WO2018113430A1 - Probiotic feed-use saccharomyces cerevisiae for producing xylo-oligosaccharide and antimicrobial peptide - Google Patents

Abstract

Provided is a recombinant Saccharomyces cerevisiae for producing a xylo-oligosaccharide and an antimicrobial peptide. The Saccharomyces cerevisiae contains a multi-gene co-expression vector, and the vector contains a specific endo-β-1,4-xylanase gene, a β-1,4-xylosidase gene, and an antimicrobial peptide gene. The recombinant Saccharomyces cerevisiae can assist in degrading the xylo-oligosaccharide generated by a xylan, facilitate proliferation of probiotics in an intestinal tract of a body, supplement the xylanase in the body, and suppress infectious microbes or pathogenic bacteria, thereby improving immunity of the body and facilitating body growth. The present invention can also be applied to kitchen waste degradation to facilitate the degradation of a hemicellulose component in the waste.

Description

Probiotic feeding Saccharomyces cerevisiae producing xylooligosaccharides and antimicrobial peptides Technical field

The present invention relates to the fields of genetic engineering and fermentation engineering, and more particularly to a probiotic feed yeast for producing xylooligosaccharides and antimicrobial peptides.

Background technique

The agricultural and sideline products such as wheat straw, chaff and bran contain anti-nutritional factor-xylan, which is a xylose polymer which is composed of β-1,4-glycosidic bonds and is one of the main components constituting the cell wall. Xylanase is one of the most important enzymes capable of degrading xylan. If the xylanase is rationally utilized, the utilization rate of lignocellulose can be improved. Xylan is one of the main anti-nutritional factors in feed. Adding xylanase to the diet is a relatively simple and effective method. Xylanase hydrolyzes xylan to xylo-oligosaccharides and xylo-oligo-xylo-oligo-xylose, as well as a small amount of xylose and arabinose, thereby eliminating the anti-nutritional effects of xylan.

Xylan-based xylo-oligosaccharides (XOs), also known as xylooligosaccharides, are composed of 2 to 7 xylose and β-1,4-glycosidic bonds. The general name of linear oligosaccharides, the active ingredients are generally xylobiose, xylotriose, xylotetraose, pentameose, etc., among which xylobiose (X2) and xylotriose (X3) Mainly. At present, the functional oligosaccharides used in feed additives mainly include oligosaccharides, galactooligosaccharides, oligo-isomaltose, soybean oligosaccharides, oligofructose, oligomannose and xylooligosaccharides, among which effects The best is xylooligosaccharides. Compared with other oligosaccharides, xylooligosaccharides have the following advantages: 1 high selectivity promotes the proliferation of bifidobacteria; 2 is not easily digested by digestive enzymes in the body; 3 is less ingested, has good compatibility, and can be distributed with other sources. Not affected; 4 is stable to acid and heat. At the same time, xylooligosaccharides also have the following probiotic effects: 1 regulate the structure of the intestinal flora; 2 inhibit the reproduction of pathogenic bacteria, reduce the production of harmful substances; 3 improve the body's immunity; 4 promote the body to synthesize other nutrients.

The invention expresses the xylan degrading enzymes through the Saccharomyces cerevisiae system, can supplement the xylanase, and assists in degrading the xylan to obtain the functional xylooligosaccharide, and exerts a probiotic effect; at the same time, it has strong alkali and heat. Stability and broad-spectrum antibacterial characteristics and antibacterial peptide genes that inhibit pathogenic bacteria, selective immune activation and regulatory functions are co-expressed to achieve synergistic promotion of multiple functions. Therefore, the recombinant Saccharomyces cerevisiae in the invention will be applied to feed addition, animal breeding and degradation of kitchen waste, and can effectively exert its synergistic effect and play a probiotic role.

Summary of the invention

The technical problem to be solved by the present invention is to provide a probiotic feed which can secrete other xylan degrading enzymes and antibacterial peptides, and can be applied in the fields of yeast culture, xylan degradation, etc., in order to overcome the above-mentioned deficiencies of the prior art. Genetically modified yeast used.

The technical problem to be solved by the present invention is to provide a fermentable yeast in order to overcome the above-mentioned deficiencies of the prior art. It can secrete β-1,4-endo-xylanase gene and β-xylosidase gene in the fields of parent culture, xylan degradation, etc. The xylan degrading enzyme and antibacterial peptide gene were simultaneously ligated into the S. cerevisiae expression vector pTEGC-BsmBI, transferred into Saccharomyces cerevisiae, and successfully secreted and expressed, and the multifunctional refractory wine produced by degrading xylan to produce xylooligosaccharide and secreting antibacterial peptide was obtained. yeast.

It is an object of the present invention to provide a Saccharomyces cerevisiae multi-gene co-expression vector capable of producing xylooligosaccharides and antimicrobial peptides and a method for constructing the same.

Another object of the present invention is to provide a recombinant Saccharomyces cerevisiae capable of producing xylooligosaccharides and antimicrobial peptides and a method for constructing the same.

The technical solution adopted by the present invention is:

A Saccharomyces cerevisiae multi-gene co-expression vector capable of producing xylooligosaccharides and antibacterial peptides, comprising β-1,4-endo-xylanase gene, β-1,4-xylosidase gene, and antibacterial Peptide gene;

The base sequence of the β-1,4-endo-xylanase gene is shown in SEQ ID NO: 1.

