CN111363709A - Genetic engineering bacterium for improving isoprene yield and construction method and application thereof - Google Patents
Genetic engineering bacterium for improving isoprene yield and construction method and application thereof Download PDFInfo
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Abstract
A genetic engineering bacterium for improving isoprene yield and a construction method and application thereof belong to the technical field of genetic engineering. In order to improve the output of isoprene synthesized by a biological method and reduce the toxicity of intermediate products to thalli, the invention provides a genetic engineering bacterium for improving the output of isoprene; the genetic engineering bacteria over-expression contains D18 packageMevalonate kinase gene ERG12 having signal peptide sequence, phosphomevalonate kinase gene ERG8 having signal peptide sequence wrapped by D18, mevalonate pyrophosphate decarboxylase gene ERG19 having signal peptide sequence wrapped by D18, isopentenyl pyrophosphate isomerase gene IDI1 having signal peptide sequence wrapped by D18, and gene IspS having isoprene synthase having signal peptide sequence wrapped by D18paAnd a recombinant plasmid containing Pdu A, Pdu B, Pdu J, Pdu K, Pdu N and Pdu gene cluster, wherein the starting strain is escherichia coli. The genetic engineering bacteria can be used for producing isoprene through fermentation.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a genetic engineering bacterium for improving isoprene yield, and a construction method and application thereof.
Background
Isoprene (isoprene) is an important chemical raw material and a platform compound, and is mainly used for synthesizing rubber. In addition, the isoprene-containing compound is also used for synthesizing other isoprene-containing compounds and derivatives thereof in fine chemical engineering, the application covers the fields of medicines, pesticides, spices, biofuels and the like, and the worldwide demand reaches millions of tons every year. Isoprene is currently produced primarily by chemical means from petroleum-based feedstocks. However, with the increasing exhaustion of fossil resources and the rising price, raw material problems and environmental problems have become important bottlenecks in the production of isoprene. Therefore, the search for new production routes of isoprene is an important development trend of industrial production of isoprene in the future.
At present, it has become possible to produce isoprene using engineered microorganisms such as E.coli, yeast, etc. However, there are two problems to be solved in the biological synthesis of isoprene: firstly, the exogenous MVA pathway is longer, the catalytic efficiency of the MVA downstream pathway is low, for example, the yield of an intermediate product mevalonic acid in the current MVA pathway can reach 84g/L, the yield of isoprene is only 28% of the theoretical conversion rate, and the promotion of the catalytic efficiency is not obvious by screening a new source of enzyme or performing directional modification on the existing enzyme; secondly, some intermediate products such as isopentenyl pyrophosphate (IPP) and dimethylallyl Diphosphate (DMAPP) are toxic to the bacteria and affect the normal growth of the bacteria.
Disclosure of Invention
In order to improve the output of isoprene synthesized by a biological method and reduce the toxicity of intermediate products to thalli, the invention provides a genetic engineering bacterium for improving the output of isoprene; the genetically engineered bacteria overexpress mevalonate kinase gene ERG12 containing a D18 wrapping signal peptide sequence, phosphomevalonate kinase gene ERG8 containing a D18 wrapping signal peptide sequence, mevalonate pyrophosphate decarboxylase gene ERG19 containing a D18 wrapping signal peptide sequence, isopentenyl pyrophosphate isomerase gene IDI1 containing a D18 wrapping signal peptide sequence, and IspS gene of isoprene synthetase containing a D18 wrapping signal peptide sequencepaAnd a recombinant plasmid containing Pdu A, Pdu B, Pdu J, Pdu K, Pdu N and Pdu gene cluster, wherein the starting strain is escherichia coli.
Further limited, the amino acid sequence of the D18 wrapping signal peptide is shown in SEQ ID NO: 1 is shown.
More particularly, the mevalonate kinase gene ERG12 is derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) ERG12, GeneBank accession No. 855248; the phosphomevalonate kinase gene ERG8 is derived from Saccharomyces cerevisiae ERG8, GeneBank accession number is 855260; the mevalonate pyrophosphate decarboxylase gene ERG19 is derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) and ERG19, and GeneBank accession number is 100195467; the isopentenyl pyrophosphate isomerase gene is an isopentenyl pyrophosphate isomerase gene IDI1 derived from saccharomyces cerevisiae (Saccharomyces cerevisiae), and the GeneBank accession number is 855986; the isoprene synthase gene IspSpaThe isoprene synthase gene IspS derived from Populus alba (Pop. mu. Lus alba)paGeneBank accession number AB198180, and the recombinant plasmid containing the PduA, PduB, PduJ, PduK, PduN and PduU gene clusters is pLysS-PduABJKNU.
The invention also provides a construction method of the genetic engineering bacteria, which comprises the following steps:
1) construction of the first recombinant plasmid: respectively adding D18 wrapping signal peptide sequences to the upstream of a mevalonate kinase gene ERG12, a phosphomevalonate kinase gene ERG8, a mevalonate pyrophosphate decarboxylase gene ERG19 and an isopentenyl pyrophosphate isomerase gene IDI1 to obtain a mevalonate kinase gene ERG12 containing a D18 wrapping signal peptide sequence, a phosphomevalonate kinase gene ERG8 containing a D18 wrapping signal peptide sequence, a mevalonate pyrophosphate decarboxylase gene ERG19 containing a D18 wrapping signal peptide sequence and an isopentenyl pyrophosphate isomerase gene IDI1 containing a D18 wrapping signal peptide sequence, and then connecting the genes to a plasmid pETduet-1 to obtain a first recombinant plasmid pETduet-D18-IDI1-D18-ERG19-D18-ERG8-D18-ERG 12;
2) second recombinant plasmid construction: the IspS gene containing the isoprene synthase ligated to the plasmid pYJM21 with the sequence of the packaging signal peptide D18paUpstream of the gene, a second recombinant plasmid pYJM21-D18 was obtained;
3) introducing the first recombinant plasmid obtained in the step 1), the second recombinant plasmid obtained in the step 2) and a recombinant plasmid pLysS-PduABJKNU containing gene clusters of PduA, PduB, PduJ, PduK, PduN and PduU into an escherichia coli competent cell to obtain a genetically engineered bacterium.
Further defined, the mevalonate kinase gene ERG12, phosphomevalonate kinase gene ERG8, mevalonate pyrophosphate decarboxylase gene ERG19 and isopentenyl pyrophosphate isomerase gene IDI1 of step 1) are obtained by chemical synthesis, amplification from a microorganism or amplification from a recombinant plasmid.
