CN110964678A

CN110964678A - Genetically engineered bacterium for synthesizing farnesene and construction method and application thereof

Info

Publication number: CN110964678A
Application number: CN201811144959.7A
Authority: CN
Inventors: 咸漠; 门潇; 张海波; 许子燕
Original assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Current assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority date: 2018-09-29
Filing date: 2018-09-29
Publication date: 2020-04-07
Anticipated expiration: 2038-09-29
Also published as: CN110964678B

Abstract

The invention provides a genetically engineered bacterium for synthesizing farnesene, which aims to solve the problems that a heterologous MVA (multi-domain vertical multi-domain) downstream path in a farnesene synthesizing process of escherichia coli is low in catalytic efficiency, intermediate metabolites such as IPP (isopropyl-propylene phosphate)/DMAPP (dimethyl propyl methacrylate) are toxic to a host and the farnesene yield is further improved.

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, and a construction method and application thereof.

Background

Farnesene (farnesene), also called farnesene and sesquicitronellaene, is a chain sesquiterpene compound with molecular formula C₁₅H₂₄The farnesene has 6 different configurations such as cis/trans, - α, - β and the like, and is usually a mixture of a plurality of isomers.

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 and the terpenoid 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 catalytic efficiency of a heterologous MVA downstream pathway is low, 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 escherichia coli, the technical scheme adopted by the invention is as follows:

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(DE3) 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-phosphate kinase ERG8, mevalonate kinase ERG12, mevalonate-5-diphosphate decarboxylase ERG19, β -farnesene synthetase AaFS gene, isopentenyl diphosphate isomerase idi gene and farnesyl diphosphate synthetase ispA gene, wherein the copy number of the β -farnesene synthetase AaFS gene, idi gene and ispA gene is2, and the starting strain is escherichia coli.

The β -farnesene synthetase AaFS gene is derived from artemisia apiacea and is obtained after optimization according to codon preference of escherichia coli, the nucleotide sequence of the gene is shown as SEQ ID No. 1, the ispA gene is derived from escherichia coli Top10, the nucleotide sequence of the gene is shown as SEQ ID No. 2, the idi gene is derived from escherichia coli BL21(DE3), and the nucleotide sequence of the gene is shown as 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) constructing plasmid pACYC-mvaE-mvaS-ispA-AaFS by optimizing gene sequence of β -farnesene synthetase AaFS according to codon preference of escherichia coli and constructing the plasmid pACYC-mvaE-mvaS-ispA carrier in a enzyme digestion-connection mode to obtain 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 Gibsonassambly method; the pET28a vector sequence is shown in 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 genetically engineered bacterium for synthesizing farnesene.

Further limiting, after the β -farnesene synthetase AaFS gene is optimized in step 1), restriction enzyme BglII and XhoI restriction sites are respectively added at two ends of the gene, the gene is synthesized and cloned on a pUC57-simple vector, the obtained plasmid pUC57-AaFS is subjected to double enzyme digestion by the restriction enzyme BglII and XhoI, the AaFS gene restriction product is recovered, pACYC-mvaE-mvaS-ispA is subjected to double enzyme digestion by the restriction enzyme BglII and XhoI, the 8260bp fragment is recovered, and the two recovered fragments are subjected to T₄And connecting the DNA ligase to obtain a plasmid pACYC-mvaE-mvaS-ispA-AaFS.

Further limiting, the sequence of an upstream primer for amplifying the pET28a vector sequence in the step 2) is shown as SEQ ID No. 5, and the sequence of a downstream primer 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 K₂HPO₄5g/L beef extract, 0.3g/L ferric ammonium citrate, 2.1g/L citric acid monohydrate, 0.06g/L MgSO₄1mL/L of a trace element solution containing (NH)₄)₆Mo₇O₂₄·4H₂O 0.37g/L、ZnSO₄·7H₂O 0.29g/L、H₃BO₃2.47g/L、CuSO₄·5H₂O0.25g/L and MnCl₂·4H₂O1.58 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 1.5 mL/L.

