CN104561082B - One plant of yeast Candida jeffriesii that can utilize xylose expression system - Google Patents

Abstract

The present invention relates to an expression system of yeast Candida jeffriesii capable of utilizing xylose, comprising an expression vector, which is operably connected with the following elements sequentially from 5'-3': pMD19-Tsimple plasmid backbone, rDNA homologous recombination sequence , exogenous gene expression cassette and screening marker gene expression cassette; exogenous gene expression cassette includes promoter, exogenous gene insertion restriction site and transcription terminator from upstream to downstream; screening marker gene expression cassette includes promoter, antibiotic Resistance genes, transcription terminators. The expression vector of the present invention can realize integrated and stable expression in Candida jeffriesii, and has important significance for basic theoretical research and product development of xylose utilization yeast Candida jeffriesii.

Description

Expression system of a xylose-utilizing yeast Candida jeffriesii

technical field

The invention relates to the technical field of genetic engineering, in particular to an expression system of yeast Candidajeffriesii capable of utilizing xylose, a construction method and application thereof.

Background technique

On the basis of the development of molecular biology, genetics, biochemistry, microbiology and other disciplines, genetic engineering technology appeared in the 1970s. This technology constitutes a new combination of genetic material by inserting nucleic acid molecules into plasmids or other carrier molecules in vitro, and is stably amplified and expressed in a new genetic background after being introduced into host cells. The expression of functional genes is very important for the study of the structure and function of the encoded protein and its practical application, and it can be expressed in different expression systems. At present, a variety of expression systems for gene function research and protein expression have been constructed, including E. coli expression systems, yeast expression systems, and mammalian cell expression systems. Whether the target gene can be expressed successfully depends on the selection of an appropriate expression system. Yeast is a low-level single-celled eukaryotic organism. It not only has the characteristics of prokaryotic organisms such as easy cultivation, fast reproduction, and convenient genetic engineering operations, but also has the functions of eukaryotic protein processing, folding, and post-translational modification. Therefore, yeast has become the most important tool and model for modern molecular biology research, especially suitable for expressing eukaryotic genes and preparing functional proteins.

Lignocellulose is one of the most abundant renewable resources on the earth, which can be converted into clean fuel ethanol, butanol, bulk chemical lactic acid, high value-added product xylitol, organic acids and microbial cell protein, etc. It can even be used to produce various industrial enzymes such as cellulase. Its effective use can solve the problem of lack of resources encountered in the process of social development, so it has attracted widespread attention. Xylose is a monosaccharide that is second only to glucose in lignocellulose hydrolyzate, and its high-efficiency biotransformation and utilization is one of the key factors affecting the development of lignocellulose industry.

At present, a variety of yeasts have been found to be able to utilize and even ferment xylose, among which Pichia stipitis, Candida shohata, and Pachysomyces tannophilus have been studied more. After years of research, the metabolic mechanism of xylose utilization by yeast has gradually become clear, and the corresponding genetic transformation system has also been continuously improved, making it possible to apply genetic engineering technologies based on gene expression and gene knockout in this type of yeast. On the one hand, the conversion efficiency of substrates such as xylose can be improved by constructing engineered bacteria, and the yield of target metabolites can be increased; on the other hand, it can be used to produce important bio-based products such as amino acids and enzymes by constructing engineered bacteria that use cellulosic raw materials as substrates Wait.

Candida jeffriesii is a newly isolated xylose-utilizing yeast. Phylogenetic analysis shows that this yeast is a sister group with another highly efficient xylose-utilizing yeast, Spathaspora passalidarum (NGUYEN N H, SUH S O, MARSHALL C J, et al. and ecological similarities: wood-boring beetles associated with novel xylose-fermenting yeasts, Spathaspora passalidarum gen. sp. nov. and Candida jeffriesii sp. nov. Mycol Res, 2006, 110(Pt10): 1232-1241). At present, the research on the gene level of Candida jeffriesii is still in its infancy, and an applicable genetic expression system is urgently needed for further research. Although there are a variety of expression vectors used in expression systems such as Saccharomyces cerevisiae and Pichia pastoris on the market, this yeast belongs to xylose-utilizing yeast and uses a special gene coding system. The codon CUG codes for serine instead of leucine (Wohlbach DJ, Kuo A, Sato TK, Potts KM, Salamov AA, Labutti KM, Sun H, Clum A, Pangilinan JL, Lindquist EA, Lucas S, Lapidus A, Jin M, Gunawan C, Balan V, Dale BE, Jeffries TW, Zinkel R,Barry KW,Grigoriev IV,GaschAP.Comparative genomics of xylose-fermenting fungi for enhanced biofuel production.Proceedings of the National Academy of Sciences of the United States of America,2011,108(32):13212-13217.), so the above Conventional expression vectors are difficult to adapt to the yeast expression system through modification.

Therefore, it is necessary to construct a gene expression system suitable for Candida jeffriesii itself. On this basis, the gene expression system can be used to carry out genetic engineering of Candida jeffriesii or the excellent gene information of Candida jeffriesii can be excavated on the basis of the expression system, so as to construct New yeast genetically engineered bacteria and screening of new excellent genes, such as cellobiase gene, etc., lay the foundation. At the same time, through genetic engineering means, Candida jeffriesii can use xylose to convert xylose to produce useful products for human beings, and establish a bioprocessing technology for the recycling of renewable resources. possible.

Contents of the invention

To solve the above-mentioned problems in the prior art, the applicant provides an expression system of yeast Candida jeffriesii that can utilize xylose. The expression vector of the present invention can realize integrated and stable expression in Candida jeffriesii, and has important significance for basic theoretical research and product development of xylose utilization yeast Candida jeffriesii.

Technical scheme of the present invention is as follows:

In one aspect, the present invention relates to an expression system of yeast Candida jeffriesii capable of utilizing xylose, including a novel expression vector, which is circular and operably connected with the following elements sequentially from 5'-3':

pMD19-Tsimple plasmid backbone, rDNA homologous recombination sequence, exogenous gene expression cassette and selection marker gene expression cassette;

The exogenous gene expression cassette includes a promoter, an exogenous gene insertion restriction site and a transcription terminator sequentially from upstream to downstream;

The selection marker gene expression box includes a promoter, an antibiotic resistance gene, and a transcription terminator.

The yeast capable of utilizing xylose is Candida jeffriesii.