The base sequence of the β-1,4-xylosidase gene is shown in SEQ ID NO: 2.

Further, the antibacterial peptide gene is selected from the group consisting of an oriental bell pepper antibacterial peptide BLP-2 mutant BLP-2-T, a heterochromatic ladybug antibacterial peptide Haxy-Col1 mutant Haxy-Col1-T, a salmon antibacterial peptide mutant, and a crisp Frog frog antibacterial peptide mutant Lf-cath-T;

The base sequence of the oriental bell antibacterial peptide BLP-2 mutant BLP-2-T is shown in SEQ ID NO: 3;

The base sequence of the Hazel-Col1 mutant Haxy-Col1-T of the S. cerevisiae antibacterial peptide is shown in SEQ ID NO: 4;

The base sequence of the salmon antibacterial peptide mutant is as shown in SEQ ID NO: 5;

The base sequence of the crispy frog antibacterial peptide mutant Lf-cath-T is shown in SEQ ID NO: 6.

Further, an α-signal peptide gene sequence is present upstream of the antimicrobial peptide gene, and the base sequence of the α-signal peptide gene is as shown in SEQ ID NO: 7.

Further, the promoter of the β-1,4-endo-xylanase gene is pgk1-1, the base sequence thereof is shown in SEQ ID NO: 8, and the terminator is pgkt1-1, and the base sequence thereof As shown in SEQ ID NO:9;

The promoter of the β-1,4-xylosidase gene is pgk1-2, the base sequence thereof is shown in SEQ ID NO: 10, the terminator is pgkt1-2, and the base sequence thereof is SEQ ID NO: 11. Shown

The promoter of the antimicrobial peptide gene is pgk1-3, the base sequence thereof is shown in SEQ ID NO: 12, the terminator is pgkt1-3, and the base sequence thereof is shown in SEQ ID NO: 13.

Further, the screening gene of the above vector contains a G418 resistance gene.

Further, the backbone of the above vector is a pGAPZaA plasmid.

Further, the above vector contains a 25s rDNA gene fragment of Saccharomyces cerevisiae, and its base sequence is SEQ ID NO:15.

A Saccharomyces cerevisiae capable of producing xylooligosaccharides and an antimicrobial peptide, wherein the recombinant Saccharomyces cerevisiae genome is inserted with the multi-gene co-expression vector of any of the above.

A method for constructing a Saccharomyces cerevisiae multi-gene co-expression vector capable of producing xylooligosaccharides and antimicrobial peptides according to any of the above, comprising the steps of:

The S1 integrated expression vector pTEGC-BsmBI was constructed:

S1.1 ligated the G418 resistance gene between the multiple cloning sites Msc I and EcoR V of the pGAPZaA plasmid vector to obtain the vector pGAPZaA-G418;

S1.2, the base sequence is ligated into the vector pGAPZaA-G418 multiple cloning site BamHI and EcoRI, and the vector pGAPZaA-G418-rDNA is obtained;

S1.3 The vector pGAPZaA-G418-rDNA was digested with Bgl II and EcoRI, and the large fragment product was recovered to obtain a linearized vector pTEGC, and the BsmBI-2 fragment represented by SEQ ID NO: 16 was linear. The vector pTEGC was ligated to obtain the integrated expression vector pTEGC-BsmBI;

S2 promoter, terminator amplification

Amplification of S2.1 promoter: using S. cerevisiae genomic DNA as a template, primers were used to amplify pgk1- by PGK1F1-BsmBI and PGK1R1-BsmBI, PGK1F2-BsmBI and PGK1R2-BsmBI, PGK1F3-BsmBI and PGK1R3-BsmBI, respectively. 1. pgk1-2, pgk1-3 promoter fragment;

Amplification of S2.2 terminator: using S. cerevisiae genomic DNA as a template, primers were used to amplify pgkt1-, PGKT1F1-BsmBI and PGKT1R1-BsmBI, PGKT1F2-BsmBI and PGKT1R2-BsmBI, PGKT1F3-BsmBI and PGKT1R3-BsmBI, respectively. 1. pgkt1-2, pgkt1-3 terminator fragment;

Acquisition of S3α-signal peptide gene, α-amylase gene, glucoamylase gene and antimicrobial peptide gene

S3.1 Acquisition of β-1,4-endo-xylanase gene containing BsmBI cleavage site: using T-vector containing β-1,4- endoxylanase gene sequence as template, by primer XynF-BsmBI and xynR-BsmBI are amplified to obtain a fragment of xynA1 gene, ie, a fragment containing a β-1,4-endo-xylanase gene;

S3.2 Acquisition of β-1,4-xylosidase gene containing BsmBI cleavage site: T-vector containing β-1,4-xylosidase gene sequence as template, by primers xylF-BsmBI and xylR- BsmBI is amplified to obtain a fragment of xyl-1 gene, that is, a fragment containing a β-1,4-xylosidase gene;