Further limiting, the construction of the first recombinant plasmid in the step 1) is to obtain a mevalonate kinase gene ERG12, a phosphomevalonate kinase gene ERG8, a mevalonate pyrophosphate decarboxylase gene ERG19 and an isopentenyl pyrophosphate isomerase gene IDI1 by PCR amplification with pYJM14 plasmid as a template;
the PCR products of the above genes are cut by restriction enzyme and inserted into the downstream of D18 sequence in plasmid pET14b-D18-GFP-SsrP vector respectively to obtain intermediate vector pET14b-D18-ERG12 of mevalonate kinase gene ERG12 containing D18 wrapping signal peptide sequence, intermediate vector pET14b-D18-ERG8 of phosphomevalonate kinase gene ERG8 containing D18 wrapping signal peptide sequence, intermediate vector pET14b-D18-ERG19 of mevalonate pyrophosphate decarboxylase gene ERG19 containing D18 wrapping signal peptide sequence and intermediate vector pET14b-D18-IDI1 of isopentenyl pyrophosphate isomerase gene IDI1 containing D18 wrapping signal peptide sequence;
then, taking pET14b-D18-ERG12 as a template, and carrying out PCR amplification to obtain a mevalonate kinase gene ERG12 containing a D18 wrapped signal peptide sequence;
using pET14b-D18-ERG8 as a template, and carrying out PCR amplification to obtain a phosphomevalonate kinase gene ERG8 containing a D18 wrapped signal peptide sequence;
performing PCR amplification by using pET14b-D18-ERG19 as a template to obtain a mevalonate pyrophosphate decarboxylase gene ERG19 containing a D18 wrapping signal peptide sequence;
the isopentenyl pyrophosphate isomerase gene IDI1 containing a signal peptide sequence wrapped by D18 is obtained by PCR amplification by taking pET14b-D18-IDI as a template, and then each gene containing the signal peptide sequence wrapped by D18 is connected to pETDuet-1 plasmid In sequence by an enzyme digestion connection or In-Fusion cloning method to obtain pETduet-D18-IDI1-D18-ERG19-D18-ERG8-D18-ERG 12.
More specifically, the nucleotide sequence of the primer used for amplifying the mevalonate kinase gene ERG12 in the step 1) is shown as SEQ ID NO: 2 and SEQ ID NO: 3 is shown in the specification; the nucleotide sequence of a primer used for amplifying the phosphomevalonate kinase gene ERG8 is shown as SEQ ID NO: 4 and SEQ ID NO: 5 is shown in the specification; the nucleotide sequence of a primer used for amplifying the mevalonate pyrophosphate decarboxylase gene ERG19 is shown as SEQ ID NO: 6 and SEQ ID NO: 7 is shown in the specification; the nucleotide sequence of a primer used for amplifying the isopentenyl pyrophosphate isomerase gene IDI1 is shown as SEQ ID NO: 8 and SEQ ID NO: 9 is shown in the figure;
the nucleotide sequence of a primer used for amplifying the mevalonate kinase gene ERG12 containing a D18 wrapping signal peptide sequence is shown as SEQ ID NO: 10 and SEQ ID NO: 11 is shown in the figure;
the nucleotide sequence of a primer used for amplifying the phosphomevalonate kinase gene ERG8 containing a D18 wrapping signal peptide sequence is shown as SEQ ID NO: 12 and SEQ ID NO: 13 is shown in the figure;
the nucleotide sequence of a primer used for amplifying the mevalonate pyrophosphate decarboxylase gene ERG19 containing a D18 wrapping signal peptide sequence is shown as SEQ ID NO: 14 and SEQ ID NO: 15 is shown in the figure;
the nucleotide sequence of a primer used for amplifying isopentenyl pyrophosphate isomerase gene IDI1 containing a D18 wrapping signal peptide sequence is shown as SEQ ID NO: 16 and SEQ ID NO: shown at 17.
The invention also provides application of the genetic engineering bacteria in fermentation production of isoprene.
Further limited, the gene engineering bacteria are utilized to produce isoprene through in vitro enzyme catalysis reaction: preparing a protein small body by the genetic engineering bacteria through induction expression, adding the protein small body into an in vitro enzyme catalysis reaction system, and incubating to prepare isoprene; the in-vitro enzyme catalysis reaction system contains the following components in each 2mL system: 10mM PBS buffer pH7.4, 4mM ATP, 30mM KCl, 8mM MgCl2、0.2mM MnCl2、0.01mM ZnSO44mM β -mercaptoethanol and 1mL of 0.25mM mevalonate, 0.2g/L protein bodies 100. mu.L.
More particularly, the method of making the proteosome is: culturing the genetically engineered bacteria to OD600After the concentration is 0.6-1.0, the bacterial liquid is induced and expressed for 9 hours by IPTG, then the bacterial liquid is centrifuged, and bacterial precipitation obtained by every 100mL of strain culture liquid is centrifuged to obtain a protein corpuscle by 15mL of lysis solution BPER-II, 1mL of lysozyme, 100 muold bacterial protease inhibitor, 1 muL of DNase I and 5.2 muL of β -mercaptoethanol with the final concentration of 4mM at the room temperature of 60rpm for 30 min.
Advantageous effects
BMCs are complex protein-based subcellular organelles that are composed of proteins, are selectively permeable, and contain small uncharged molecules such as acetaldehyde and CO2、O2Large molecules such as coenzyme B12NADPH, ATP, etc., can freely pass through. The invention utilizes BMCs to integrate the MVA downstream related pathway gene and recombinant plasmids containing Pdu A, Pdu B, Pdu J, Pdu K, Pdu N and Pdu gene clusters into escherichia coli to construct genetic engineering bacteria, the BPER-II is utilized to break the bacteria in an in vitro reaction, then the nano bioreactor containing isoprene downstream pathway is obtained by ultra-high speed centrifugation, and reaction substrate mevalonic acid solution is added into the reactor liquidThe method provided by the invention realizes the synthesis of isoprene by utilizing mevalonic acid in vitro through a reactor, and an in vitro enzyme activity detection experiment proves that the gene engineering bacteria constructed by the invention can produce isoprene through in vitro reaction, thereby reducing the influence of toxic metabolic intermediate products on the growth of bacteria and realizing the construction of an isoprene protein synthesis machine in vitro.
In shake flask fermentation experiments, isoprene production was five to six fold higher for all strains expressing the fused D18-tagged metabolic pathway (regardless of whether PDU coat protein was expressed) compared to the control strain expressing the fused His-tagged metabolic pathway.
Drawings
FIG. 1 is a schematic diagram of the construction process of an isoprene synthesis pathway expression vector with a signal peptide wrapped by D18 fused to the N-terminal of a wrapping protein.
FIG. 2 shows a plasmid map for expression of the PDU coat protein encoding gene and the D18 or His tag-added isoprene synthesis pathway gene.