The DNA sequence of the exogenous gene expression box promoter and transcription terminator in the described expression vector comes from yeast Spathaspora passalidarum, and the complete genome sequence of this yeast is in NCBI (http://www.ncbi.nlm.nih.gov/) No. NZ_AEIK00000000, wherein the yeast may be the cell strain NRRL Y-27907 from the American Agricultural Research Type Culture Collection.

Preferably, the rDNA homologous recombination sequence is 18s rDNA, the sequence of which is shown in SEQ ID NO:1. Another option is that the similarity between the rDNA sequence and SEQ ID NO: 1 is not less than 90%, preferably 95%, more preferably 98%.

The promoters in the exogenous gene expression cassette include SpADH _P , SpXYL _P ; the transcription terminators include SpCYC1 _T , SpXYL _T .

The promoter in the expression cassette of the screening marker gene includes _SpTEF1P ; the antibiotic resistance gene can be commonly used in the expression vector, including hygromycin resistance gene, bleomycin resistance gene, G418; the transcription terminator includes SpCYC1 _T , ScCYC1 _T .

The SpADH _P promoter is the alcohol dehydrogenase gene ADH1 promoter of Spathaspora passalidarum source, and the DNA sequence of the promoter is shown in SEQ ID NO:2;

The SpXYL _P promoter is a xylose reductase gene XYL promoter derived from Spathaspora passalidarum, and the DNA sequence of the promoter is shown in SEQ ID NO:3;

The SpTEF1 _P promoter is a transcription initiation factor gene TEF1 promoter derived from Spathaspora passalidarum, and the DNA sequence of the promoter is shown in SEQ ID NO:4;

The SpCYC1 _T terminator is the cytochrome C gene CYC1 terminator derived from Spathaspora passalidarum, and the DNA sequence of the terminator is shown in SEQ ID NO:5;

The SpXYL _T terminator is the XYL terminator of the xylose reductase gene derived from Spathaspora passalidarum, and the DNA sequence of the terminator is shown in SEQ ID NO:6.

The ScCYC1 _T terminator is the cytochrome C gene CYC1 terminator derived from Saccharomyces cerevisiae, and the DNA sequence of the terminator is shown in SEQ ID NO:7.

The DNA fragment of the exogenous gene may include the exogenous gene and a selection marker gene, and the exogenous gene is inserted between the promoter and the transcription terminator in the expression cassette of the exogenous gene. In one embodiment of the present invention, the exogenous gene is gfp gene, the DNA sequence of which is shown in SEQ ID NO:11.

More than one marker gene can be included in the described screening marker gene expression cassette, and the marker gene can be hygromycin B resistance gene, bleomycin resistance gene and/or G418; in one embodiment of the present invention In , the marker gene is the hygromycin B resistance gene.

The DNA sequence of the hygromycin B resistance gene is shown in SEQ ID NO:8.

The DNA sequence of the bleomycin resistance gene is shown in SEQ ID NO:9.

The DNA sequence of G418 is shown in SEQ ID NO:10.

The yeast used in the present invention is a strain of yeast Candida jeffriesii capable of utilizing xylose. Specifically, the yeast is purchased from the Agricultural Research Culture Collection of the United States, and the deposit number is NRRL Y-27738.

Specifically, the inventors have prepared a novel expression vector according to the following technical scheme:

Using the genomic DNA of Spathaspora passalidarum as a template, the 18s rDNA homologous recombination sequence was obtained by PCR amplification with primers P1 and P2; the SpTEF1 _P promoter was obtained by PCR amplification with primers P23 and P24; the PCR amplification with primers P25 and P26 was obtained SpADH1 _P promoter; SpXYL _P promoter was obtained by PCR amplification with primers P27 and P28; SpCYC1 _T terminator was obtained by PCR amplification with primers P29 and P30; SpXYL _T terminator was obtained by PCR amplification with primers P31 and P32. The fragment hph _m -ScCYC1 _T was obtained by PCR amplification using PMD-hph _m plasmid DNA as a template and primers P21 and P22. The nucleotide sequences of the primers P1-P2 are shown in SEQ ID NO.13-14, and the nucleotide sequences of the P21-P32 are shown in SEQ ID NO.33-44.

Using pMD19-Tsimple as the backbone, the above fragments were ligated, and the ligation was performed at appropriate restriction enzyme cutting sites. The specific connection method is: after connecting the 18s rDNA at the EcoR V restriction site of pMD19-Tsimple, connect the exogenous gene expression cassette promoter, exogenous gene expression cassette transcription terminator, and SpTEF1 _P promoter in sequence along the direction of the 18s rDNA , hph _m -ScCYC1 _T .

The regulation of gene expression in yeast is a very complex process, and the selection of appropriate promoters and transcription terminators is crucial for the expression of foreign proteins. The present invention provides combinations of promoters and transcription terminators for exogenous protein expression, including SpADH _P -SpCYC1 _T , SpXYLP -SpCYC1 _T , SpADH _P -SpXYL _T , SpXYLP _-SpXYLT , _{SpTEF1 P -SpCYC1 T ,} , SpXYL _P -SpXYL _T .

The promoter was predicted according to the following method:

(1) According to the Spathaspora passalidarum genome sequence, select all nucleotide fragments between the gene of the promoter commonly used in the yeast expression system and the open reading frame of the previous gene;

(2) Using online promoter evaluation software to predict possible promoter sequences online;

(3) According to the potential promoter sequence with high scoring value, design primers to amplify to obtain the promoter sequence to be tested.

The terminator is obtained according to the following method:

(1) According to the Spathaspora passalidarum genome sequence, select about 300 bp behind the open reading frame of the gene to which the terminator commonly used in the yeast expression system belongs as the terminator sequence to be tested.

(2) Design primers to amplify to obtain the terminator to be detected.

The promoter and transcription terminator for exogenous protein expression are used in combination for the functional expression of the exogenous gene. In an embodiment, the gfp gene is inserted between the promoter for expression of the foreign protein and the transcription terminator.

The gene expression vector is integrated into the genome of the Candida jeffriesii host bacterium.

The Candida jeffriesii host bacteria is (but not limited to) the cell strain NRRL Y-27738 from the American Agricultural Research Culture Collection.