The gene engineering bacteria for synthesizing farnesene provided by the invention are used in the construction process:

the plasmid pACYC-mvaE-mvaS-ispA is constructed by adopting pACYCDuet-1 as an original empty vector, and is described in Zhang H, Liu Q, Cao Y, Feng X, Zheng Y, Zou H, Liu H, YangJ, Xian M.2014.Microbiological process of sabinene-a new depending-based precorred precursorof advanced biological.Microbiological Cell Factories 13: 20.

The plasmid pTrc-low, vector map is shown in fig. 2 c, the original empty vector adopted during construction is pTrcHIS2b, the plasmid pTrc-low is described in Zhang H, Liu Q, Cao Y, Feng X, Zheng Y, Zou H, Liu H, Yang J, Xian M.2014. Microbiological process of combining gene-a new depending-based plasmid of advanced biological of Microbiological cells 13:20, the plasmid contains a 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(DE3) engineering bacteria introduced with heterologous MVA ways containing one gene respectively, 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 synthesis pathway for 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, wherein a is the shake flask fermentation result of genetically engineered bacteria containing two plasmids, the abscissa is fermentation time (h), and the ordinate (left) is OD₆₀₀The ordinate (right) of the absorbance of (A) is the farnesene yield (mg/L); b is the shake flask fermentation result containing the three-plasmid genetic engineering bacteria, the abscissa is fermentation time (h), and the ordinate (left) is OD₆₀₀The 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, E.coli BL21(DE3), competent cells, primers, reagents and the like used in the examples were 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 #: FD 0083.

Restriction enzyme XhoI was purchased from Thermo Scientific, cat #: FD 0694.

T₄Ligase purchaseBought from NEB, cat number: M0202S.

The primers were synthesized from: qingdao Zhixi Biotechnology Limited.

Coli Top10, purchased from Huada Gene.

Cm^rRepresents chloramphenicol resistance; kan^rRepresents kanamycin resistance; amp^rRepresents ampicillin. The strain provided by the invention is Escherichia coli (Escherichia coli) BL21(DE3), the contained plasmids are pACYC-mvaE-mvaS-ispA-AaFS, pTrc-low and pET28a-AaFS-ispA-idi, and the plasmids are respectively constructed by 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) Plasmid pACYC-mvaE-mvaS-ispA-AaFS is constructed by optimizing the codon preference of escherichia coli on β -farnesene synthetase AaFS gene sequence from sweet wormwood, respectively adding restriction enzyme BglII and XhoI restriction sites at two ends, synthesizing the sequence from Huada gene, wherein the synthesized sequence is shown as SEQ ID No. 1, cloning AaFS gene with BglII and XhoI restriction sites after synthesis to pUC57-simple vector, and obtaining plasmid by enzyme digestion-connection method according to the instruction of the vector use, wherein the plasmid is marked as pUC57-AaFS, the plasmid pACYC-mvaE-mvaS-ispA-FS (shown as a in the vector map 2) is constructed by adopting enzyme digestion-connection method, firstly, and the restriction enzyme BglII and XhoI are respectively used for carrying out double digestion on plasmid pACYC-mvaE-pUCA-pUCa and ISpA 57-FS, and 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 DH5 α competent cells and plated with LB Cm^rPlates were incubated overnight at 37 ℃. Observing the colony condition on the plate the next day, selecting single bacteria to fall into a liquid culture medium, culturing at 37 ℃ until the single bacteria are concentrated, carrying out colony 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 figure 2) by Gibson Assembly method, firstly, pET28a vector and three gene fragments of AaFS, ispA and idi are amplified, and pET28a plasmid, pUC57-AaFS plasmid, pACYC-mvaE-mvaS-ispA plasmid and Escherichia coli BL21(DE3) bacterial liquid are respectively used 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 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.

