CN110241103B - Method for producing isoprene by biological enzyme method - Google Patents

Abstract

The invention provides a dandelion IspS gene, and the obtained recombinant Pichia pastoris can efficiently express the IspS gene by constructing a recombinant vector of the gene and introducing the recombinant vector into the Pichia pastoris, so that isoprene is efficiently catalytically produced, and the yield of isoprene is obviously improved compared with that of isoprene in the prior art.

Description

Method for producing isoprene by biological enzyme method

Technical Field

The invention relates to the field of fermentation, in particular to a method for producing isoprene by a biological enzyme method.

Background

Isoprene (Isoprene), known under the generic name 2-methyl-1, 3-butadiene, has the molecular formula C₅H₈It is an oily liquid which has no color, pungent odor and is easy to volatilize. Isoprene can be burned in air or oxygen and can form an explosive mixture with air, with an explosion limit of > 1.6%. The structure of the material has covalent double bonds, so the material has active chemical properties, can be oxidized, added and polymerized with various substances, is easy to generate homopolymerization and copolymerization, and is an important platform chemical raw material. The application of isoprene in China is mainly focused on the rubber industry and the fine chemical industry. Among them, the application in the rubber industry accounts for 90%. Isoprene is also widely used in the production of agricultural chemicals, medicines, perfumes, and adhesives.

Due to the wide range of uses of isoprene, the demand for isoprene has increased accordingly. At present, industrial production methods of isoprene can be classified into 3 types: c5 fraction separation method, chemical synthesis method, and dehydrogenation method. The prior industrial production methods of isoprene have a plurality of disadvantages. Mainly focuses on the aspects of long process flow, high equipment requirement, low product yield, low purity, environmental pollution and the like. And the raw materials of the methods are mostly from petroleum cracking C₅The increasing exhaustion of petroleum resources in the distillate will affect the production of isoprene. Therefore, in order to protect the environment and reasonably utilize petroleum resources, a green and effective isoprene production pathway is soughtThe method is carried out as follows.

Many organisms in nature have the ability to produce isoprene, such as green plants like poplar, willow, conifer and the like, and some microorganisms like fungi and bacteria. The isoprene compounds play an important role in plants, and as primary metabolites, the isoprene compounds can maintain the fluidity of cell membranes, assist electron transportation, participate in respiration and photosynthesis, and regulate growth and development; as a stimulus metabolite may participate as a signal against threats from the environment, pests, etc. However, plants produce very little isoprene and are difficult to collect, and therefore, it is difficult to mass-produce isoprene by means of plants in nature. Different from plants, the microorganism has the advantages of small size, fast propagation, no influence of natural environment on growth and the like, and is more suitable for large-scale production of isoprene. At present, microorganisms for synthesizing isoprene by a biological method mainly comprise yeast, bacillus subtilis, escherichia coli and the like, wherein the genetic background of the escherichia coli is clear, and a receptor system is complete and is suitable for introduction and expression of exogenous genes. In 2012, two companies in japan planned to jointly develop isoprene biosynthesis processes. The biosynthesis of isoprene in domestic research is started late, and is still in the research stage of laboratories at present, and the aspects of further research on the aspects of selecting appropriate raw materials, improving the conversion rate of isoprene and establishing an effective isoprene separation system are needed. There are two metabolic pathways for synthesizing isoprene precursors, isopentenyl diphosphate (IPP) and dimethylallyl Diphosphate (DMAPP), in vivo, namely the 2-C-methyl-D-erythritol-4-phosphate pathway and the mevalonate pathway (MEP pathway and MVA pathway).

The in vitro multi-enzyme catalytic system also has application in the production of isoprene. Tyrer p.korman et al constructed a cell-free, multi-enzyme catalyzed route to isoprene containing 12 enzymes, and the glycolytic pathway provided sufficient ATP for the MVA pathway, NADPH and acetyl coa, with molar yields of isoprene approaching 100%. Thus, by inserting the isoprene pathway into the glycolysis pathway, it is possible to produce isoprene and other acetyl-CoA derived isoprenoid compounds from glucose in vitro.

CN103789293B discloses a method for producing isoprene by using blue algae, which comprises the following steps: a fusion protein comprising prenyl pyrophosphate isomerase and isoprene synthase. The invention carries out genetic modification on the blue-green algae, expresses isoprene synthetase (IspS) from plant sources in the blue-green algae, and obviously improves the yield of isoprene produced by the genetically engineered blue-green algae through a plurality of methods.

CN103233044B A production method of isoprene, and provides a Pichia pastoris engineering strain for producing isoprene, with the preservation number of CCTCC NO: M2013167. The invention constructs a pichia pastoris engineering strain which can express isoprene synthetase through transforming isoprene synthetase gene from kudzu (Pueraria montana var. lobata) into pichia pastoris, and further can produce isoprene through high-efficiency fermentation, and can be widely applied to the production of isoprene.