In one embodiment of the present invention, the Candida jeffriesii host bacterium is Candida jeffriesii NRRL Y-27738 from the American Agricultural Research Culture Collection.

Another aspect of the present invention provides a method for constructing a Candida jeffriesii gene expression system, comprising the following steps:

The expression vector construction of described Candida jeffriesii gene expression system:

According to the whole genome sequence of yeast Spathaspora passalidarum, the partial sequence of Spathaspora passalidarum 18s rDNA was retrieved as the homologous recombination site. Using the genomic DNA of Spathaspora passalidarum as a template, the 18s rDNA homologous recombination sequence was obtained by PCR amplification with primers P1 and P2. At the same time, the restriction site EcoR I was introduced upstream of the fragment, and the restriction sites Bgl II, BamH I and Kpn were introduced downstream I. The PCR product was purified and cloned into the plasmid pMD19-Tsimple to obtain the recombinant plasmid pMD-18srDNA.

Using PMD-hph _m plasmid DNA as a template, the fragment hph _m -ScCYC1 _T was obtained by PCR amplification with primers P21 and P22, and BamH I and Pst I were introduced upstream of the fragment, and Kpn I was introduced downstream. The purified PCR product and the recombinant plasmid pMD-18srDNA were digested with BamH I and Kpn I respectively, and the digested products were ligated to obtain the recombinant plasmid PR-hph _m .

Spathaspora passalidarum genomic DNA was used as a template, and primers P23 and P24 were used for PCR amplification to obtain the SpTEF1 _P promoter, and restriction sites BamH I and Pst I were introduced at both ends of the fragment. The purified PCR product and the recombinant plasmid PR-hph _m were double digested with BamH I and Pst I, respectively, and the digested products were ligated to obtain the recombinant plasmid PRTH.

Using Spathaspora passalidarum genomic DNA as a template, the SpADH _P promoter was obtained by PCR amplification with primers P25 and P26; the SpXYL _P promoter was obtained by PCR amplification with primers P27 and P28; at the same time, the restriction site Bgl II was introduced upstream of the fragment , the restriction sites Sal I and BamH I were introduced downstream. The purified PCR product and recombinant plasmid PRTH were double-digested with Bgl II and Sal I, respectively, and the digested recombinant plasmid and PCR product were ligated to obtain recombinant plasmids PRATH and PRXTH.

SpCYC1 _T terminator was obtained by PCR amplification with primers P29 and P30 using Spathaspora passalidarum genomic DNA as a template; SpXYL _T terminator was obtained by PCR amplification with primers P31 and P32. Restriction sites Sal I and Not I were introduced upstream of the fragment, and restriction site BamH I was introduced downstream of the fragment. The purified PCR product and recombinant plasmids PRATH and PRXTH were double-digested with Sal I and BamH I respectively, and the digested products were ligated to obtain PR series recombinant plasmids PRACTH, PRAXTH, PRXCTH and PRXXTH.

The nucleotide sequences of the primers P1-P2 are shown in SEQ ID NO.13-14.

The nucleotide sequences of P21-P32 are shown in SEQ ID NO.33-44.

Activation and cultivation of host bacteria Candida jeffriesii.

Genetic transformation of PR series recombinant plasmids in the host strain Candida jeffriesii.

Screening of positive transformants of the host strain Candida jeffriesii.

The construction method of the Candida jeffriesii gene expression system, wherein the expression vector is a recombinant plasmid PRACTH, wherein the promoter and terminator of the foreign gene expression cassette are SpADH _P and SpCYC1 _T respectively.

The construction method of the Candida jeffriesii gene expression system, wherein the expression vector is a recombinant plasmid PRAXTH, wherein the promoter and terminator of the foreign gene expression cassette are SpADH _P and SpXYL _T respectively.

The construction method of the Candida jeffriesii gene expression system, wherein the expression vector is a recombinant plasmid PRXCTH, wherein the promoter and terminator of the foreign gene expression cassette are _SpXYLP and SpCYC1 _T respectively.

The construction method of the Candida jeffriesii gene expression system, wherein the expression vector is a recombinant plasmid PRXXTH, wherein the promoter and terminator of the foreign gene expression cassette are SpXYL _P and SpXYL _T respectively.

The construction method of the Candida jeffriesii gene expression system, wherein the genetic transformation method includes PEG-LiAC transformation method, electric transformation method and protoplast transformation method, and the preferred transformation method is PEG-LiAC transformation method.

The construction method of the Candida jeffriesii gene expression system, wherein, the screening method of the host bacteria Candidajeffriesii transformants is resistance plate screening, and the initially screened transformants are subjected to colony PCR or genome PCR detection, and finally through the detection of foreign protein Transformants were identified by methods of activity or metabolites.

The invention provides a method for expressing foreign genes, comprising the steps of:

(1) providing the integrated Candida jeffriesii gene expression system;

(2) Inserting the exogenous gene into the expression vector by inserting the exogenous gene into the restriction site to obtain a recombinant expression vector;

(3) Transforming the recombinant expression vector into a host bacterium Candida jeffriesii and expressing the foreign gene in the host bacterium.

The present invention also provides a method for metabolic engineering host bacterium Candida jeffriesii, comprising the steps of:

(1) providing the integrated Candida jeffriesii expression system described in claim 1;

(2) inserting the foreign gene of the target gene into the expression vector into the restriction site to obtain the recombinant expression vector;

(3) transforming the recombinant expression vector into the host bacterium Candida jeffriesii and expressing the target gene in the host bacterium;

(4) Cultivate the recombinant bacteria, and detect the metabolites of the recombinant bacteria.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

The beneficial technical effects of the present invention are:

1. The yeast used in the present invention is a novel xylose-utilizing yeast, which has high-efficiency xylose utilization ability and strong ethanol production ability, and has good industrial application prospect and value. However, this yeast belongs to the xylose-utilizing Candida, and this type of yeast uses a special gene coding system, the codon CUG codes for serine instead of leucine, so it is used in the expression of expression systems such as Saccharomyces cerevisiae and Pichia pastoris The vector was not suitable for this xylose-utilizing yeast. In view of this, the inventors constructed a series of novel expression vectors suitable for the yeast itself, and the expression of exogenous proteins and metabolic engineering of the yeast can be conveniently carried out by using the series of expression vectors.

