set.

Control group: selecting the single colony obtained in comparative example 1, synthesizing farnesene by the shake flask fermentation method, sampling at 24h, 48h and 72h respectively, and measuring OD by spectrophotometry ₆₀₀ Farnesene production was determined by gas chromatography in triplicate.

As shown in the attached figure 3, in the shake flask fermentation level, when the engineering bacteria containing two plasmids in the comparative example 1 are fermented for 48 hours, the farnesene yield is only at the milligram level and is close to 6mg/L (shown in a figure 3), while the engineering bacteria containing three plasmids constructed by the invention (the copy numbers of the beta-farnesene synthetase AaFS gene, the idi gene and the ispA gene in the engineering bacteria are all 2) are fermented for 48 hours, the yield is close to 2g/L, the yield is improved by about 300 times, and when the fermentation time is up to 72 hours, the farnesene yield is further improved to 2.01g/L (shown in b in a figure 3).

2. The application of the genetically engineered bacterium constructed in example 1 in the synthesis of farnesene is described by taking a fed-batch fermentation method as an example.

First-stage seed liquid: selecting a single colony obtained in the step 3) in the example 1 into 5mL of LB culture medium containing corresponding resistance, and carrying out shake culture at 37 ℃ for 8-12 h to obtain a first-stage seed solution.

Secondary seed liquid: 1mL of the primary seed solution was transferred to 100mL of LB medium containing the corresponding resistance (Cm/Amp/Kan) and shake-cultured overnight at 37 ℃. Preparing 2L of fermentation medium in the morning of the first day, pouring into a fermentation tank with the volume of 5L, sterilizing at 115 ℃ for 30min, connecting a temperature electrode, a pH electrode, an oxygen dissolving electrode and supply pipes of the tank to a fermentation controller, opening an air valve, adjusting the stirring speed to 300r/min, opening condensed water to reduce the temperature of the medium to 37 ℃, adjusting the pH to about 6.9, and adding 100mL and 3mL of secondary seed liquid, trace elements and corresponding antibiotics (Cm, amp and Kan). And observing whether the dissolved oxygen and the pH value are normal or not, and if foams exist, adding a defoaming agent dropwise properly. OD was measured every three hours ₆₀₀ OD after about 9 hours ₆₀₀ When 11 was reached, the temperature was first reduced to 30 ℃ and then IPTG and 400mL of n-dodecane were added to a final concentration of 0.1mM, after which glucose was supplemented at a rate of 3% and incubated overnight. And gradually adjusting the stirring speed to 600r/min in the morning, adding IPTG every eight hours, frequently checking the dissolved oxygen and pH change, and accelerating the sugar adding rate if the dissolved oxygen is suddenly increased. The yields (aqueous, organic, off-gas) were measured every 24 hours. As shown in the attached figure 4, the yield of the farnesene reaches 12.73g/L in 48h, which exceeds the highest yield of 8.74g/L of farnesene synthesized horizontally by an escherichia coli fermentation tank reported in the current literature.

SEQUENCE LISTING

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

<120> genetic engineering bacterium for synthesizing farnesene, construction method and application thereof