From the above results, it can be seen that the production of isoprene by microorganisms has been a matter that can be industrially achieved. But the yield is low and there is room for improvement.

In the previous research, the inventor screens plants capable of producing natural rubber aiming at the advantages of various Mongolian plateau plants and the like, so that the limitation of a single source of the natural rubber is relieved. Selecting six kinds of milk plants, namely Dandelion (Taraxacum officinale, Dandelion), pachyrhizus (Cynanchum thesioides (Freyn) K.Schum), goose down vine (Cynanchum chinenseR.Br), humifuse Euphorbia herb (Euphorbia humifusa Willd), lactuca sativa (Linn.) DC and Sonchus brachyanus DC, and performing polymer extraction, polymer characterization, chemical component analysis and rubber hydrocarbon content determination on four experimental samples of Dandelion, goose down vine, pachyrhizus and humifuse Euphorbia herb; and the relation between the amount of the samples of the chicory and the common sow thistle is only subjected to a polymer qualitative experiment, and the result shows that the average rubber hydrocarbon content of the dandelion is 12.29 percent, and the yield is the highest, and the inventor conjectures that the activity of the enzyme for synthesizing the isoprene is higher necessarily. Therefore, corresponding studies were conducted on the isoprene synthase gene related to dandelion.

Disclosure of Invention

The invention provides a method for improving the capacity of synthesizing isoprene by saccharomyces cerevisiae, which clones an IspS gene sequence SEQ ID NO 1 from dandelion to a plasmid through enzyme digestion connection, and transforms the IspS gene sequence SEQ ID NO 1 to a saccharomyces cerevisiae body so as to realize the synthesis of isoprene in the saccharomyces cerevisiae.

Further, the da-IspS is encoded by a gene in SEQ ID NO.1, and the amino acid sequence of the da-IspS is that of SEQ ID NO.2 of the sequence Listing.

The invention also provides a method for efficiently expressing a target protein by using pichia pastoris, which comprises the following steps:

(1) constructing a pichia pastoris vector containing a target protein gene;

(2) transforming pichia pastoris with the target protein gene-containing pichia pastoris vector in the step (C), and screening out successfully transformed pichia pastoris;

(3) culturing the successfully transformed pichia pastoris screened in the step (2) in a yeast culture medium, and carrying out corresponding isoprene production expression;

(4) the amount of isoprene produced was measured.

In a specific form, the pichia pastoris is pichia pastoris.

In a specific form, the expression vector may be any one of pHILD2, pPIC9K, pPIC3,5K, pPICZA, pPICZB and pPICZaB.

In another aspect, the invention provides a pichia pastoris expression vector.

The recombinant vector is constructed by the following method:

artificially synthesizing or amplifying the da-IspS gene by PCR, adding an EcoRI enzyme cutting site at the upstream of the 5 'end of the da-IspS gene, and adding a NotI enzyme cutting site at the downstream of the 3' end of the da-IspS gene; treating the gene fragment with XhoI and EcoRI double digestion, and then connecting the treated gene fragment with pPIC3.5 vector which is also digested with EcoRI and NotI double digestion to obtain a recombinant vector pPIC3.5-da-IspS containing the da-IspS gene;

the synthesis method is preferably a PCR amplification method, and comprises the following specific steps: extracting chromosome DNA of the dandelion, and carrying out PCR by using the chromosome DNA as a template and upstream and downstream primers shown by SEQ ID NO.1 and SEQ ID NO.2 in a sequence table to obtain a target DNA fragment;

a recombinant bacterium for efficiently secreting and expressing da-IspS is a recombinant Pichia pastoris (Pichia pastoris) containing the da-IspS.

The Pichia pastoris is Pichia pastoris GS 115.

The invention also provides application of the recombinant strain for expressing da-IspS in preparation of isoprene.

The invention has the beneficial effects that: through the recombinant vector constructed by the invention, the obtained recombinant pichia pastoris can efficiently express the IspS gene, so that isoprene is efficiently expressed, and the yield of the isoprene is obviously improved compared with that of isoprene in the prior art.

Drawings

FIG. 1 is a schematic diagram of a secretory expression vector pPIC3.5.

FIG. 2 is an isoprene yield chart, CK is a blank control without introduced gene; the right column indicates the expression level of the gene-introduced yeast strain.