Obtainment of S3.3α-signal peptide-antibacterial peptide gene: The T-vector containing the α-signal peptide gene sequence and the T-vector containing the antimicrobial peptide were used as templates, and the α-signal peptide sequence was directionally linked to no signal by overlap extension PCR. The 5' end of the antibacterial peptide gene of the peptide, the mfa-amp gene fragment, ie, the fragment containing the α-signal peptide gene sequence and the antimicrobial peptide gene sequence, is amplified; the overlap During the extension PCR, both ends of the amplification product mfa-amp are introduced into the cleavage sequence of BsmBI having opposite cutting directions by primers;

Construction of S4 Saccharomyces Cerevisiae Multi-gene Co-expression Vector

The β-1,4-endoxylanase gene expression cassette element pgk1-1, xynA1, pgkt1-1 obtained above; β-1,4-xylosidase gene expression cassette element pgk1-2, xyl-1 , pgkt1-2; antibacterial peptide gene expression cassette elements pgk1-3, mfa-amp, pgkt1-3 were digested with the type IIs restriction endonuclease BsmBI, purified and recovered; meanwhile, cut with the type IIs restriction endonuclease BsmBI The above integrated expression vector pTEGC-BsmBI is linearized; the fragments used are ligated into the linearized integrated expression vector pTEGC-BsmBI by a one-step method to obtain a Saccharomyces cerevisiae multi-gene co-expression vector;

The base sequences of the above primers are as follows:

PGK1F1-BsmBI: CGTTCCAPidc GAAGTACCTTCAAAG

PGK1R1-BsmBI: CGTCTCGGctaTATATTTGTTGTAAA

PGK1F2-BsmBI: CGTCTCAgtcaGAAGTACCTTCAAAG

PGK1R2-BsmBI: CGTCTCGGcatTATATTTGTTGTAAA

PGK1F3-BsmBI: CGTCTCAtgcaGAAGTACCTTCAAAG

PGK1R3-BsmBI: CGTCTCGtcgaTATATTTGTTGTAAA

PGKT1F1-BsmBI: CGTCTCAtgtacGATCTCCCATCGTCTCTACT

PGKT1R1-BsmBI: CGTCTCGGgtcaAAGCTTTTTCGAAACGCAG

PGKT1F2-BsmBI: CGTCTCAtacgGATCTCCCATCGTCTCTACT

PGKT1R2-BsmBI: CGTCTCGtgcaAAGCTTTTTCGAAACGCAG

PGKT1F3-BsmBI: CGTCTCAatcgGATCTCCCATCGTCTCTACT

PGKT1R3-BsmBI: CGTCTCGagtcAAGCTTTTTCGAAACGCAG

xynF-BsmBI: CCTTCCAgcta ATGAAGGTTACTGCTGCT

xynR-BsmBI: CGTCTCAgtac TTAAGAAGAGATAGTAACA

xylF-BsmBI: CGTCTCAgcatATGCCAGGTGCTGCTTCTATCGTTGCT

xylR-BsmBI: CGTCTCAtacg TTATTGTGGAGCGATCAATTGTTCT.

A method for constructing recombinant Saccharomyces cerevisiae capable of producing xylooligosaccharides and antibacterial peptides, and transforming the Saccharomyces cerevisiae multi-gene co-expression vector constructed above into a Saccharomyces cerevisiae host, screening positive monoclonal colonies, and verifying the correct sequencing, that is, obtaining A recombinant Saccharomyces cerevisiae producing xylooligosaccharides and antimicrobial peptides.

The beneficial effects of the invention are:

The recombinant Saccharomyces cerevisiae of the present invention can simultaneously secrete xylan degradation related enzymes, such as β-1,4-endopoly A multifunctional recombinant yeast of carbohydrase, β-1,4-xylosidase, and antimicrobial peptides. Among them, β-1,4-endo-xylanase and β-1,4-xylosidase can assist in the degradation of xylan-derived xylo-oligosaccharide (xylose), which is an important prebiotic and can promote the intestinal tract in the body. Proliferation of probiotics in the Dao; can also supplement the deficiency of xylanase in the body. The selected antimicrobial peptide can also inhibit the bacteria or pathogenic bacteria. Therefore, its application products, such as yeast cultures, can more effectively promote the body's immunity and promote the growth of the body, and can also be applied to the degradation of kitchen waste to promote the degradation of hemicellulose components in waste and bacteria. Growing. It can be applied to many fields such as feed addition, animal breeding, and degradation of kitchen waste.

DRAWINGS

Figure 1 is a Congo red staining method to verify the xylanase activity of recombinant S. cerevisiae constructed in Example 2.

detailed description

The present invention will be further described below in conjunction with specific embodiments, but is not limited thereto.

Example 1 Construction of integrated expression vector pTEGC-BsmBI

First, the integrated expression vector pTEGC-BsmBI construction

1) Acquisition of G418 resistance gene

The gene of interest was amplified by PCR, and the G418 resistance gene was amplified using the G418F-MscI and G418R-EcoRV primers (Table 1) using the vector pPIC9k as a template. PCR reaction conditions: 98 ° C for 10 s, 55 ° C for 15 s, 72 ° C for 50 s, 30 cycles, 72 ° C for 10 min. It was verified by 2% agarose gel electrophoresis.