2. The present invention provides a method for transforming Candida jeffriesii with other DNA molecules, said DNA molecules can come from the library or synthetic DNA molecules formed by linking Candida jeffriesii genome fragments between the above vector promoter and terminator. The present invention thus provides techniques that can be used to express gene diversity libraries to produce products from which new biologically active substances can be screened.

3. In the integrated expression vector of the present invention, multiple rDNA sequences were tried to be used as integration sites, but only one of the sequences showed an integration effect, and this sequence was a partial sequence of 18s rDNA, and this site showed The characteristics of high copy number and good stability make this series of integrated expression vectors a yeast multi-copy integrated expression vector suitable for the expression of foreign proteins and metabolic engineering of Candida jeffriesii.

4. In the novel expression vector constructed by the present invention, multiple CUG codons in the open reading frames of resistance genes such as the hygromycin B resistance gene and bleomycin resistance gene used are subjected to site-directed mutation, and the mutation is UUG, the site-directed mutation of the CUG codon in the open reading frame of the reporter gene gfp was mutated into UUG, thus realizing the effective transformation and functional expression of this series of novel expression vectors in yeast Candida jeffriesii.

Description of drawings

Fig. 1 is hph gene CDS sequence ( Indicate site-directed mutagenesis site);

Fig. 2 is the gfp gene CDS sequence after the site-directed mutation of embodiment 4 Indicate site-directed mutagenesis site);

Fig. 3 is the plasmid map of the green fluorescent protein recombinant plasmid PRACTH-gfp _m of embodiment 4;

Fig. 4 is that the bacteria green fluorescent protein recombinant plasmid transformant colony PCR verification figure of embodiment 4 (M is Maker, swimming lane 1-9 is positive transformant amplification result, and swimming lane 10 is negative control);

Fig. 5 shows the transformation of Candida jeffriesii with the green fluorescent protein recombinant plasmid PRACTH-gfp _m in Example 4 and the GFP fluorescence microscope detection (the left picture is dark field, and the right picture is bright field).

Detailed ways

The technical solutions of the present invention will be described in detail below in conjunction with the embodiments. It should be understood that these examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. Any modifications or substitutions made to the methods, steps or conditions of the present invention without departing from the spirit and essence of the present invention are deemed to fall within the scope of the present invention.

The experimental methods that do not indicate the specific conditions in the following examples are basically carried out in accordance with the commonly used cloning manuals or the conditions suggested by the manufacturer; the reagents or instruments used that do not indicate the manufacturer are all commercially available. product.

Unless defined otherwise herein, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In the present invention, the term "operably linked" refers to the functional spatial arrangement of two or more nucleic acid sequences. For example: the promoter sequence is placed at a specific position relative to the nucleic acid sequence of the target gene, so that the transcription of the target gene is guided by the promoter and expression becomes feasible, thus, the promoter sequence is "operably linked" to on the nucleic acid sequence. In general, the term "operably linked" means that the DNA sequences being linked are contiguous by ligation at appropriate restriction sites. If the site does not exist, oligonucleotide adapters or linkers synthesized according to conventional methods can be used.

Example 1: Construction of PR series integrated expression vectors

1. Construction of recombinant plasmid pMD-18s rDNA

(1) According to the whole genome sequence of Spathaspora passalidarum (GenBank accession NZ_AEIK00000000), two primers were designed to intercept the partial sequence of Spathaspora passalidarum 18s rDNA as the homologous recombination site. The primer sequences are as follows: the underlined part of primer P1 is the recognition site of EcoR I, and the underlined part of primer P2 is the recognition site of Bgl II, BamH I and Kpn I respectively from the 5' to 3' ends.

P1: 5'GCCGGAATTCTGCCAGTAGTCATATGCTTGTCTC3 '

P2: 5'ATATTAGG GGTACC CG GGATCC GA AGATCT GTTGAAGAGCAATAAT3'

(2) Cultivate Spathaspora passalidarum overnight, collect cells, separate and extract genomic DNA.

(3) Spathaspora passalidarum genomic DNA was used as a template, and PCR amplification was performed with primers P1 and P2. The amplification conditions were pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 1.5 min, 30 cycles, and extension at 72°C for 5 min. The PCR amplified product was purified and recovered and then cloned into pMD19-Tsimple vector (purchased from Dalian TaKaRa Company) to obtain recombinant plasmid pMD-18s rDNA.

Two, the construction of recombinant plasmid PMD-hph _m

(1) According to the expression of hygromycin resistance gene in plasmid pRS303H (Taxis C, Knop M. System of centromeric, episomal, and integrative vectors based on drug resistance markers for Saccharomycescerevisiae. BioTechniques, 2006, 40(1): 73-78.) Cassette sequences Design primers P3 and P4.

P3: 5'ACATTTTGATGGCCGCACGG3'

P4: 5'AACTCCTTCCTTTTCGGTTAGAGCG3'

(2) Using the pRS303H plasmid DNA as a template, the hph expression cassette fragment was amplified with primers P3 and P4. The amplification conditions were pre-denaturation at 95°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1.5 minutes, 30 pieces Cycle, extend at 72°C for 5 min. After the fragments were purified and recovered, they were cloned into the pMD19-Tsimple vector to obtain the recombinant plasmid PMD-hph.

(3) Design mutation primers according to the CUG codon in the hygromycin resistance gene CDS, wherein the underlined part in the primer sequence is the mutation site.

P5: 5' GAGAAGTTTT TTG ATCGAAAAGTTCGACAGC 3'

P6: 5' GCTGTCGAACTTTTCGAT CAA AAACTTCTC 3'

P7: 5' GACAGCGTCTCCGAC TTG ATGCAGCTCTCG 3'

P8: 5' CGAGAGCTGCAT CAA GTCGGAGACGCTGTC 3'

P9: 5' GGGCGTGGATATGTC TTG CGGGTAAATAG 3'

P10: 5' CTATTTACCCG CAA GACATATCCACGCCC 3'

P11: 5' AATTCAGCGAGAGC TTG ACCTATTGCATCT 3'

P12: 5' AGATGCAATAGGT CAA GCTCTCGCTGAATT 3'

P13: 5' TTGCAAGAC TTG CCTGAAACCGAA TTG CCCGCTGTT 3'

P14: 5' AACAGCGGG CAA TTCGGTTTCAGG CAA GTCTTGCAA 3'

P15: 5' AATTGCCCGCTGTT TTG CAGCCGGT 3'

P16: 5'ACCGGCTG CAAAACAGCGGGCAATT 3'

P17: 5' AGGCTCTCGATGAG TTG ATGCTTTGGGCCGAG 3'

P18: 5' CTCGGCCCAAAGCAT CAA CTCATCGAGAGCCT 3'

P19: 5'GCTCCAACAATGTC TTG ACGGACAATGG 3'

P20: 5' CCATTGTCCGT CAA GACATTGTTGGAGC 3'

Using the plasmid PMD-hph as a template, Stratagene site-directed mutagenesis kit (purchased from Agilent Technologies Co., Ltd.) was used to perform PCR amplification with the above primers to obtain a circular PCR product. Amplification conditions were: pre-denaturation at 95°C for 2 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 2 minutes, extension at 68°C for 3.5 minutes, 30 cycles, and full extension at 68°C for 5 minutes.