Detailed Description

The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

Example 1 construction of recombinant expression vector containing da-IspS Gene

(1) PCR amplification of da-IspS Gene

The amino acid sequence of the isoprene synthase from dandelion is removed with an intron to obtain the isoprene synthase with the amino acid sequence of SEQ ID NO: 2, the specific sequence is as follows:

optimizing an isoprene synthase (Isps) nucleotide sequence according to pichia pastoris codon preference, wherein the optimized nucleotide sequence is SEQ ID NO:1 artificially synthesizing the sequence, wherein the specific sequence is as follows:

taking the DNA obtained in the step (1) as a template, adding specific sequences of an upstream primer and a downstream primer as follows:

Isps-F：5’-CCGGAATTCatggctggtgacagattgtc-3’

Isps-R：5’-ATTTGCGGCCGgtcccagtctggacaaccagt-3’

primestar DNA polymerase (from TaKaRa) and dNTP mixture were subjected to PCR reaction under the following conditions: pre-denaturation at 98 ℃ for 3 min; cycle amplification 32 times: 20s at 98 ℃, 25s at 55 ℃ and 65s at 72 ℃; finally extension for 90s at 72 ℃. Sequencing and identifying the product obtained after amplification to obtain a target gene fragment, wherein the target gene fragment contains a coding sequence of a natto kinase Pro-NK gene, an EcoRI enzyme cutting site is arranged on the upstream of the 5 'end of the sequence, and a NotI enzyme cutting site is arranged on the upstream of the 3' end of the sequence.

(2) Construction of recombinant expression vector containing da-IspS Gene

The PCR product was recovered using a gel recovery kit, and da-IspS and the vector pPIC3.5 were digested with restriction enzymes EcoR1 and Not1 and ligated overnight at 4 ℃ under the action of T4 ligase. Transforming Escherichia coli DH5a, screening with LB-Amp plate to obtain positive clone, sequencing to determine the correctness of its gene sequence, obtaining pPIC3.5-da-IspS expression vector, the pPIC3.5 plasmid map is shown in figure 1.

Example 2 transformation of Pichia pastoris and fermentation culture with pPIC3.5-da-IspS

The pPIC3.5-Isps is digested by utilizing restriction endonuclease SacI or SalI to be linearized, and the target gene is more favorably and homologously recombined into the chromosome of pichia pastoris. Yeast cells were treated with 1M sorbitol, mixed with the purified linear plasmid containing the da-IspS gene, and transformed using an electric transformer. The recovered yeast cells are spread on an MD plate and cultured for 3-4 days at 30 ℃. Single colonies growing on MD plates were picked and inoculated into 25mL BMGY at 30 ℃ with shaking at 300rpm to OD 600. about.3-5. Centrifugation was carried out at 1500-. Adding the above culture into 1L shake flask, covering with two layers of sterilized gauze or cheese cloth, and placing into shaking table for continuous growth. Every 24 hours, methanol was added to a final concentration of 0.5% to continue induction. The amount of medium was checked to ensure that methanol was added correctly, since evaporation would reduce the volume of the medium. After the recombinant pichia pastoris cells are induced and expressed for 72-96h in a shake flask, sealing the shake flask by using a sealing plug, continuously culturing the shake flask for 30min, treating the shake flask for 30min at 60 ℃, extracting 1mL of headspace gas for gas chromatography detection, and taking an isoprene standard as a standard and a blank yeast strain without a gene introduced as a control. And (3) detecting the yield of isoprene by GC-FID (gas chromatography-flame ionization Detector) by adopting a headspace sampling method. The column oven, the sample inlet and the detector are at 80 deg.C, 180 deg.C and 180 deg.C, respectively. As shown in FIG. 2, the yield of isoprene in the yeast strain introduced with the target gene can reach 72mg/L, and the method has a good production prospect.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

Sequence listing

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Claims (7)

1. A da-IspS protein, wherein the corresponding amino acid sequence is shown in SEQ ID NO: 2, respectively.

2. The da-IspS gene has a nucleotide sequence shown in SEQ ID NO:1 is shown.

3. A recombinant vector comprising the da-IspS gene of claim 2.

4. A recombinant bacterium comprising the recombinant vector according to claim 3.

5. A method for expressing a protein of interest, comprising the steps of SEQ ID NO:1, connecting the gene with a yeast expression vector, and then transforming yeast to perform induced expression.

6. A method for efficiently expressing a target protein by using pichia pastoris comprises the following steps:

(1) construction of a peptide containing SEQ ID NO:1 target gene of Pichia yeast carrier;

(2) transforming pichia pastoris with the target protein gene-containing pichia pastoris vector in the step (C), and screening out successfully transformed pichia pastoris;

(3) culturing the successfully transformed pichia pastoris screened in the step (2) in a yeast culture medium, and carrying out corresponding isoprene production expression;

(4) the amount of isoprene produced was measured.

7. Use of the protein of claim 1 for the production of isoprene.

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