The target gene is recovered, purified, transformed into E. coli, verified, and sent for sequencing. The purified fragment was recovered and stored at -20 ° C until use. The obtained G418 resistance gene was ligated with the T vector, transformed into Escherichia coli DH5α strain, cultured at 37 ° C, and the plasmid DNA was extracted, and the positive strain was screened by colony PCR using G418F-MscI and G418R-EcoRV primers, and the positive clone was sent to English. Jieji sequencing verified the correctness of the gene. The sequencing results showed that the G418 resistance gene and its restriction site were correctly ligated into T-load without mutation, and the base sequence of the G418 resistance gene is shown in SEQ ID NO: 14.

Table 1 Amplification of G418 resistance gene primers

Note: The letter underlined is the recognition/cleavage sequence of the restriction enzyme.

2) Construction of vector pGAPZaA-G418

The pGAPZaA plasmid was cleaved with restriction endonucleases MscI and EcoRV at 37 ° C and verified by 1.5% agarose gel electrophoresis; cleavage of MscI and EcoRV by restriction endonuclease to cleave pMD-G418 vector to obtain G418 resistance The gene was verified by 1.5% agarose gel electrophoresis; the pGAPZaA vector in the above-mentioned digested product was recovered and purified, The G418 resistance gene was ligated into the vector pGAPZaA using T4 ligase to obtain the vector pGAPZaA-G418.

3) rDNA gene amplification

The S. cerevisiae genomic DNA was used as a template, and the rDNA gene was amplified by primer rDNAF and rDNAR primers (see Table 2). The PCR amplification conditions were: 98 ° C for 10 s, 55 ° C for 15 s, 72 ° C for 60 s, 30 cycles, 72 ° C for 10 min. ; Validated on 1% agarose gel electrophoresis, and introduced EcoRI and BamHI restriction sites in the upstream and downstream.

The obtained rDNA gene was ligated with the T vector, transformed into Escherichia coli DH5α strain, cultured at 37 ° C, and the plasmid DNA was extracted, and the positive strain was screened by colony PCR using rDNAF and rDNAR primers. The sequencing results showed that the rDNA gene and its restriction site were correctly ligated into T-load without mutation, and the base sequence of the rDNA gene is shown in SEQ ID NO: 15.

Table 2 amplification of rDNA gene primers

Note: The letter underlined is the recognition/cleavage sequence of the restriction enzyme.

4) Construction of vector pGAPZaA-G418-rDNA

The rDNA fragment on the above T vector was digested with restriction endonucleases BamHI and EcoRI, and the plasmid pGAPZaA-G418 was cleaved, and the pGAPZaA-G418 vector backbone was recovered and purified. The rDNA was ligated into the linearized vector pGAPZaA-G418 by T4 ligase. The recombinant vector pGAPZaA-G418-rDNA was obtained.

5) Construction of the integrated expression vector pTEGC-BsmBI

The restriction endonuclease cleaves Bgl II and EcoRI to cleave the plasmid pGAPZaA-G418-rDNA, and excises the GAP promoter and a-signal peptide from the BglII to EcoRI restriction sites on the vector, and recovers the large fragment product to obtain linearization. Vector pTEGC.

Using the pMD19-T simple vector as a template, the primers PMDF-BsmBI and PMDR-BsmBI (see Table 3) were used to amplify a BsmBI-cleaving site recognition sequence, a fragment of about 233 bp, BsmBI-2, and ligated into the T vector. The sequence was sent to Ingreal, sequenced correctly, and no mutation occurred. The base sequence of BsmBI-2 is shown in SEQ ID NO: 16.

The recombinant T vector was excised by restriction endonuclease cleavage of Bgl II and EcoR I, and the 233 bp DNA fragment BsmBI-2 was recovered, and then correctly ligated into the linearized vector pTEGC by T4 ligase to obtain the integrated expression vector pTEGC. -BsmBI.

Table 3 Amplification of primers containing BsmBI backbone DNA

Note: The uppercase letter at the underline is the BglII or EcoRI restriction site; the lowercase letter is the recognition sequence of the IIs restriction endonuclease BsmBI enzyme.

Second, the amplification of the promoter and terminator

1) Amplification of the promoter:

Using the S. cerevisiae genomic DNA as a template, the pgk1-1 promoter fragment (the base sequence is shown in SEQ ID NO: 8) was amplified using PGK1F1-BsmBI and PGK1R1-BsmBI primers (see Table 4) for expression of β. - The promoter of the 1,4-endo-xylanase gene.

Similarly, using the S. cerevisiae gene DNA as a template, the PGK1F2-BsmBI and PGK1R2-BsmBI primers (see Table 4) were used to amplify the pgk1-2 promoter fragment (the base sequence is shown in SEQ ID NO: 10). A promoter expressing a β-1,4-xylosidase gene.

Similarly, the S. cerevisiae gene DNA was used as a template, and the pgk1-3 promoter fragment (the base sequence is shown in SEQ ID NO: 12) was amplified using PGK1F3-BsmBI and PGK1R3-BsmBI primers (see Table 4). A promoter that expresses the antimicrobial peptide gene.

The promoter gene fragments obtained by the above amplification were respectively ligated into the pMD19-T Simple vector, and verified by sequencing to retain the correct positive clone.

2) Amplification of the terminator:

The S. cerevisiae genomic DNA was used as a template, and the pgkt1-1 terminator (the base sequence is shown in SEQ ID NO: 9) was amplified using the primers PGKT1F1-BsmBI and PGKT1R1-BsmBI (see Table 4) for expression of β-1. , the terminator of the 4-endo-xylanase gene.