<130>

<160> 12

<170> PatentIn version 3.5

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<210> 3

<211> 549

<212> DNA

<213> idi Gene

<400> 3

atgcaaacgg aacacgtcat tttattgaat gcacagggag ttcccacggg tacgctggaa 60

aagtatgccg cacacacggc agacaccctc ttacatctcg cgttctccag ttggctgttt 120

aatgccaagg ggcaattatt agttacccgc cgcgcactga gcaaaaaagc atggcctggc 180

gtgtggacta actcggtttg tgggcaccca caactgggag aaagcaacga agacgcagtg 240

atccgccgtt gccgttatga gcttggcgtg gaaattacgc ctcctgaatc tatctatcct 300

gactttcgct atcgcgccac cgatccgaat ggcattgtgg aaaatgaagt gtgtccggta 360

tttgccgcac gcaccaacag tgcgttacag atcaacgatg atgaagtgat ggattatcaa 420

tggtgtgatt tagcagatgt attacacggt attgatgcca cgccgtgggc gttcagtccg 480

tggatggtaa tgcaggcagc caatagtgaa gcaagaaaat tgttgtctgc tttcgcgcag 540

cacaattaa 549

<210> 4

<211> 5279

<212> DNA

<213> pET28a vector sequence

<400> 4

ggatccgaat tcgagctccg tcgacaagct tgcggccgca ctcgagcacc accaccacca 60

ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc 120

tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct 180

gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc 240

gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc 300

gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct 360

ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa 420

aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 480

cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 540

ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat 600

tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg 660

tttacaattt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 720

ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag 780

catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag 840

ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg 900

gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc 960

aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg 1020

caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc 1080

aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa 1140

tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa 1200

cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa 1260

tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa 1320

atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc 1380

tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg 1440

cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt 1500

atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc 1560

ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat 1620

tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1680

aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1740

caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1800

ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1860

cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1920

tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1980

gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 2040

ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 2100

gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 2160

caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 2220

ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 2280

tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 2340

ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 2400

agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 2460

aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 2520

gcatatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac 2580

actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct 2640

gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc 2700

tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg 2760

cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg 2820

tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg 2880

ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc 2940

atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat 3000

gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg 3060

gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt 3120

ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct 3180

gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct 3240

caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca 3300

ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg 3360

atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt 3420

ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca 3480

agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag 3540

agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg 3600

acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc 3660

atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca 3720

ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 3780

gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac 3840

gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac 3900

gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca 3960

tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc 4020

ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc 4080

agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact 4140

ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca 4200

gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg 4260

ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa 4320

aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt 4380

gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc 4440

actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg 4500

ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc 4560

gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga 4620

ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc 4680

cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga 4740

aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac 4800

attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt 4860

gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga 4920

agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca 4980

aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac 5040

aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat 5100

aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga 5160

ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga 5220

taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat accatgggc 5279

<210> 5

<211> 38

<212> DNA

<213> upstream primer for amplifying pET28a vector sequence

<400> 5

ctttcgcgca gcacaattaa ggatccgaat tcgagctc 38

<210> 6

<211> 20

<212> DNA

<213> downstream primer for amplification of pET28a vector sequence

<400> 6

gcccatggta tatctccttc 20

<210> 7

<211> 38

<212> DNA

<213> upstream primer for AaFS Gene amplification

<400> 7

gaaggagata taccatgggc atgagcaccc tgccgatt 38

<210> 8

<211> 21

<212> DNA

<213> downstream primer for AaFS Gene amplification

<400> 8

ttaaacaacc atcgggtgca c 21

<210> 9

<211> 53

<212> DNA

<213> upstream primer for ispA Gene amplification

<400> 9

tgcacccgat ggttgtttaa aggaggttaa ttggatggac tttccgcagc aac 53

<210> 10

<211> 27

<212> DNA

<213> downstream primer for ispA Gene amplification

<400> 10

ttatttatta cgctggatga tgtagtc 27

<210> 11

<211> 53

<212> DNA

<213> upstream primer for amplification of idi Gene

<400> 11

tcatccagcg taataaataa aggaggttaa ttggatgcaa acggaacacg tca 53

<210> 12

<211> 20

<212> DNA

<213> downstream primer for idi Gene amplification

<400> 12

ttaattgtgc tgcgcgaaag 20

Claims (8)

1. The genetic engineering bacteria for synthesizing farnesene are characterized in that the genetic engineering bacteria are overexpression acetyl Co-A acyltransferase/HMG-CoA reductasemvaEGene HMG-CoA synthetasemvaSGene, mevalonate-5-phosphate kinaseERG8Mevalonate kinaseERG12Mevalonate-5-diphosphate decarboxylaseERG19Beta-farnesene synthetaseAaFSGene isopentenyl diphosphate isomeraseidiGene and farnesyl diphosphate synthaseispARecombinant strain of gene, beta-farnesene synthetaseAaFSGene, gene,idiGene, gene,ispAThe copy number of the gene is2, and the starting strain is escherichia coli; the beta-farnesene synthetase AaFS gene is obtained after optimization according to the codon preference of escherichia coli, and the optimized nucleotide sequence is shown as SEQ ID No. 1; the above-mentioned ispAThe gene nucleotide sequence is shown as SEQ ID No. 2; the above-mentionedidiThe nucleotide sequence of the gene is shown as SEQ ID No. 3.