(4) After the above PCR product was purified, it was digested with endonuclease Dpn I at 37° C. for 30 minutes, and reacted at 65° C. for 15 minutes to inactivate endonuclease Dpn I. After purification of the digested product, transform E.coli JM109 competent cells (purchased from Beijing Quanshijin Company), spread on LB (100 μg/ml ampicillin) plate, pick a single colony, extract the plasmid and send it to Sangong Bioengineering (Shanghai) Co., Ltd. conducts sequencing and selects the plasmid with the correct mutation site. After the above mutation operation, the mutant plasmid was named PMD-hph _m , where the CDS sequence of the mutated hph is shown in FIG. 1 .

3. Construction of recombinant plasmid PR-hph _m

(1) According to the hygromycin resistance gene expression cassette sequence in the PMD-hph _m plasmid, two primers were designed: the underlined part of primer P21 is the recognition site of BamH I and Pst I from the 5' to 3' ends respectively, and the underlined part of primer P22 Part of it is the recognition site of Kpn I.

P21: 5'CG GGATCC AAA CTGCAG ATGGGTAAAAAGCCTGAACTCAC3'

P22: 5'GG GGTACC AACTCCTTCCTTTTCGGTTAGAGCG3'

(2) Using PMD-hph _m plasmid DNA as a template, PCR amplification was performed with primers P21 and P22 to obtain PCR products. The amplification conditions were pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 63°C for 30 s, extension at 72°C for 1.5 min, 30 cycles, and extension at 72°C for 5 min.

(3) The purified PCR product and the recombinant plasmid pMD-18s rDNA were digested with BamH I and Kpn I respectively, and the digested products were purified and ligated overnight, transformed into E.coli JM109 competent cells, and spread on LB ( 100 μg/ml ampicillin) plate, pick a single colony, extract the plasmid and verify it by double digestion with BamH I and Kpn I, and obtain the recombinant plasmid PR-hph _m .

4. Construction of recombinant plasmid PRTH

(1) According to the complete genome sequence of Spathaspora passalidarum (GenBank accession NZ_AEIK00000000) and the sequence information provided by the online database EMBL-EBI (http://www.ebi.ac.uk/), the transcription initiation factor and last gene of Spathaspora passalidarum were obtained All DNA sequences between open reading frames. According to the promoter software (http://www.softberrv.com and http://www.cbs.dtu.dk/services/), the above sequences were predicted online, and then two restriction sites were designed according to the selected sequences. For the primers, about 700 bp upstream of the Spathasporapassalidarum transcription initiation factor was used as the promoter SpTEF1 _P : the underlined part of primer P23 is the recognition site of BamH I, and the underlined part of primer P24 is the recognition site of Pst I.

P23: 5'CG GGATCC ACCACTTACATAATAGAAAGAC 3'

P24: 5' ACGAGC CTGCAG TTTTGATTGATTGATTG 3'

(2) Using Spathaspora passalidarum genomic DNA as a template, PCR amplification was performed with primers P23 and P24 to obtain PCR products. The amplification conditions were pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 50 s, 30 cycles, and extension at 72°C for 5 min.

(3) The purified PCR product and the recombinant plasmid PR-hph _m were digested with BamH I and Pst I respectively, and the digested products were purified and ligated overnight, transformed into E.coli JM109 competent cells, and spread on LB ( 100 μg/ml ampicillin) plate, pick a single colony, extract the plasmid and verify it by double digestion with BamH I and Pst I, and obtain the recombinant plasmid PRTH.

5. Construction of recombinant plasmids PRATH and PRXTH

(1) According to the complete genome sequence of Spathaspora passalidarum (GenBank accession NZ_AEIK00000000) and the sequence information provided by the online database EMBL-EBI (http://www.ebi.ac.uk/), obtain Spathaspora passalidarum alcohol dehydrogenase and xylose reduction Enzymes respectively and all DNA sequences between the open reading frame of the previous gene. According to the promoter software (http://www.softberrv.com and http://www.cbs.dtu.dk/services/), the above sequence is predicted online, and then primers are designed according to the restriction site of the selected sequence, Take about 1000 bp upstream of the open reading frames of Spathaspora passalidarum alcohol dehydrogenase and xylose reductase as promoters, namely SpADH _P and SpXYL _P : the underlined part of primers P25 and P27 is the recognition site of Bgl II, and the underlined part of primers P26 and P28 From the 5' to the 3' end are the recognition sites of BamH I and Sal I respectively.

P25: 5' GCCGGA AGATCT GTAAATTAATGCTACATCAGTTGAGG 3'

P26: 5' CG GGATCC ACGC GTCGAC TATATTTTATTTAGGAATT 3'

P27: 5' GCCGGA AGATCT GTGACATAGTTAACTATGGC 3'

P28: 5' CG GGATCC ACGC GTCGAC TTTATTGTATTGTG 3'

(2) Using Spathaspora passalidarum genomic DNA as a template, PCR amplification was performed with primers P25 and P26 to obtain the fragment SpADH _P . The amplification conditions were pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, 30 cycles, and extension at 72°C for 5 min.

(3) Using Spathaspora passalidarum genomic DNA as a template and primers P27 and P28 for PCR amplification, the fragment _SpXYLP was obtained. The amplification conditions were pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 1 min, 30 cycles, and extension at 72°C for 5 min.