Using the Saccharomyces cerevisiae genomic DNA as a template, the pgkt1-2 terminator (the base sequence is shown in SEQ ID NO: 11) was amplified using the primers PGKT1F2-BsmBI and PGKT1R2-BsmBI (see Table 4) for expression of β-1. , the terminator of the 4-xylosidase gene.

The S. cerevisiae genomic DNA was used as a template, and the pgkt1-3 terminator (the base sequence is shown in SEQ ID NO: 13) was amplified using the primers PGKT1F3-BsmBI and PGKT1R3-BsmBI (see Table 4) for expression of the antimicrobial peptide gene. of Terminator.

The above-mentioned amplification-derived terminator gene fragments were ligated into the pMD19-T Simple vector, and verified by sequencing, and the correct positive clones were retained.

Table 4 primers for amplifying the Saccharomyces cerevisiae promoter and terminator

Note: The capital letter under the underline is the recognition sequence of the type IIs restriction endonuclease BsmBI, and the underlined lower case letter is the cleavage sequence of the type IIs restriction endonuclease BsmBI.

3. Acquisition of α-signal peptide gene, β-1,4-endoxylanase gene (xynA1), β-1,4-xylosidase gene (xyl-1) and antimicrobial peptide gene (amp)

1) Obtainment of α-signal peptide gene: Mfa-BsmBI fragment was amplified by using Saccharomyces cerevisiae genomic DNA as a template and MfaF and MfaR primers (see Table 5). The amplification procedure was as follows: 98 ° C for 10 s, 55 ° C for 15 s, 72 °C30s, 30 cycles, 72 °C for 10 min; ligated into the T vector, sent samples for sequencing, and selected the correct positive clones, thereby storing the α-signal peptide gene (the base sequence is shown in SEQ ID NO: 7) to T In the carrier.

Table 5 Primers for amplifying α-signal peptide gene, β-1,4-endoxylanase gene, β-1,4-xylosidase gene and antimicrobial peptide gene

Note: The capital letter under the underline is the recognition sequence of the type IIs restriction endonuclease BsmBI, and the lowercase bold letter under the underline is the cleavage sequence of the type IIs restriction endonuclease BsmBI.

2) Obtainment of β-1,4-endo-xylanase gene: the base sequence of the β-1,4-endo-xylanase gene xynA1 optimized by artificial synthesis is shown in SEQ ID NO: 1. .

3) Obtaining the β-1,4-xylosidase gene xyl-1: the base sequence of the β-1,4-xylosidase gene xyl-1 optimized by artificial synthesis is shown in SEQ ID NO: 2;

4) Obtainment of antibacterial peptide genes: The following four antibacterial peptides were selected for this study:

The artificially optimized antibacterial peptide BLP-2 mutant BLP-2-T of Bombina orientalis (base sequence is shown in SEQ ID NO: 3), and the amino acid sequence of BLP-2-T is GIGSKILSAGKGALKGLAKGLAEHFAN (SEQ ID NO:45);

The artificially optimized Harmonia axyridis antibacterial peptide Haxy-Col1 mutant Haxy-Col1-T (base sequence is shown in SEQ ID NO: 4), the amino acid sequence of Haxy-Col1-T is SLQGGAPNFPQPGQEKQEGWKFDPSLTRGEDGNTRGSINIHHTGPNHEVGANWDKVIRGPNKAKPTYSIHGSWRW (SEQ ID NO: 46);

The artificially optimized squid antibacterial peptide mutant has an amino acid sequence of KGRGKQGGKVRKSS (SEQ ID NO: 47) and a base sequence as shown in SEQ ID NO: 5;

The antimicrobial peptide mutant Lf-cath-T (base sequence is shown in SEQ ID NO: 6) of the optimized crispy frog was artificially synthesized, and the amino acid sequence of Lf-cath-T was GKCNVLGQRKQLLRSIGSGSHIGSVVLPRG (SEQ ID NO: 48).

In this example, the antibacterial peptide BLP-2 mutant BLP-2-T of oriental bell was used as an antibacterial peptide for the subsequent construction of a multi-gene co-expression vector of Saccharomyces cerevisiae.

The β-1,4-endo-xylanase gene (xynA1), the β-1,4-xylosidase gene (xyl-1), and the antibacterial peptide gene sequence obtained above were each stored in a pMD19-T Simple plasmid. spare.

5) β-1,4-endo-xylanase gene fragment containing BsmBI cleavage site: using T-vector containing β-1,4-endoxylanase gene fragment (xynA1) as template The specific primers xynF-BsmBI and xynR-BsmBI (see Table 5) were amplified to obtain a β-1,4-endo-xylanase gene fragment xynA1 containing a cleavage site of BsmBI.

6) β-1,4-xylosidase gene fragment containing BsmBI cleavage site: T-vector containing β-1,4-xylosidase gene fragment as template, specific primers xylF-BsmBI and xylR-BsmBI (See Table 5) Amplification was carried out to obtain a β-1,4-xylosidase gene fragment xyl-1 containing a cleavage site of BsmBI.