FIG. 3 SDS-PAGE of purified samples of MVA downstream pathway gene alone or co-expressed with PDU fused with D18 encapsulated signal peptide. Wherein M is protein marker (kD); PET-D18-low is pETDuet-D18-lower plasmid; the Pdu is pLysS-Pdu plasmid.
FIG. 4 is an electron microscope observation of protein bodies, wherein A is a control group of Escherichia coli containing pLysS plasmid, and B is a control group of Escherichia coli containing pLysS-PduABJKNU plasmid; c is an experimental group, the genetic engineering bacteria constructed by the invention contains three recombinant plasmids of pYJM21-D18, pLOW8 and pLysS-PduABJKNU.
FIG. 5 is an in vitro enzyme activity assay of BMCs purified by adding mevalonate as a substrate, wherein "+" represents that the plasmid is transformed into a recipient strain and "-" represents that the plasmid is not transformed into the corresponding strain; pLysS-Pdu represents pLysS-PduABJKNU plasmid; d18-low (pETduet-D18-IDI-D18-ERG19-D18-ERG8-D18-ERG12) represents the first recombinant plasmid; D18-IspSpaRepresents a second recombinant plasmid; his-low (pETduet-His-IDI-His-ERG19-His-ERG8-His-ERG12) represents the first recombinant control plasmid, His-IspSpa(pYJM21) represents a second recombinant control plasmid;coli BL 03, E.coli BL02, E.coli BL 01 represent the number of strains transformed with different plasmids, respectively, and the ordinate represents the isoprene yield in ug/L.
Fig. 6 detection of isoprene production by shake flask fermentation of e.coli strains containing different plasmids, wherein "+" represents that the plasmid was transformed into the recipient strain and "-" represents that the plasmid was not transformed into the corresponding strain; pLysS-Pdu represents pLysS-PduABJKNU plasmid; d18-low (pETduet-D18-IDI-D18-ERG19-D18-ERG8-D18-ERG12) represents the first recombinant plasmid; his-low (pETduet-His-IDI-His-ERG19-His-ERG8-His-ERG12) represents the first recombinant control plasmid, D18-IspSpaRepresents a second recombinant plasmid; His-IspSpa(pYJM21) represents a second recombinant control plasmid; the abscissa represents the number of strains transformed with different plasmids, respectively, and the ordinate represents the peak area of isoprene.
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.
The reagents, materials, methods and apparatus used in the following examples, unless otherwise specified, are conventional in the art and are commercially available to those skilled in the art.
The pLysS-PdiABJKNU, pET14b-D18-GFP-SsrP and pLysS plasmids described in the present invention are all described in Lee MJ, Brown I R, Juodeikis R, et al.
pYJM21 and pYJM14 are described in Yang J, Xiaoan M, Su S, et al.
The public is available from the national academy of sciences Qingdao institute of energy and processes.
pETduet-1 was purchased from Novagen.
The terms mentioned in the present invention are explained as follows:
a nano bioreactor: in the present invention meansThe recombinant plasmid pLysS-PduABJKNU containing PduA, PduB, PduJ, PduK, PduN and PduU gene clusters is expressed in colibacillus to form nanometer level protein corpuscle. The protein corpuscle is in a nanometer level and has a spatial structure, so the protein corpuscle can be used for a nanometer bioreactor. In this experiment, the recombinant plasmid pLysS-PduABJKNU was found to contain a mevalonate kinase gene ERG12 having a signal peptide sequence wrapped by D18, a phosphomevalonate kinase gene ERG8 having a signal peptide sequence wrapped by D18, a mevalonate pyrophosphate decarboxylase gene ERG19 having a signal peptide sequence wrapped by D18, an isopentenyl pyrophosphate isomerase gene IDI1 having a signal peptide sequence wrapped by D18, and an isoprene synthase gene IspS having a signal peptide sequence wrapped by D18paAfter the combination of the genes, the protein bodies were expressed in E.coli.
D18 encapsidation signal peptide: the amino acid sequence is MEINEKLLRQIIEDVLSEPMGSSHHHHHHS SGLVPRGSH, the first 18 amino acids are from Pdu, and have the function of signal peptide, the following "PMGSS" amino acid sequence as connecting chain, and the following "HHHHHHSSGLVPRGSH" amino acid sequence as His-tag.
Enzyme digestion connection: the method is a common experimental method in the vector construction technology, wherein the gene segment or the vector is cut by restriction endonuclease, and then the target gene segment is connected with the target vector through DNA ligase.
IPTG isopropyl- β -D-thiogalactoside.
In the following examples pETduet-D18-IDI1-D18-ERG19-D18-ERG8-D18-ERG12 is also called pLOW-8 or pETDuet-D18-lower or shortly D18-low.
pET-His-IDI1-His-ERG19-His-ERG12-His-ERG8 is also called pLOW-4 or pETDuet-His-lower, or simply His-low.
pYJM21-D18 is also called D18-IspSpaOr pACYC-mvaE-mvaS-D18-IspSpa。
pYJM21 also known as His-IspSpaOr pACYC-mvaE-mvaS-His-IspSpa。
Embodiment 1. construction method of genetic engineering bacteria for improving isoprene yield.
Increased isoprene production prepared in this exampleThe genetically engineered bacterium (a) overexpresses a mevalonate kinase gene ERG12 containing a signal peptide sequence wrapped by D18, a phosphomevalonate kinase gene ERG8 containing a signal peptide sequence wrapped by D18, a mevalonate pyrophosphate decarboxylase gene ERG19 containing a signal peptide sequence wrapped by D18, an isopentenyl pyrophosphate isomerase gene IDI1 containing a signal peptide sequence wrapped by D18, and an IspS gene for isoprene synthase containing a signal peptide sequence wrapped by D18paAnd a recombinant plasmid containing Pdu A, Pdu B, Pdu J, Pdu K, Pdu N and Pdu gene cluster, wherein the starting strain is escherichia coli. The amino acid sequence of the D18 wrapping signal peptide is shown in SEQ ID NO: 1 is shown. The mevalonate kinase gene ERG12 is derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) ERG12, and GeneBank accession number is 855248; the phosphomevalonate kinase gene ERG8 is derived from Saccharomyces cerevisiae ERG8, GeneBank accession number is 855260; the mevalonate pyrophosphate decarboxylase gene ERG19 is derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) and ERG19, and GeneBank accession number is 100195467; the isopentenyl pyrophosphate isomerase gene IDI1 is derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) IDI1, and GeneBank accession number is 855986; the isoprene synthase gene IspSpaThe gene IspSpa derived from Populus alba (Populus alba) isoprene synthase, GeneBank accession number AB198180, the recombinant plasmid containing PluA, PdiB, PdiJ, PdiK, PdiN and PdiU gene clusters is pLysS-PdiABJKNU. The construction method of the genetic engineering bacteria comprises the following steps:
(1) construction of the first recombinant plasmid: adding a sequence containing a D18 wrapping signal peptide to the upstream of a mevalonate kinase gene ERG12, a phosphomevalonate kinase gene ERG8, a mevalonate pyrophosphate decarboxylase gene ERG19 and an isopentenyl pyrophosphate isomerase gene IDI1 respectively to obtain a mevalonate kinase gene ERG12 containing a D18 wrapping signal peptide sequence, a phosphomevalonate kinase gene ERG8 containing a D18 wrapping signal peptide sequence, a mevalonate pyrophosphate decarboxylase gene ERG19 containing a D18 wrapping signal peptide sequence and an isopentenyl pyrophosphate isomerase gene IDI1 containing a D18 wrapping signal peptide sequence, and then connecting the genes to a plasmid pETduet-1 to obtain a first recombinant plasmid pETduet-D18-ID1I-D18-ERG19-D18-ERG8-D18-ERG 12.