(4) Digest the purified fragment SpADH _P and the recombinant plasmid PRTH with Bgl II and BamH I respectively, purify the digested products and connect them overnight, transform E.coli JM109 competent cells, and spread on LB (100 μg/ ml ampicillin) plate, pick a single colony, extract the plasmid and verify it by double enzyme digestion with Bgl II and BamH I, and gel electrophoresis shows that the band of about 1000bp is positively connected to obtain the recombinant plasmid PRATH.

(5) Digest the purified fragment _SpXYLP and the recombinant plasmid PRTH with Bgl II and BamH I, respectively, purify the digested products and connect them overnight, transform E.coli JM109 competent cells, and spread on LB (100 μg/ ml ampicillin) plate, pick a single colony, extract the plasmid and verify by double enzyme digestion with Bgl II and BamH I, after gel electrophoresis, the band of about 1000bp is forward connected, and the recombinant plasmid PRXTH is obtained.

6. Construction of PR series integrated expression vector

(1) According to the complete genome sequence of Spathaspora passalidarum (GenBank accession NZ_AEIK00000000) and the sequence information provided by the online database EMBL-EBI (http://www.ebi.ac.uk/), the cytochrome C1 and xylose reductase of Spathaspora passalidarum were obtained The downstream sequence of the open reading frame. Design primers to transfer Spathaspora passalidarum cytochrome C1 and about 300bp downstream of the open reading frame of xylose reductase as transcription terminators, namely SpCYC1 _T and SpXYL _T : the underlined parts of primers P29 and P31 are Sal I and The recognition site of Not I, the underlined part of primers P30 and P32 is the recognition site of BamH I.

P29: 5'ACGC GTCGA CATAAGAAT GCGGCCGCGCTAACTTCAATTAGAAT3 '

P30: 5'CG GGATCC CATCACTATAAGCGAAATCGGGTTTC 3'

P31: 5' ACGC GTCGAC ATAAGAAT GCGGCCGC GTTTGATTTCTAGTTTATAT3'

P32: 5' GCGC GGATCC ATAGTTAACTATGTCACTTGAACTC 3'

(2) Using Spathaspora passalidarum genomic DNA as a template, PCR amplification was performed with primers P29 and P30 to obtain the fragment SpCYC1 _T . The amplification conditions were pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 63°C for 30 s, extension at 72°C for 30 s, 30 cycles, and extension at 72°C for 5 min.

(3) Using Spathaspora passalidarum genomic DNA as a template, PCR amplification was performed with primers P31 and P32 to obtain the fragment SpXYL _T . The amplification conditions were pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 61°C for 30 s, extension at 72°C for 30 s, 30 cycles, and extension at 72°C for 5 min.

(4) The purified fragment SpCYC1 _T and the recombinant plasmid PRATH were digested with Sal I and BamH I respectively, and the digested products were purified and ligated overnight, transformed into E.coli JM109 competent cells, and spread on LB (100 μg/ ml ampicillin) plate, pick a single colony, extract the plasmid and verify it by double digestion with Sal I and BamH I, and obtain the recombinant plasmid PRACTH.

(5) The purified fragment SpCYC1 _T and the recombinant plasmid PRXTH were digested with Sal I and BamH I respectively, and the digested products were purified and ligated overnight, transformed into E.coli JM109 competent cells, and spread on LB (100 μg/ ml ampicillin) plate, pick a single colony, extract the plasmid and verify it by double digestion with Sal I and BamH I, and obtain the recombinant plasmid PRXCTH.

(6) Digest the purified fragment SpXYL _T and the recombinant plasmid PRATH with Sal I and BamH I, respectively, purify the digested products and connect them overnight, transform E.coli JM109 competent cells, and spread on LB (100 μg/ ml ampicillin) plate, pick a single colony, extract the plasmid and verify it by double digestion with Sal I and BamH I to obtain the recombinant plasmid PRAXTH.

(7) The purified fragment SpXYL _T and the recombinant plasmid PRXTH were digested with Sal I and BamH I, respectively, and the digested products were purified and ligated overnight, transformed into E.coli JM109 competent cells, and spread on LB (100 μg/ ml ampicillin) plate, pick a single colony, extract the plasmid and verify it by double enzyme digestion with Sal I and BamH I, and obtain the recombinant plasmid PRXXTH.

Example 2: Establishment of PEG/LiAc-mediated transformation method of Candida jeffriesii.

Using Candida jeffriesii NRRL Y-27738 as the host bacterium, the method of transforming yeast mediated by PEG/LiAc is implemented as follows:

1. Preparation of Competent Candida jeffriesii NRRL Y-27738

(1) Inoculate the Candida jeffriesii NRRL Y-27738 stored in the cryopreservation tube into the YPD medium, and activate the shake flask for 48 hours.

(2) Streak the activated bacterial solution on the YPD plate and store it at 4°C.

(3) Pick a single colony of Candida jeffriesii NRRL Y-27738 from the YPD plate, inoculate it in 20ml of YPD medium, and culture it overnight at 30°C in a 100ml shake flask.

(4) Inoculate 50ml of YPD culture medium with the fresh bacterial solution cultured overnight, and culture it in a 250ml shake flask at 30°C and 200rpm until the OD600 of the bacterial solution reaches about 1.2.

(5) Centrifuge at 5000 rpm for 5 minutes at room temperature to collect bacterial cells.

(6) The cells were resuspended in 500 μl of 0.1 mol/L LiAc, centrifuged, and the supernatant was discarded to obtain competent cells.

2. Preparation of linearized recombinant plasmid

(1) Inoculate E.coli containing the recombinant expression plasmid in LB medium and culture overnight.

(2) Collect E. coli bacterial cells, and extract recombinant plasmids by alkaline lysis method. For specific methods, refer to the Biotech Plasmid Extraction Kit.

(3) Single-digest the recombinant plasmid with restriction endonuclease Stu I, enzyme digestion reaction system (50 μL): 40 μL DNA, 5 μL buffer, 1.5 μL restriction endonuclease Stu I, make up 50 μL with double distilled water, mix well Afterwards, place in a 37°C incubator for enzyme digestion for 2 hours.

(4) Purify the digested product to obtain a linearized recombinant plasmid.