7) Obtainment of α-signal peptide-antibacterial peptide gene: using T vector containing α-signal peptide gene sequence and T vector containing Brinelp peptide mutant BLP-2 mutant BLP-2-T gene as template Primers MfaF3-BsmBI, Mfa-ampR, Mfa-ampF, and Mfa-ampR-BsmBI (see Table 5) were ligated into the anti-peptide peptide of the signal-free peptide by overlap extension PCR (SOE-PCR). At the 'end, the mfa-blp gene fragment (containing the α-signal peptide gene sequence and the antimicrobial peptide BLP-2 mutant BLP-2-T gene) was amplified.

The above amplified gene fragments were ligated into the pMD19-Simple vector, and verified by sequencing, and the correct positive clones were retained.

Construction of a multi-gene co-expression vector capable of producing xylooligosaccharides and antimicrobial peptides

The β-1,4-endo-xylanase gene expression cassette element pgk1-1 (promoter) and xynA1 (containing β-1,4-endo-xylan) obtained in the above-mentioned "two" and "three" Enzyme gene xynA1), pgkt1-1 (terminator); β-1,4-xylosidase gene expression cassette element pgk1-2 (promoter), xyl-1 (containing β-1,4-xylosidase gene xyl -1), pgkt1-2 (terminator); antibacterial peptide gene expression cassette element pgk1-3 (promoter), mfa-blp (containing α-signal peptide gene sequence and antimicrobial peptide BLP-2 mutant BLP-2-T Gene), pgkt1-3 (terminator) were digested from the T vector by the type IIs restriction endonuclease BsmBI, and purified and recovered. At the same time, the integrated expression constructed in the above "one" was cleaved by the type IIs restriction endonuclease BsmBI. The vector pTEGC-BsmBI was linearized. The above-mentioned fragment was ligated into the integrated expression vector pTEGC-BsmBI by T4 ligase, and the S. cerevisiae multi-gene co-expression vector pTEGC-xynA1-xyl-1-blp was obtained, transformed into E. coli DH5a, and the transformants were selected and verified by sequencing. A positively ligated positive transformant was obtained and the plasmid was extracted to obtain a multi-gene co-expression vector pTEGC-xynA1-xyl-1-blp capable of producing xylooligosaccharides and antimicrobial peptides.

Example 2 Recombinant Saccharomyces cerevisiae capable of producing xylooligosaccharides and antimicrobial peptides

I. Screening and verification of recombinant Saccharomyces Cerevisiae

Before the electroporation of Saccharomyces cerevisiae, the sensitivity test of resistance screening marker G418 was carried out on Saccharomyces cerevisiae. It was found that yeast was inhibited from growing on YPD plates with G418 concentration of 200 μg/ml, and the concentration of resistance above 200 μg/ml was selected. Screening, such as 300 μg/ml.

The Saccharomyces cerevisiae multi-gene co-expression vector pTEGC-xynA1-xyl-1-blp constructed in Example 1 was linearized with restriction endonuclease HpaI and transferred into S. cerevisiae using lithium acetate-mediated electroporation transformation in G418. The cells were cultured on a YPD plate at a concentration of 300 μg/ml for more than 48 hours, and the grown single colonies were picked as transformants. The PCR-converted transformants were screened in YPD liquid medium containing 300 μg/ml, 500 μg/ml, and 600 μg/ml of G418 to obtain positive monoclonal colonies, which were verified by sequencing to obtain correctly linked positive recombinant yeast transformants. That is, a recombinant Saccharomyces cerevisiae capable of producing xylooligosaccharides and antimicrobial peptides, referred to as Glam-x1.

2. Detection of xylanase activity of recombinant Saccharomyces cerevisiae Glam-x1

1) Identification of xylanase activity of recombinant Saccharomyces cerevisiae by Congo red staining

experimental method:

The recombinant Saccharomyces cerevisiae Glam-x1 constructed in this example was transferred to a YP containing 1% xylan (formulation: 5 g/l yeast extract, 10 g/l tryptone) agar plate, and cultured for 72 hours or more. Then, 10 mL of 0.1% Congo red staining solution was added, and the mixture was stained at room temperature for 40 min, and then decolorized with 1 M NaCl solution for 30 min to observe the hydrolysis circle.

Experimental results:

The observation results are shown in Fig. 1. It can be seen that there are obvious hydrolyzed circles around the different monoclonals of the recombinant Saccharomyces cerevisiae constructed in this example, indicating that the recombinant Saccharomyces cerevisiae constructed in this example has good xylan. Enzyme activity.

2) Determination of xylanase activity of recombinant Saccharomyces cerevisiae by DNS method and determination of β-xylosidase activity of recombinant Saccharomyces cerevisiae by pNP method

experimental method:

The xylanase activity of the recombinant Saccharomyces cerevisiae Glam-x1 constructed in this example was determined by reference to GB/T 23874-2009 feed additive xylanase activity assay; the β-xylosidase activity of recombinant yeast Glam-x1 was determined. To determine the reference Lacke AH method: Pipette 200 μL of 5mmol/L substrate (pNP-X) prepared with 50mmol/L pH 7.0 phosphate buffer solution, preheat for 5min at 55°C, add 50μL of appropriately diluted enzyme solution, react for 10min, then The reaction was terminated by adding 750 μL of a 2 mol/L sodium carbonate solution, and after cooling, A410 was measured, and the enzyme activity was calculated based on pNP.