The above genes are known genes, and the method for obtaining the gene is a PCR method using genome as template, or a direct gene chemical method, wherein the nucleotide sequence of D18 wrapping signal peptide sequence is not limited to the nucleotide sequence in the vector pET14b-D18-GFP-SsrP used in the following examples, and the nucleotide sequence coding for "MEINEKLLRQIIEDVLSEPMGSSHHHHHHSSGLVPRGSH" is used as the signal peptide sequence, and the above method belongs to a mature molecular biological method, and has no specificity.
The process of constructing the first recombinant plasmid (pLOW-8) containing D18-tagged mevalonate MVA downstream pathway-related genes is described below (the construction process is shown in FIG. 1, in which the IDI gene is the IDI1 gene).
a) Amplification of target Gene
Carrying out Polymerase Chain Reaction (PCR) by taking pYJM14 as a template and respectively using the following primers F and R to respectively amplify the fragments of the gene ERG12, the gene ERG8, the gene ERG19 and the gene IDI1, wherein the nucleotide sequence of the primer used for amplifying the mevalonate kinase gene ERG12 is shown as SEQ ID NO: 2 and SEQ ID NO: 3 is shown in the specification; the nucleotide sequence of a primer used for amplifying the phosphomevalonate kinase gene ERG8 is shown as SEQ ID NO: 4 and SEQ ID NO: 5 is shown in the specification; the nucleotide sequence of a primer used for amplifying the mevalonate pyrophosphate decarboxylase gene ERG19 is shown as SEQ ID NO: 6 and SEQ ID NO: 7 is shown in the specification; the nucleotide sequence of a primer used for amplifying the isopentenyl pyrophosphate isomerase gene IDI1 is shown as SEQ ID NO: 8 and SEQ ID NO: shown at 9.
The PCR amplification system is shown as follows:
the PCR procedure was 94 ℃ for 3min, 30 × (94 ℃ for 10s, 55 ℃ for 30s, 72 ℃ for 1min/kb), 72 ℃ for 5min, 4 ℃ ∞
The sequences of the primers are shown below:
TABLE 1 primer sequence Listing
The PCR product was recovered and purified by using a gel recovery and purification kit (Biyuntian, cat # D0056).
b) Intermediate support preparation
After obtaining each target gene, the obtained target gene was ligated to pET14b plasmid (shown in fig. 1) by using restriction enzymes Nde I and Spe I as a restriction enzyme, as follows:
pET14b-D18-GFP-SsrP was simultaneously double-digested with restriction enzyme 1(Nde I) and restriction enzyme 2(Spe I).
Plasmid and PCR product, the enzyme cutting system is:
the enzyme system was incubated at 37 ℃ for 1 h. Gel recovery and purification are carried out, DNA ligase is used for ligation, and a ligation system is shown as follows:
and (3) incubating the connection system at 22 ℃ for 30min, respectively transforming E.coli DH5 α competence by using the connection products, coating the competence on an LB solid plate containing 34mg/mL ampicillin, screening positive clones by PCR, respectively extracting recombinant plasmids pET14b-D18-ERG12, pET14b-D18-ERG8, pET14b-D18-ERG19 and pET14b-D18-IDI from the positive clones, and identifying by restriction enzyme cutting and sequencing.
c) pETduet-D18-IDI1-D18-ERG19-D18-ERG8-D18-ERG 12.
Using pET14b-D18-ERG12 as a template, and carrying out PCR amplification to obtain a mevalonate kinase gene ERG12 containing a D18 wrapped signal peptide sequence; the primer is pET-D18-ERG12-F, pET-ERG12-R, and the nucleotide sequence is shown as SEQ ID NO: 10 and SEQ ID NO: 11 is shown in the figure;
using pET14b-D18-ERG8 as a template, and carrying out PCR amplification to obtain a phosphomevalonate kinase gene ERG8 containing a D18 wrapped signal peptide sequence; the primer is pET-D18-ERG8-F, pET-ERG8-R nucleotide sequence shown in SEQ ID NO: 12 and SEQ ID NO: 13 is shown in the figure;
performing PCR amplification by using pET14b-D18-ERG19 as a template to obtain a mevalonate pyrophosphate decarboxylase gene ERG19 containing a D18 wrapping signal peptide sequence; the primer is pET-D18-ERG19-F, pET-ERG19-R nucleotide sequence shown in SEQ ID NO: 14 and SEQ ID NO: 15 is shown in the figure;
and (2) performing PCR amplification by taking pET14b-D18-IDI as a template to obtain an isopentenyl pyrophosphate isomerase gene IDI1 containing a D18 wrapping signal peptide sequence, wherein the used primer is pET-D18-IDI1-F, pET-IDI1-R, and the nucleotide sequence is shown as SEQ ID NO: 16 and SEQ ID NO: shown at 17.