3. Conversion of Candida jeffriesii NRRL Y-27738 by PEG/LiAc method

(1) Add the following transformation mixture in order to the competent cells: 240 μL PEG3350, 36 μL 1.0mol/LLiAc, 25 μL salmon sperm DNA, 50 μL linear DNA to be transformed, wherein the salmon sperm DNA is boiled in water for 10 minutes and immediately ice-bathed;

(2) Shake each reaction tube vigorously until the cells are completely mixed;

(3) Incubating at 30°C for 1 hour;

(4) Heat shock in a metal bath at 42°C for 22 minutes;

(5) After cooling down to room temperature, centrifuge at 5000rpm for 5min, discard the supernatant;

(6) Add 1ml of YPD medium and incubate at 30°C for 2 hours;

(7) Centrifuge at 5000 rpm for 5 min, discard 800 μl of supernatant, mix the cells and spread on a hygromycin B (100 mg/mL) resistant plate, culture at 30°C for 3-4 days to obtain transformants.

Example 3: Establishment of the yeast Candida jeffriesii transformation method mediated by electroporation

Using Candida jeffriesii NRRL Y-27738 as the host bacterium, the electroporation method was used to mediate the transformation of yeast as follows:

1. Preparation of Candida jeffriesii NRRL Y-27738 electroporation competent cells

(1) Inoculate the Candida jeffriesii NRRL Y-27738 stored in the cryopreservation tube into the YPD medium, and activate the shake flask for 48 hours;

(2) Streak the activated bacterial solution on the YPD plate and store it at 4°C;

(3) Pick a single colony of Candida jeffriesii NRRL Y-27738 from the YPD plate, inoculate it in 20ml of YPD medium, and culture it overnight at 30°C in a 100ml shake flask;

(4) Inoculate the overnight cultured fresh bacterial solution into 50ml YPD medium, and culture it in a 250ml shake flask at 30°C and 200rpm until the OD600 of the bacterial solution reaches about 1.2;

(5) Place the bacterial solution on ice for 30 minutes, centrifuge at 5000 rpm, 4°C for 5 minutes, and collect bacterial cells;

(6) Add pre-cooled 20ml double distilled water to wash the bacteria twice, centrifuge at 5000rpm, 4°C for 5min, and collect the bacteria cells;

(7) Add 20ml of pre-cooled 1.0mol/L sorbitol to wash the cells twice, centrifuge at 5000rpm, 4°C for 5min, and collect the cells;

(8) Add 200 μl of pre-cooled 1.0 mol/L sorbitol to mix the bacterial cells to obtain competent cells.

2. Preparation of linearized recombinant plasmid

(1) Inoculate E.coli containing the recombinant expression plasmid in LB medium and culture overnight.

(2) Collect E. coli bacterial cells, and extract recombinant plasmids by alkaline lysis method. For specific methods, refer to the Biotech Plasmid Extraction Kit.

(3) Single-digest the recombinant plasmid with restriction endonuclease Stu I, enzyme digestion reaction system (50 μL): 40 μL DNA, 5 μL buffer, 1.5 μL restriction endonuclease Stu I, make up 50 μL with double distilled water, mix well Afterwards, place in a 37°C incubator for enzyme digestion for 2 hours.

(4) Purify the digested product to obtain a linearized recombinant plasmid.

3. Electroconversion of Candida jeffriesii NRRL Y-27738

(1) Mix 100 μl of electroporation competent cells and 10 μl of DNA into a 0.2 cm electroporation cup;

(2) The electroporation cup was bathed in ice for 5 minutes, the setting condition was 1500v, and the electroporation was performed for 5s;

(3) Immediately add 1ml of pre-cooled 1.0mol/L sorbitol to the electroporation cup, transfer to the incubator at 30°C and incubate for 1h;

(4) centrifuge at 5000rpm for 5min, discard the supernatant;

(5) Add 1ml of YPD medium, mix the cells, and incubate in an incubator at 30°C for 2 hours;

(6) Centrifuge at 5000 rpm for 5 min, discard 800 μl of supernatant, mix the cells and spread on a hygromycin B (100 mg/mL) resistant plate, culture at 30°C for 3-4 days to obtain transformants.

Example 4: Application of PR series integrative expression vectors in expressing foreign genes.

1. Construction of recombinant expression vector for green fluorescent protein

(1) Design primers according to the gene sequence of green fluorescent protein: the underlined part of primer P33 is the recognition site of Sal I, and the underlined part of primer P34 is the recognition site of Not I.

P33: 5' ACGC GTCGAC ATGGGTAAGGGAGAAGAACTTTTCAC 3'

P34: 5'ATAAGAATGCGGCCGCTTATTTGTATAGTTCATCCATGCCATG3 '

(2) Using the DNA of the green fluorescent protein gene as a template, PCR amplification was performed with primers P33 and P34 to obtain the gene gfp. The amplification conditions were pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 45 s, 30 cycles, and extension at 72°C for 5 min.

(3) Cloning the purified gfp fragment into the pMD19-Tsimple vector, transforming E.coli JM109 competent cells, spreading it on LB (100 μg/ml ampicillin) plate, picking a single colony, and extracting the plasmid Sal I and Not I Double digestion verification, the recombinant plasmid pMD-gfp was obtained.

(4) Since the codon CUG exists in the open reading frame of the gfp gene sequence, the yeast Candida jeffriesii uses a special coding system, that is, the codon CUG encodes serine instead of leucine. Therefore, it is necessary to carry out site-directed mutation on the gfp gene, and mutate the codon CUG to the codon UUG.

(5) Using base C at position 601 in the CDS of the gfp gene sequence as the mutation site, design single-point mutation primers: the underlined parts of primers P35 and P36 are the mutation sites.

P35: 5' TACCAGACAACCATTAC TTG TCCACACAATCTGCC3'

p36: 5' GGCAGATTGTGTGGA CAA GTAATGGTTGTCTGGTA 3'

(6) Using the recombinant plasmid pMD-gfp as a template, Stratagene site-directed mutagenesis kit was used to perform PCR amplification with primers P35 and P36 to obtain a circular PCR product. The amplification conditions are: pre-denaturation at 95°C for 2 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 2 minutes, extension at 68°C for 3.5 minutes, 30 cycles, and full extension at 68°C for 5 minutes;

(7) After the above PCR product was purified, it was digested with endonuclease Dpn I at 37° C. for 30 minutes, and reacted at 65° C. for 15 minutes to inactivate endonuclease Dpn I. After the digestion product was purified, it was transformed into E.coli JM109 competent cells, spread on LB (100 μg/ml ampicillin) plate, picked a single colony, extracted the plasmid and sent it to Sangon Bioengineering (Shanghai) Co., Ltd. Sequence, select the plasmid with the correct mutation site, and name it pMD-gfp _m . The DNA sequence after site-directed mutation of the gfp gene is SEQ ID NO: 11, as shown in FIG. 2 .