Experimental results:

The results of the xylanase activity assay of the recombinant Saccharomyces cerevisiae constructed in Example 2 are shown in Table 6. As can be seen, the total xylanase activity of the recombinant S. cerevisiae Glam-x1 of Example 2 was about 0.5 U/ Above ml, significantly higher than the control host S. cerevisiae.

Table 6 Determination of xylanase activity of recombinant Saccharomyces cerevisiae constructed in Example 2

Group	Xylanase enzyme activity (U/ml)
Host Saccharomyces	0.01
Example 2 Recombinant Saccharomyces Cerevisiae Glam-x1	0.53

The results of the detection of β-xylosidase activity of the recombinant Saccharomyces cerevisiae constructed in Example 2 are shown in Table 7, from which it can be seen that the β-xylosidase enzyme activity of the recombinant Saccharomyces cerevisiae Glam-x1 of Example 2 was about 3.58 U. Above /ml, significantly higher than the control host S. cerevisiae.

Table 7 Determination of β-xylosidase activity of recombinant Saccharomyces cerevisiae constructed in Example 2

Group	--xylosidase enzyme activity (U/ml)
Host Saccharomyces	0.01
Example 2 Recombinant Saccharomyces Cerevisiae Glam-x1	3.58

Third, the antibacterial effect detection of recombinant Saccharomyces Cerevisiae

experimental method:

1 Escherichia coli CICC10899 and Pseudomonas aeruginosa CICC10419 were used as test bacteria, and the test bacteria were cultured in liquid medium until OD ₆₀₀ nm=0.4, appropriately diluted, mixed and uniformly coated onto the plate in MH medium. (The medium formulation was: 5 g/l beef extract, 17.5 g/l casein hydrolysate, 1.5 g/l starch, agar powder 20 g/l).

2 A suitable volume of the fermentation broth of the recombinant Saccharomyces cerevisiae obtained in this example was added to an Oxford cup, and the sterile water was used as a negative control, and ampicillin (1.5 μg) was used as a positive control, and cultured at 37 ° C for 16-18 h.

3 observation, statistical inhibition zone situation.

Experimental results:

The experimental results are shown in Table 8. It can be seen that the fermentation broth of the recombinant Saccharomyces cerevisiae constructed in this example has obvious inhibition zones against Escherichia coli CICC10899 and Pseudomonas aeruginosa CICC10419, indicating that the recombinant Saccharomyces cerevisiae is obtained. Glam-x1 successfully secretes antibacterial peptides.

Table 8 antibacterial effect of recombinant bacteria

Note: “-” has no bacteriostatic effect, “+” inhibition zone diameter is 7 to 14mm, and “++” inhibition zone diameter is above 14mm.

Example 3 Construction of a multi-gene co-expression vector capable of producing xylooligosaccharides and antimicrobial peptides

The method for constructing the Saccharomyces cerevisiae multi-gene co-expression vector of the present embodiment is the same as that of Example 1, except that the antimicrobial peptide gene ligated into the vector is replaced with the Haxy-Col1 mutant Haxy-Col1-T gene of the Harmonia axyridis. The Saccharomyces cerevisiae multi-gene co-expression vector constructed in this example was named pTEGC-xynA1-xyl-col except that the base sequence is as shown in SEQ ID NO: 4.

Example 4 Construction of a multi-gene co-expression vector capable of producing xylooligosaccharides and antimicrobial peptides

The method for constructing the Saccharomyces cerevisiae multi-gene co-expression vector in the present embodiment is the same as in Example 1, except that the antibacterial peptide gene ligated into the vector is replaced with the squid antibacterial peptide mutant gene (base sequence is shown in SEQ ID NO: 5), and the like. The same as in Example 1, the Saccharomyces cerevisiae multi-gene co-expression vector constructed in this example was named pTEGC-xynA1-xyl-1-par.

Example 5 Construction of a multi-gene co-expression vector capable of producing xylooligosaccharides and antimicrobial peptides

The method for constructing the Saccharomyces cerevisiae multi-gene co-expression vector of the present embodiment is the same as that of Example 1, except that the antimicrobial peptide gene ligated into the vector is replaced with the antimicrobial peptide mutant Lf-cath-T of the crispy frog (base sequence is SEQ ID NO: The other is the same as Example 1, except that the Saccharomyces cerevisiae multi-gene co-expression vector constructed in this example was named pTEGC-xynA1-xyl-cath.

Example 6 Construction of recombinant Saccharomyces cerevisiae capable of producing xylooligosaccharides and antimicrobial peptides

The Saccharomyces cerevisiae multi-gene co-expression vector pTEGC-xynA1-xylcol constructed in Example 3 was linearized with restriction endonuclease and transferred into S. cerevisiae using lithium acetate-mediated electroporation transformation at a G418 concentration of 300 μg/ml. The YPD plate was cultured for more than 48 hours, and the single colony that grew out was picked as a transformant. The PCR-converted transformants were screened in YPD liquid medium containing 300 μg/ml, 500 μg/ml, and 600 μg/ml of G418 to obtain positive monoclonal colonies, which were verified by sequencing to obtain correctly linked positive recombinant yeast transformants. The construction of recombinant Saccharomyces cerevisiae capable of producing xylooligosaccharides and antimicrobial peptides can be obtained, and the recombinant Saccharomyces cerevisiae obtained in this example is simply referred to as Glam-x2.