Table 2 primer sequences are shown below:
the genes containing the signal peptide sequence wrapped by D18 were ligated to pETDuet-1 plasmid (as shown in FIG. 2) by enzyme digestion to obtain pETduet-D18-IDI1-D18-ERG19-D18-ERG8-D18-ERG 12. The specific method comprises the following steps:
① PCR product of isopentenyl pyrophosphate isomerase gene IDI1 containing D18 wrapped signal peptide sequence is cut by restriction enzyme NcoI and SacI and then is connected to pETDuet-1 fragment cut by restriction enzyme NcoI and SacI to obtain pETDuet-D18-IDI;
② contains D18 package signal peptide sequence mevalonate pyrophosphate decarboxylase gene ERG19 PCR products through SacI and SalI restriction enzyme digestion, connected to pETDuet-D18-IDI1 vector through SacI and SalI restriction enzyme digestion, obtain pETduet-D18-IDI1-D18-ERG 19;
③ contains D18 package signal peptide sequence phosphomevalonate kinase gene ERG8 PCR products through FseI and AvaI restriction endonuclease cut and connected to pETduet-D18-IDI1-D18-ERG19 carrier cut by FseI and AvaI restriction endonuclease cut, obtain pETduet-D18-IDI1-D18-ERG19-D18-ERG 8;
④ contains D18 package signal peptide sequence mevalonate kinase gene ERG12 PCR products through AvaI and PacI restriction enzyme cutting, connected to the same AvaI and PacI restriction enzyme cutting pETduet-D18-IDI1-D18-ERG19-D18-ERG8 carrier, to obtain pETduet-D18-IDI1-D18-ERG19-D18-ERG8-D18-ERG12, as the first recombinant plasmid, short pLOW-8.
The PCR product recovery, plasmid and PCR product enzyme digestion, connection system, method for transforming escherichia coli and positive clone screening method are the same as the above, and the positive clones obtained by screening in the above steps are identified by restriction enzyme digestion and sequencing.
2) Second recombinant plasmid construction: the IspS gene for isoprene synthase containing a signal peptide sequence wrapped with D18 can be constructed by reference to the procedure described in step 1paThe recombinant plasmid pET14b-D18-IspSpaThen the gene IspS of isoprene synthetase with a signal peptide sequence wrapped by D18 is subjected to enzyme digestion connection or Infusion methodpaThe gene was constructed into pYJM21 vector, and in this example, IspS, which is a gene for isoprene synthase and chemically synthesized in plasmid pYJM21, containing the signal peptide sequence of D18paUpstream of the gene, a second recombinant plasmid pYJM21-D18 was obtained, and its vector map was represented by pACYC-mvaE-mvaS-D18-IspS in FIG. 2paThis part is done by huada corporation.
3) Introducing the first recombinant plasmid obtained in the step 1), the second recombinant plasmid obtained in the step 2) and a recombinant plasmid pLysS-PduABJKNU containing gene clusters of PduA, PduB, PduJ, PduK, PduN and PduU into a competent cell of escherichia coli BL21(DE3) to obtain a genetically engineered bacterium.
The plasmid construction method relates to a PCR technology, a nucleic acid synthesis technology, a plasmid restriction enzyme cutting method, an enzyme section recovery method, an enzyme section connection method, an escherichia coli transformation method and the like, belongs to a mature molecular biology method and has no specificity if no special description exists.
Comparative example 1: constructing a His-tag-containing vector of the MVA downstream pathway-related gene.
pET-His-IDI1-His-ERG19-His-ERG12-His-ERG8(pLOW-4)。
The difference from example 1 is that the upstream of each over-expressed gene in the genetically engineered bacteria described in this comparative example is histidine-tag His-tag whose amino acid sequence is "HHHHHHSSGLVPRGSH".
1) Construction of plasmid pET-His-IDI1-His-ERG19-His-ERG12-His-ERG8(pLOW-4)
A) Preparation of target gene:
since the D18 tag contains a His tag, a method for obtaining a target gene is employed
The recombinant plasmids pET14b-D18-ERG12, pET14b-D18-ERG8, pET14b-D18-ERG19 and pET14b-D18-IDI1 are used as templates, and the following primers F and R are used for carrying out Polymerase Chain Reaction (PCR) to respectively amplify ERG12, ERG8, ERG19 and IDI1 fragments, wherein the PCR amplification system is shown as follows:
the PCR procedure was 94 ℃ for 3min, 30 × (94 ℃ for 10s, 55 ℃ for 30s, 72 ℃ for 1min/kb), 72 ℃ for 5min, 4 ℃ ∞
TABLE 3 primer sequences
B) Preparation of intermediate Carrier
The PCR product was recovered and purified by using a gel recovery and purification kit (Biyuntian, cat # D0056).
Plasmid and PCR product, the enzyme cutting system is:
the enzyme system was incubated at 37 ℃ for 1 h. Gel recovery and purification are carried out, DNA ligase is used for ligation, and a ligation system is shown as follows:
the ligation system was incubated at 22 ℃ for 30min, the ligation products were transformed into E.coli DH5 α competent, spread onto LB solid plates of 34mg/mL ampicillin, positive clones were screened by PCR, and recombinant plasmids pET-His-IDI1, pET-His-IDI1-His-ERG19, pET-His-IDI1-His-ERG19-His-ERG12, pET-His-IDI1-His-ERG19-His-ERG12-His-ERG8(pLOW-4) were extracted from the positive clones and identified by restriction enzyme digestion and sequencing (FIG. 2).
C) pETduet-His-IDI-His-ERG19-His-ERG8-His-ERG 12.
Using pET14b-D18-ERG12 as a template, and obtaining a mevalonate kinase gene ERG12 containing a His sequence through PCR amplification; the primer is pET-ERG12-F, pET-ERG12-R, and the nucleotide sequence is shown in SEQ ID NO: 18 and SEQ ID NO: 11 is shown in the figure;
using pET14b-D18-ERG8 as a template, and obtaining a phosphomevalonate kinase gene ERG8 containing a His sequence through PCR amplification; the primer is pET-ERG8-F, pET-ERG8-R nucleotide sequence shown as SEQ ID NO: 19 and SEQ ID NO: 13 is shown in the figure;
performing PCR amplification by using pET14b-D18-ERG19 as a template to obtain a mevalonate pyrophosphate decarboxylase gene ERG19 containing a His sequence; the primer is pET-ERG19-F, pET-ERG19-R nucleotide sequence shown as SEQ ID NO: 20 and SEQ ID NO: 15 is shown in the figure;
and (2) performing PCR amplification by taking pET14b-D18-IDI1 as a template to obtain isopentenyl pyrophosphate isomerase gene IDI1 containing a His sequence, wherein the used primer is pET-IDI-F, pET-IDI-R, and the nucleotide sequence is shown as SEQ ID NO: 21 and SEQ ID NO: shown at 17.