Subjecting the PCR product to agarose gel electrophoresis and target band gel cutting recovery, determining the concentration of gel recovery product, performing Gibson assembly using NEBuilder kit, calculating the proportion of fragments and the amount of each component according to the instruction, performing ligation reaction at 50 deg.C for 60min, diluting the product with equal volume of sterile water, taking 5 μ L of heat shock-transformed DH5 α competent cells and coating LBKan^rPlate, incubated at 37 deg.CAnd (4) at night. Observing the colony condition on the plate the next day, selecting single bacteria to fall into a liquid culture medium, culturing at 37 ℃ until the single bacteria are concentrated, 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(DE3) competent cells are transformed by thermally shocking plasmids pACYC-mvaE-mvaS-ispA-AaFS, pTrc-low and pET28a-AaFS-ispA-idi (triplasmid) with correct sequencing, corresponding triple-antibody (Cm, Amp and Kan) LB culture medium plates are coated, wherein the final concentration of Cm in LB culture medium is 34mg/L, the final concentration of Amp in LB culture medium is 100mg/L, and the final concentration of Kan in LB culture medium is 50mg/L, and the E.coli is cultured at 37 ℃ until a single colony grows out to obtain the farnesene-synthesizing genetically engineered bacteria.

In the embodiment, recombinant bacteria of genetically engineered bacteria over-expressing 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-diphospho decarboxylase ERG19 gene, β -farnesene synthetase AaFS gene, isopentenyl diphosphate isomerase idi gene and farnesyl diphosphate synthetase ispA gene are obtained, wherein the copy number of the β -farnesene synthetase AaFS gene, idi gene and ispA gene is 2.

Comparative example 1. example 1 was repeated, except that in this comparative example, in step 3), E.coli BL21(DE3) competent cells were transformed by heat shock using plasmids pACYC-mvaE-mvaS-ispA-AaFS and pTrc-low (two plasmids in total) whose sequencing was correct, and LB medium plates were plated 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 quantitative detection of farnesene is carried out by using gas chromatography, a chromatographic column is an Agilent DB-5MS (30m multiplied by 0.25mm multiplied by 0.25 mu m) capillary column, and the temperature rise program of the column is that the initial temperature is kept at 60 ℃ for 0.75min, the temperature is raised to 180 ℃ at the speed of 10 ℃/min, then the temperature is lowered to the initial temperature, and β -farnesene standard substance is used for making a standard curve for quantification.

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 K₂HPO₄5g/L beef extract, 0.3g/L ferric ammonium citrate, 2.1g/L citric acid monohydrate, 0.06g/L MgSO₄1mL/L of a trace element solution containing (NH)₄)₆Mo₇O₂₄·4H₂O 0.37g/L、ZnSO₄·7H₂O 0.29g/L、H₃BO₃2.47g/L、CuSO₄·5H₂O0.25g/L and MnCl₂·4H₂O1.58g/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 trace element solution is 1.5 mL/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 a 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 the primary seed solution 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 ℃ until OD is achieved₆₀₀Adding IPTG (isopropyl thiogalactoside) with a final concentration of 0.1mM and 10mL of n-dodecane into the mixture at about 0.6-0.8, performing shake culture at 30 ℃, sampling for 24h, 48h and 72h respectively, and measuring OD (optical density) by a spectrophotometer₆₀₀The fermentation product is β -farnesene identified by gas chromatography-mass spectrometry, and the farnesene yield is determined by gas chromatography and three repetitions are set.

Control group: the one obtained in comparative example 1 was picked upSingle colony is subjected to shake flask fermentation to synthesize farnesene, sampling for 24h, 48h and 72h respectively, and measuring OD with spectrophotometer₆₀₀Farnesene production was determined by gas chromatography in triplicate.