(8) Digest the recombinant plasmids pMD-gfp _m and PRACTH with Sal I and Not I, respectively. After digestion and purification of the gfp fragment, it was ligated overnight with the enzyme digestion product of the plasmid PRACTH, transformed into E.coli JM109 strain, spread on LB (100 μg/ml ampicillin) plate, picked a single colony, extracted the plasmid and doubled Sal I and Not I. After enzyme digestion verification, the green fluorescent protein recombinant expression vector PRACTH-gfp _m was obtained, and the plasmid map is shown in Figure 3.

2. Construction of Candida jeffriesii recombinant bacteria

The plasmid PRACTH-gfp _m linearized by Stu I was transformed into Candida jeffriesii NRRL Y-27738 according to the method described in Example 2 or Example 3 to obtain transformants.

3. Screening and verification of transformants

(1) Transformants were screened by a high-concentration hygromycin B resistance plate. The transformant obtained after transforming the recombinant expression vector of green fluorescent protein into Candida jeffriesii NRRL Y-27738 was transferred to a higher concentration hygromycin B (200 μg/mL) resistance plate, cultured at 30°C for 2-3 days, and the same method was used Transplanted 3 times in a row to obtain pure culture transformant strains.

(2) Colony PCR verification of positive transformants. A PCR amplification system was prepared with primers P21 and P22, and a small amount of microwave-treated transformant cells were added to the system. PCR amplification conditions were pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 63°C for 30 s, extension at 72°C for 1.5 min, 30 cycles, and extension at 72°C for 5 min. The PCR product was subjected to agarose gel electrophoresis to show a band of about 1100 bp, as shown in Figure 4, which was consistent with the size of the DNA fragment hph _m -ScCYC1T band.

(3) Fluorescent detection of positive transformants. The purely cultured transformant strain obtained in step (1) was inoculated in YPD medium, and after shaking the flask for 18 hours at 30°C, the bacteria were collected, washed twice with double distilled water and resuspended in double distilled water. Draw a small amount of suspension and place it on a glass slide, cover it with a cover glass, observe and take pictures under a fluorescent microscope with an oil lens. As shown in Figure 5, the cells can be observed to emit green fluorescence.

Claims (6)

1. an expression system of the yeast Candida jeffriesii that can utilize xylose, it is characterized in that comprising a kind of expression vector, and described expression vector comprises following operable element successively from 5 '-3': pMD19-Tsimple plasmid backbone, rDNA homologous recombination sequence, exogenous gene expression cassette and selection marker gene expression cassette; The exogenous gene expression cassette includes a promoter, an exogenous gene insertion restriction site and a transcription terminator sequentially from upstream to downstream; The selection marker gene expression box includes a promoter, an antibiotic resistance gene, and a transcription terminator; The yeast capable of utilizing xylose is Candida jeffriesii; The rDNA homologous recombination sequence is a 18s rDNA sequence; The promoters in the exogenous gene expression cassette are SpADH _P , SpXYL _P ; the transcription terminators are SpCYC1 _T , SpXYL _T ; The promoter in the expression cassette of the screening marker gene is SpTEF1 _P ; the antibiotic resistance gene is selected from hygromycin resistance gene, bleomycin resistance gene, G418; the transcription terminator is SpCYC1 _T , ScCYC1 _T ; The 18s rDNA sequence is shown in SEQ ID NO:1; The SpADH _P promoter is the alcohol dehydrogenase gene ADH1 promoter of Spathaspora passalidarum source, and the DNA sequence of the promoter is shown in SEQ ID NO:2; The SpXYL _P promoter is a xylose reductase gene XYL promoter derived from Spathaspora passalidarum, and the DNA sequence of the promoter is shown in SEQ ID NO:3; The SpTEF1 _P promoter is a transcription initiation factor gene TEF1 promoter derived from Spathaspora passalidarum, and the DNA sequence of the promoter is shown in SEQ ID NO:4; The SpCYC1 _T terminator is the cytochrome C gene CYC1 terminator derived from Spathaspora passalidarum, and the DNA sequence of the terminator is shown in SEQ ID NO:5; The SpXYL _T terminator is the xylose reductase gene XYL terminator derived from Spathaspora passalidarum, and the DNA sequence of the terminator is shown in SEQ ID NO:6; The ScCYC1 _T terminator is the cytochrome C gene CYC1 terminator derived from Saccharomyces cerevisiae, and the DNA sequence of the terminator is shown in SEQ ID NO:7; The DNA sequence of the hygromycin resistance gene is shown in SEQ ID NO: 8; The DNA sequence of the bleomycin resistance gene is shown in SEQ ID NO:9; The DNA sequence of G418 is shown in SEQ ID NO:10. 2. The expression system according to claim 1, characterized in that, the host yeast Candida jeffriesii is preserved in the American Agricultural Culture Collection, and the preservation number is NRRL Y-27738. 3. The application of the expression system according to any one of claims 1 to 2 in the genetic transformation of the host bacterium Candida jeffriesii. 4. The use of the expression system according to claim 1, characterized in that it is used for expressing foreign proteins and for metabolic engineering of host bacteria. 5. A method for expressing a foreign gene, characterized in that it comprises the steps of: (1) preparing the expression system according to claim 1; (2) Inserting the exogenous gene into the expression vector by inserting the exogenous gene into the restriction site to obtain a recombinant expression vector; (3) Transforming the recombinant expression vector into a host bacterium Candida jeffriesii and expressing the foreign gene in the host bacterium. 6. A method for metabolic engineering transformation host bacterium Candida jeffriesii, characterized in that it comprises the steps: (1) preparing the expression system according to claim 1; (2) inserting the foreign gene of the target gene into the expression vector into the restriction site to obtain the recombinant expression vector; (3) transforming the recombinant expression vector into the host bacterium Candida jeffriesii and expressing the target gene in the host bacterium; (4) Cultivate the recombinant bacteria obtained in step (3), and detect the metabolites of the recombinant bacteria.