Each of the above genes containing His sequence was ligated to pETDuet-1 plasmid (as shown in FIG. 2) in sequence by enzyme digestion to obtain pETduet-His-IDI1-His-ERG19-His-ERG8-His-ERG 12. The specific method comprises the following steps:
① the PCR product of isopentenyl pyrophosphate isomerase gene IDI containing His sequence is cut by NcoI and SacI restriction endonuclease and then is connected to pETDuet-1 fragment cut by NcoI and SacI restriction endonuclease to obtain pETDuet-His-IDI 1;
② the PCR product of mevalonate pyrophosphate decarboxylase gene ERG19 containing His sequence is cut by SacI and SalI restriction enzymes and then connected to pETDuet-His-IDI1 carrier cut by SacI and SalI restriction enzymes to obtain pETduet-His-IDI1-His-ERG 19;
③ the PCR product of phosphomevalonate kinase gene ERG8 containing His sequence is cut by FseI and AvaI restriction endonuclease and then connected to pETduet-His-IDI-His-ERG19 carrier cut by FseI and AvaI restriction endonuclease to obtain pETduet-His-IDI1-His-ERG19-His-ERG 8;
④ contains the sequence of His mevalonate kinase gene ERG12 PCR products through AvaI and PacI restriction endonuclease were connected to pETduet-His-IDI1-His-ERG19-His-ERG8 vector through AvaI and PacI restriction endonuclease to obtain pETduet-His-IDI1-His-ERG19-His-ERG8-His-ERG12, short for pLOW-4.
The PCR product recovery, plasmid and PCR product enzyme digestion, connection system, method for transforming escherichia coli and positive clone screening method are the same as the above, and the positive clones obtained by screening in the above steps are identified by restriction enzyme digestion and sequencing.
2) Introducing the pLOW-4 recombinant plasmid obtained in the step 1), the pYJM21 plasmid and the recombinant plasmid pLysS-PduABJKNU containing the gene clusters of PduA, PduB, PduJ, PduK, PduN and PduU into a competent cell of escherichia coli BL21(DE3) to obtain a genetically engineered bacterium.
The plasmid construction method relates to a PCR technology, a nucleic acid synthesis technology, a plasmid restriction enzyme cutting method, an enzyme section recovery method, an enzyme section connection method, an escherichia coli transformation method and the like, belongs to a mature molecular biology method and has no specificity if no special description exists.
Example 2. application of the genetically engineered bacteria prepared in example 1 in fermentation production of isoprene.
This example illustrates that the genetically engineered bacterium prepared in example 1 is used to produce isoprene through an in vitro enzyme-catalyzed reaction, in which the genetically engineered bacterium is induced to express to prepare a proteosome, and then the proteosome is added to an in vitro enzyme-catalyzed reaction system and incubated at room temperature to prepare isoprene. The specific method comprises the following steps:
1. validation of protein bodies
(1) The recombinant E.coli constructed in example 1 was inoculated into 100mL of LB medium and cultured on a shaker at 37 ℃ and 180rpm to OD6000.6-1.0, adding inducer IPTG to a final concentration of 0.4mM, and culturing at 30 deg.C for 9 hr.
(2) The bacterial liquid was centrifuged at 5000rpm for 10min, washed twice with Buffer A, 10mL of Buffer A and 15mLBPER-II (1 mL of lysozyme, 100. mu.L of bacterial protease inhibitor, 1. mu.L of DNase I and 5.2. mu.L of β -mercaptoethanol (final concentration: 4mM)) were added thereto, centrifuged at room temperature for 60rpm, 30min, ice-cooled at 12000rpm for 1min, centrifuged at 20000rpm for 20min, and the precipitate was dissolved in Buffer B and stored at 4 ℃.
Buffer A：50mM Tris-HCl(pH8.0)、500mM KCl、12.5mM MgCl21.5% 1,2-PD (propylene glycol)
Buffer B：50mM Tris-HCl(pH8.0)、50mM KCl、5mM MgCl21% 1,2-PD (propylene glycol)
(3)SDS-PAGE
After 9h of induction expression, the thalli are collected by centrifugation, ultrasonic waves and lysate BPER-II are respectively used for treating and breaking the thalli, and purified BMCs or corresponding control samples are obtained by centrifugal cell debris removal and ultracentrifugation. The samples were subjected to SDS-PAGE and Coomassie blue staining, and the molecular weight and the electrophoretic results in the literature compared prove that the PDU coat protein and the downstream pathways of MVA, namely ERG8, ERG12, ERG19 and IDII are expressed, and the specific results are shown in FIG. 3.
(4) Treatment of electron microscopy
Approximately 1.5mL of the cells were collected on a 0.05-0.1 scale from EP tube, 500mL of 2.5% glutaraldehyde was added, the pipette was blown evenly, and the cell was refrigerated overnight at 4 ℃. And centrifuging for 2min at 1000g to collect thalli, discarding supernatant, washing with PBS for three times, wherein each time is half an hour, and uniformly blowing cells during each washing. Osmate 1% was added, fixed for 70min, and then washed three times with PBS, half an hour each time. Acetone dehydration is carried out according to the gradient of 30 percent, 50 percent, 70 percent, 80 percent, 90 percent and 95 percent of volume fraction, and then pure acetone dehydration is carried out for three times, wherein each dehydration lasts for 20 min. A mixture was prepared according to the ratio of 7 volumes of acetone to 3 volumes of Spurr resin, and added to the cells for 10 hours of permeation. A mixture was prepared by mixing 3 volumes of acetone and 7 volumes of Spurr resin, and the mixture was added to the cells and allowed to permeate for 10 hours. The pure sprr resin was allowed to permeate overnight. And (3) placing the thalli into an embedding mold, adding Spurr resin, and placing in a 65-degree oven for 24 hours. After the resin is trimmed, the resin is sliced by an ultrathin slicer, and observed by an electron microscope after dyeing. As observed by TEM, we found bacterial micro-chamber BMCs in the experimental group similar to the control strain (pLysS-PduABJKNU). The specific results are shown in FIG. 4.
2. Detection of in vitro enzyme Activity
(1) The recombinant E.coli constructed in example 1 was inoculated into 100mL of LB medium and cultured on a shaker at 37 ℃ and 180rpm to OD6000.6-1.0, adding inducer IPTG to a final concentration of 0.4mM, and culturing at 30 deg.C for 9 hr.
(2) The bacterial liquid was centrifuged at 5000rpm for 10min, washed twice with Buffer A, 10mL of Buffer A and 15mLBPER-II (1 mL of lysozyme, 100. mu.L of bacterial protease inhibitor, 1. mu.L of DNase I and 5.2. mu.L of β -mercaptoethanol (final concentration: 4mM)) were added thereto, centrifuged at room temperature for 60rpm, 30min, ice-cooled at 12000rpm for 1min, centrifuged at 20000rpm for 20min, and the precipitate was dissolved in Buffer B and stored at 4 ℃.
(3) Preparation of mevalonate
0.5mM KOH solution and 0.5mM mevalonolactone in a volume ratio of 1.05: 1, at 37 ℃ for 30min, and then bathing at 30 ℃ for 4h to obtain 0.25mM mevalonic acid.