As shown in the attached figure 3, at the shake flask fermentation level, when the engineering bacteria containing two plasmids in the comparative example 1 is fermented for 48 hours, the farnesene yield is only at the milligram level and is close to 6mg/L (shown as a in the attached figure 3), while the engineering bacteria containing three plasmids (the copy numbers of β -farnesene synthetase AaFS gene, idi gene and ispA gene in the engineering bacteria are all 2) constructed by the invention 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 as b in the attached 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: picking the single colony obtained in the step 3) in the embodiment 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-level 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). Observing whether the dissolved oxygen and the pH value are normal or not, and if foam exists, adding a defoaming agent dropwise. OD was measured every three hours₆₀₀OD after about 9 hours₆₀₀When 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 cultured 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. Every 24 th hourThe yield (aqueous phase, organic phase, tail gas) was measured at one time. 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 in 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

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<170>PatentIn version 3.5

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<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

1. A genetically engineered bacterium for synthesizing farnesene is characterized in that the genetically engineered bacterium is a recombinant bacterium for over-expressing acetyl Co-A acyltransferase/HMG-CoA reductase mvaE gene, HMG-CoA synthetase mvaS gene, mevalonate-5-phosphate kinase ERG8, mevalonate kinase ERG12, mevalonate-5-diphosphate decarboxylase ERG19, β -farnesene synthetase AaFS gene, isopentenyl diphosphate isomerase idi gene and farnesyl diphosphate synthetase ispA gene, the copy number of the β -farnesene synthetase AaFS gene, the idi gene and the ispA gene is2, and the starting strain is escherichia coli.

2. The genetically engineered bacterium for synthesizing farnesene according to claim 1, wherein the β -farnesene synthetase AaFS gene is obtained by optimizing the codon preference of escherichia coli, the optimized nucleotide sequence is shown as SEQ ID No. 1, the ispA gene nucleotide sequence is shown as SEQ ID No. 2, and the idi gene nucleotide sequence is shown as SEQ ID No. 3.

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

4. The method for constructing genetically engineered bacteria for synthesizing farnesene according to any one of claims 1 to 3, which comprises the following steps:

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 in SEQ ID No. 4;

5. The method for constructing genetically engineered bacteria for synthesizing farnesene according to claim 4, wherein restriction enzyme BglII and XhoI cleavage sites are respectively added to two ends of the gene after the gene of β -farnesene synthetase AaFS is optimized in step 1), the gene is synthesized and cloned to a pUC57-simple vector to obtain a plasmid pUC57-AaFS, the restriction enzyme BglII and XhoI double-cleavage is carried out on the plasmid AaFS, an AaFS gene cleavage product is recovered, a 8260bp fragment is recovered from CYpAC-mvaE-mvaS-ispA, and the two cleavage recovery fragments are subjected to T double-cleavage₄And connecting the DNA ligase to obtain a plasmid pACYC-mvaE-mvaS-ispA-AaFS.

6. The method for constructing genetically engineered bacteria for synthesizing farnesene according to claim 4, wherein the upstream primer sequence for amplifying the pET28a vector sequence 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.

7. The use of the genetically engineered bacteria of any one of claims 1 to 3 in the synthesis of farnesene.

8. The application of the genetically engineered bacteria in the synthesis of farnesene according to claim 7, wherein the genetically engineered bacteria are cultured in a primary seed culture medium, and the 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.

9. The use of the synthetic farnesene according to claim 8, wherein 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.

10. The use of the synthetic farnesene according to claim 8, wherein the secondary seed medium comprises: 20g/L glucose, 9.8g/L K₂HPO₄5g/L beef extract, 0.3g/L ferric ammonium citrate, 2.1g/L citric acid monohydrate, 0.06g/L MgSO₄1mL/L of a trace element solution containing (NH)₄)₆Mo₇O₂₄·4H₂O0.37g/L、ZnSO₄·7H₂O 0.29g/L、H₃BO₃2.47g/L、CuSO₄·5H₂O0.25g/L and MnCl₂·4H₂O1.58g/L; fermentation cultureThe nutrient 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 1.5 mL/L.