2. The genetically engineered bacterium of claim 1, wherein the escherichia coli is BL21 (DE 3).

3. The method for constructing genetically engineered bacteria for synthesizing farnesene according to claim 1 or 2, which comprises the following steps:

1) Construction of plasmid pACYC-mvaE-mvaS-ispA-AaFS: after the gene sequence of the beta-farnesene synthetase AaFS is optimized according to the codon preference of escherichia coli, constructing a pACYC-mvaE-mvaS-ispA carrier in a digestion-connection mode to obtain a plasmid pACYC-mvaE-mvaS-ispA-AaFS;

2) Construction of plasmid pET28 a-AaFS-ispA-idi:

respectively amplifying pET28a vector sequences,AaFSGene, gene,ispAGenes andidia gene sequence, and a pET28a-AaFS-ispA-idi plasmid is constructed and obtained by a Gibson Assembly method; the pET28a vector sequence is shown as SEQ ID No. 4; the upstream primer sequence for pET28a vector sequence amplification is shown as SEQ ID No. 5, and the downstream primer sequence is shown as SEQ ID No. 6;AaFSthe sequence of the upstream primer for gene amplification is shown as SEQ ID No. 7, and the sequence of the downstream primer is shown as SEQ ID No. 8;ispAthe sequence of the upstream primer for gene amplification is shown as SEQ ID No. 9, and the sequence of the downstream primer is shown as SEQ ID No. 10; idithe sequence of the upstream primer for gene amplification is shown as SEQ ID No. 11,the sequence of the downstream primer is shown as SEQ ID No. 12;

3) And (3) plasmid transformation: and (3) transforming the plasmids constructed in the steps 1) and 2) and the pTrc-low plasmid into an escherichia coli host cell together to obtain the genetically engineered bacterium for synthesizing farnesene.

4. The method for constructing genetically engineered bacteria for synthesizing farnesene according to claim 3, wherein after the gene of the beta-farnesene synthetase AaFS in the step 1) is optimized, restriction enzymes are respectively added to two ends of the geneBglII、XhoI restriction enzyme site, synthesizing, cloning to pUC57-simple vector to obtain plasmid pUC57-AaFS with restriction enzymeBglII、XhoI, double enzyme digestion, namely recovering an AaFS gene enzyme digestion product; subjecting pACYC-mvaE-mvaS-ispA to restriction enzymeBglII、XhoI, double enzyme digestion, and recycling 8260bp segments; the two enzyme digestion fragments are recovered through T ₄ And connecting the DNA ligase to obtain a plasmid pACYC-mvaE-mvaS-ispA-AaFS.

5. The use of the genetically engineered bacterium of claim 1 or 2 in the synthesis of farnesene.

6. The application of the genetically engineered bacteria in synthesizing farnesene according to claim 5, wherein the genetically engineered bacteria is cultured by a primary seed culture medium, and an obtained seed solution is inoculated into a shake flask fermentation culture medium for fermentation to obtain farnesene; or after the genetically engineered bacteria are sequentially cultured by a first-stage seed culture medium and a second-stage seed culture medium, inoculating the obtained second-stage seed liquid into a fermentation culture medium of a fermentation tank for fermentation to obtain farnesene.

7. The use of the synthetic farnesene according to claim 6, wherein the primary seed culture medium is an LB culture medium comprising: 10g/L NaCl, 10g/L peptone, 5g/L yeast extract, and the balance water.

8. Use of the synthetic farnesene according to claim 6, characterized in thatThe secondary seed culture medium comprises the following components: 20g/L glucose, 9.8g/L K ₂ HPO ₄ 5g/L beef extract, 0.3g/L ferric ammonium citrate, 2.1g/L citric acid monohydrate, 0.06g/L MgSO ₄ 1mL/L of trace element solution, wherein the trace element solution is prepared from (NH) ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O 0.37 g/L、 ZnSO ₄ ·7H ₂ O 0.29 g/L、H ₃ BO ₃ 2.47 g/L、CuSO ₄ ·5H ₂ O0.25 g/L and MnCl ₂ ·4H ₂ O1.58 g/L; the fermentation medium is added with betaine with the final concentration of 1g/L on the basis of the secondary seed culture medium, and the final concentration of the trace element solution is 1.5mL/L.

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