(4) In vitro enzyme reaction
In vitro enzyme activation reaction was carried out in a stoppered glass vial, reaction system (2 mL): 50mM phosphate buffer (pH7.4),4mM ATP, 30mM KCl, 8mM MgCl2、0.2mM MnCl2、0.01mM ZnSO44mM β -mercaptoethanol, 2.5mM mevalonic acid, and 100. mu.L recombinant protein corpuscle (0.2 g/L). after reaction at room temperature for three hours, 1mL headspace gas was taken for gas chromatographic analysis, and the amount was determined by using the standard as the standard curve, and setting the standard curveThree replicates were placed.
The isoprene nano bioreactor (namely the protein corpuscle prepared by the gene engineering bacteria constructed by the invention) generates 500ug/L of isoprene in vitro reaction, thereby confirming the successful construction of the isoprene nano bioreactor. The specific results are shown in FIG. 5. Therefore, the invention uses the protein bodies as a nano reactor, wraps the signal peptide through D18, and successfully prepares mevalonate pyrophosphate decarboxylase gene ERG19, mevalonate kinase gene ERG12, phosphomevalonate kinase gene ERG8, isopentenyl pyrophosphate isomerase gene IDI1 and isoprene synthase gene IspSpaThe gene expressed protein is gathered in the nano reactor, and the concentration of the substrate, the enzyme and the cofactor is concentrated, so that the catalytic efficiency is improved. This study also demonstrated that small molecules such as isoprene, ATP, mevalonate can freely pass through the protein bodies. In addition, some intermediates in the isoprene metabolic pathway, such as isopentenyl pyrophosphate (IPP), dimethylallyl Diphosphate (DMAPP), etc., are toxic to the bacterial cells and can affect the normal growth of the bacterial cells. If the problem cannot be effectively solved, the further improvement of the yield of the isoprene is influenced, and the industrial process of synthesizing the isoprene by a biological method is hindered. The protein corpuscle can be used as a nano reactor, and can isolate the damage of toxic intermediate products to host cells by wrapping a metabolic pathway.
Example 3. application of the genetically engineered bacteria prepared in example 1 in the production of isoprene by fermentation.
This example illustrates the synthesis of isoprene by shake flask fermentation in genetically engineered bacteria obtained by the preparation.
And (3) mixing the activated engineering escherichia coli according to the proportion of 1: 100 percent of the total weight600When the concentration is 0.6-0.8, IPTG with the final concentration of 0.4mM is added into the bacterial liquid, and then the bacterial liquid is transferred to 30 ℃ and cultured continuously under the condition of 180 rpm. After the engineering strain is induced and cultured for 24 hours, taking 1mL headspace gas, and carrying out quantitative detection by using gas GC, wherein the detection method refers to Yang J, Xian M,Su S,Zhao G,Nie Q,JiangX,et al.Enhancing production of bio-isoprene using hybrid MVA pathway andisoprene synthase in E.coli.PLoS one.2012；7:e33509.。
through preliminary shake flask fermentation experiments, it was detected that all strains expressing the fused D18-tagged metabolic pathway (the strain constructed in example 1) (regardless of whether PDU coat protein is expressed) had five to six-fold increased isoprene production compared to the control expression fused His-tagged metabolic pathway (the strain constructed in comparative example 1), and the specific results are shown in FIG. 6.
As can be seen, the invention enables mevalonate pyrophosphate decarboxylase gene ERG19, mevalonate kinase gene ERG12, phosphomevalonate kinase gene ERG8, isopentenyl pyrophosphate isomerase gene IDI1 and isoprene synthase gene IspS by wrapping signal peptide with D18paThe protein expressed by the gene is gathered together, and the concentration of the substrate, the enzyme and the cofactor is improved, so that the catalytic efficiency is improved.
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.
Nucleotide sequence listing
<110> institute of bioenergy and Process in Qingdao, China academy of sciences
<120> genetic engineering bacterium for improving isoprene yield and construction method and application thereof
<130>
<160>22
<170>PatentIn version 3.5
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Met Glu Ile Asn Glu Lys Leu Leu Arg Gln Ile Ile Glu Asp Val Leu
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Leu Val Pro Arg Gly Ser His
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1 5 10 15
Claims (10)
1. A genetically engineered bacterium for improving the yield of isoprene is characterized in that the genetically engineered bacterium overexpresses a mevalonate kinase gene ERG12 containing a signal peptide sequence wrapped by D18, a phosphomevalonate kinase gene ERG8 containing a signal peptide sequence wrapped by D18, a mevalonate pyrophosphate decarboxylase gene ERG19 containing a signal peptide sequence wrapped by D18, an isopentenyl pyrophosphate isomerase gene IDI1 containing a signal peptide sequence wrapped by D18, and an isoprene synthetase gene IspS containing a signal peptide sequence wrapped by D18paAnd a recombinant plasmid containing Pdu A, Pdu B, Pdu J, Pdu K, Pdu N and Pdu gene cluster, wherein the starting strain is escherichia coli.
2. The genetically engineered bacterium of claim 1, wherein the amino acid sequence of the D18-wrapped signal peptide is as shown in SEQ ID NO: 1 is shown.
3. The genetically engineered bacterium of claim 1, wherein the mevalonate kinase gene ERG12 is mevalonate kinase gene ERG12 derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae), GeneBank accession No. 855248; the phosphomevalonate kinase gene ERG8 is derived from Saccharomyces cerevisiae ERG8, and GeneBank accession number is 855260; the mevalonate pyrophosphate decarboxylase gene ERG19 is derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) and ERG19, and GeneBank accession number is 100195467; the isopentenyl pyrophosphate isomerase gene IDI1 is derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) IDI1, and GeneBank accession number is 855986; the isoprene synthase gene IspSpaThe isoprene synthase gene IspS derived from Populus alba (Populus alba)paGeneBank accession number AB198180, and the recombinant plasmid containing the PduA, PduB, PduJ, PduK, PduN and PduU gene clusters is pLysS-PduABJKNU.
4. The method for constructing a genetically engineered bacterium according to any one of claims 1 to 3, comprising the steps of:
1) construction of the first recombinant plasmid: respectively adding D18 wrapping signal peptide sequences to the upstream of a mevalonate kinase gene ERG12, a phosphomevalonate kinase gene ERG8, a mevalonate pyrophosphate decarboxylase gene ERG19 and an isopentenyl pyrophosphate isomerase gene IDI1 to obtain a mevalonate kinase gene ERG12 containing a D18 wrapping signal peptide sequence, a phosphomevalonate kinase gene ERG8 containing a D18 wrapping signal peptide sequence, a mevalonate pyrophosphate decarboxylase gene ERG19 containing a D18 wrapping signal peptide sequence and an isopentenyl pyrophosphate isomerase gene IDI1 containing a D18 wrapping signal peptide sequence, and then connecting the genes to a plasmid pETduet-1 to obtain a first recombinant plasmid pETduet-D18-IDI1-D18-ERG19-D18-ERG8-D18-ERG 12;
2) second recombinant plasmid construction: the IspS gene containing the isoprene synthase ligated to the plasmid pYJM21 with the sequence of the packaging signal peptide D18paUpstream of the gene, a second recombinant plasmid pYJM21-D18 was obtained;
3) introducing the first recombinant plasmid obtained in the step 1), the second recombinant plasmid obtained in the step 2) and a recombinant plasmid pLysS-PduABJKNU containing gene clusters of PduA, PduB, PduJ, PduK, PduN and PduU into an escherichia coli competent cell to obtain a genetically engineered bacterium.
5. The method of constructing according to claim 4, wherein the mevalonate kinase gene ERG12, phosphomevalonate kinase gene ERG8, mevalonate pyrophosphate decarboxylase gene ERG19 and isopentenyl pyrophosphate isomerase gene IDI1 of step 1) are obtained by chemical synthesis, amplification from a microorganism or amplification from a recombinant plasmid.
6. The method for constructing the plasmid of claim 5, wherein the first recombinant plasmid in step 1) is constructed by using pYJM14 plasmid as a template and obtaining mevalonate kinase gene ERG12, phosphomevalonate kinase gene ERG8, mevalonate pyrophosphate decarboxylase gene ERG19 and isopentenyl pyrophosphate isomerase gene IDI1 through PCR amplification respectively;
the PCR products of the above genes are cut by restriction endonuclease, and inserted into the downstream of D18 sequence in plasmid pET14b-D18-GFP-SsrP vector respectively to obtain intermediate vector pET14b-D18-ERG12 of mevalonate kinase gene ERG12 containing D18 wrapping signal peptide sequence, intermediate vector pET14b-D18-ERG8 of phosphomevalonate kinase gene ERG8 containing D18 wrapping signal peptide sequence, intermediate vector pET14b-D18-ERG19 of mevalonate pyrophosphate decarboxylase gene ERG19 containing D18 wrapping signal peptide sequence and intermediate vector pET14b-D18-IDI1 of isopentenyl pyrophosphate isomerase gene IDI1 containing D18 wrapping signal peptide sequence;
then, taking pET14b-D18-ERG12 as a template, and carrying out PCR amplification to obtain a mevalonate kinase gene ERG12 containing a D18 wrapped signal peptide sequence;
using pET14b-D18-ERG8 as a template, and carrying out PCR amplification to obtain a phosphomevalonate kinase gene ERG8 containing a D18 wrapped signal peptide sequence;
performing PCR amplification by using pET14b-D18-ERG19 as a template to obtain a mevalonate pyrophosphate decarboxylase gene ERG19 containing a D18 wrapping signal peptide sequence;
the isopentenyl pyrophosphate isomerase gene IDI1 containing a signal peptide sequence wrapped by D18 is obtained by PCR amplification by taking pET14b-D18-IDI1 as a template, and then each gene containing the signal peptide sequence wrapped by D18 is connected to pETDuet-1 plasmid In sequence by an enzyme digestion connection or In-Fusion cloning method to obtain pETduet-D18-IDI1-D18-ERG19-D18-ERG8-D18-ERG 12.
7. The construction method according to claim 6, wherein the nucleotide sequence of the primer used for amplifying the mevalonate kinase gene ERG12 in step 1) is as shown in SEQ ID NO: 2 and SEQ ID NO: 3 is shown in the specification; the nucleotide sequence of a primer used for amplifying the phosphomevalonate kinase gene ERG8 is shown as SEQ ID NO: 4 and SEQ ID NO: 5 is shown in the specification; the nucleotide sequence of a primer used for amplifying the mevalonate pyrophosphate decarboxylase gene ERG19 is shown as SEQ ID NO: 6 and SEQ ID NO: 7 is shown in the specification; the nucleotide sequence of a primer used for amplifying the isopentenyl pyrophosphate isomerase gene IDI1 is shown as SEQ ID NO: 8 and SEQ ID NO: 9 is shown in the figure;
the nucleotide sequence of a primer used for amplifying the mevalonate kinase gene ERG12 containing a D18 wrapping signal peptide sequence is shown as SEQ ID NO: 10 and SEQ ID NO: 11 is shown in the figure;
the nucleotide sequence of a primer used for amplifying the phosphomevalonate kinase gene ERG8 containing a D18 wrapping signal peptide sequence is shown as SEQ ID NO: 12 and SEQ ID NO: 13 is shown in the figure;
the nucleotide sequence of a primer used for amplifying the mevalonate pyrophosphate decarboxylase gene ERG19 containing a D18 wrapping signal peptide sequence is shown as SEQ ID NO: 14 and SEQ ID NO: 15 is shown in the figure;
the nucleotide sequence of a primer used for amplifying isopentenyl pyrophosphate isomerase gene IDI1 containing a D18 wrapping signal peptide sequence is shown as SEQ ID NO: 16 and SEQ ID NO: shown at 17.
8. Use of the genetically engineered bacterium of any one of claims 1 to 3 for the fermentative production of isoprene.
9. The use of claim 8, wherein the genetically engineered bacteria are used to produce isoprene by in vitro enzymatic reaction: the genetic engineering bacteria are induced to express to prepare protein bodies, then the protein bodies are added into an in vitro enzyme catalysis reaction system, and isoprene is obtained through incubation preparation; the in-vitro enzyme catalysis reaction system contains the following components in each 2mL system: 10mM PBS buffer pH7.4, 4mM ATP, 30mM KCl, 8mM MgCl2、0.2mM MnCl2、0.01mMZnSO44mM β -mercaptoethanol and 1ml of 0.25mM mevalonate, 0.2g/L proteosome 100 uL.
10. The use according to claim 9, wherein the proteosome is prepared by: culturing the genetically engineered bacteria to OD600After the concentration is 0.6-1.0, the bacterial liquid is induced and expressed for 9 hours by IPTG, then the bacterial liquid is centrifuged, and bacterial precipitation obtained by every 100mL of strain culture liquid is centrifuged to obtain a protein corpuscle by 15mL of lysis solution BPER-II, 1mL of lysozyme, 100 times of bacterial protease inhibitor, 1uL of DNase I and 5.2uL of β -mercaptoethanol with the final concentration of 4mM at the room temperature of 60rpm for 30 min.
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