Example 7 Construction of recombinant Saccharomyces cerevisiae capable of producing xylooligosaccharides and antimicrobial peptides

The Saccharomyces cerevisiae multi-gene co-expression vector pTEGC-xynA1-xyl-par constructed in Example 4 was linearized with restriction endonuclease and transferred into S. cerevisiae using lithium acetate-mediated electroporation transformation at a G418 concentration of 300 μg. The cells were cultured on /ml YPD plates for more than 48 hours, and the single colonies grown were picked as transformants. The PCR-converted transformants were screened in YPD liquid medium containing 300 μg/ml, 500 μg/ml, and 600 μg/ml of G418 to obtain positive monoclonal colonies, which were verified by sequencing to obtain correctly linked positive recombinant yeast transformants. The construction of recombinant Saccharomyces cerevisiae capable of producing xylooligosaccharides and antimicrobial peptides can be obtained, and the recombinant Saccharomyces cerevisiae obtained in this example is simply referred to as Glam-x3.

Example 8 Construction of recombinant Saccharomyces cerevisiae capable of producing xylooligosaccharides and antimicrobial peptides

The Saccharomyces cerevisiae multi-gene co-expression vector pTEGC-xynA1-xyl-cath constructed in Example 5 was linearized with restriction endonuclease and transferred into S. cerevisiae using lithium acetate-mediated electroporation transformation at a G418 concentration of 300 μg. /ml The YPD plate was cultured for more than 48 hours, and the single colony that grew out was picked as a transformant. The PCR-converted transformants were screened in YPD liquid medium containing 300 μg/ml, 500 μg/ml, and 600 μg/ml of G418 to obtain positive monoclonal colonies, which were verified by sequencing to obtain correctly linked positive recombinant yeast transformants. That is, the construction of recombinant Saccharomyces cerevisiae which is likely to produce xylooligosaccharides and antimicrobial peptides, and the recombinant Saccharomyces cerevisiae obtained in this example is simply referred to as Glam-x4.

Example 9 Comparison of Antibacterial Properties of Antibacterial Peptide Mutants and Original Antibacterial Peptides

The antibacterial peptide BLP-2 of Bombina orientalis and its mutant BLP-2-T (base sequence as shown in SEQ ID NO: 3) and the antibacterial peptide Haxy-Col1 of Harmonia axyridis Its mutant Haxy-Col1-T (base sequence as shown in SEQ ID NO: 4), salmon antibacterial peptide and mutant thereof (base sequence as shown in SEQ ID NO: 5), antimicrobial peptide of crispy frog Lf-cath and its mutant Lf-cath-T (base sequence is shown as SEQ ID NO: 6). Salmonella CMCC50071, Escherichia coli CICC10899 and Staphylococcus aureus ATCC22023 were used as indicator bacteria to detect the minimum inhibitory concentration (MIC) of the above-mentioned indicator bacteria before and after mutation of various antimicrobial peptides.

The test results are shown in Table 9, and the modified antimicrobial peptide mutants were superior to the unmutated antimicrobial peptides. Among them, the antibacterial peptide BLP-2 of the oriental bell and its mutant BLP-2-T, the antibacterial peptide of the heterochromatic ladybug and its mutant Haxy-Col1-T have better antibacterial performance (low MIC).

Table 9 antibacterial peptide antibacterial performance comparison table

The recombinant S. cerevisiae prepared in the different examples described above was further tested for performance.

I. Detection of xylan activity by different recombinant Saccharomyces cerevisiae

experimental method:

The monoclonal clones of the largest hydrolyzed circle in each of the examples identified by Congo red staining were selected, and the recombinant Saccharomyces cerevisiae (Glam-x1, Glam-x2) constructed in the same manner as in Examples 2, 6, 7, and 8 were respectively taken. , Glam-x3, Glam-x4) and unmodified original host Saccharomyces cerevisiae, respectively, were placed in YPD liquid culture containing 0.15% xylan, inoculated according to 10% (v/v) inoculum, culture The conditions were 30 ° C, 200 rpm, and the culture time was 96 h or more. Samples were taken at 0h, 48h and 96h, the sample was centrifuged at 10000 rpm for 15 min, and the supernatant was taken. The content of xylose and xylooligosaccharide in the assay medium was determined by HPLC (mobile phase 0.05 M H ₂ SO ₄ , injection volume 20 μl). Take xylobiose and xylotriose as examples.

Experimental results:

The test results are shown in Table 10. It can be seen that the supernatant was aspirated at 48h and 96h for HPLC analysis. It was found that the concentration of Glam-x1 by fermentation to produce xylobiose and xylotriose was significantly higher than Glam-x2. Glam-x3 and Glam-x4 produced, in which Glut-x1, Glam-x3, Glam-x4, and Glam-x2 produced reduced amounts of xylobiose and xylotriose. The amount of xylose produced, Glam-x1 was also higher than that of the recombinant strains of other examples at 48 h, and was absorbed and consumed by S. cerevisiae at 96 h.

Table 10 Activity detection of xylan from different recombinant Saccharomyces cerevisiae

Second, the effect of different recombinant Saccharomyces cerevisiae on solid-state fermentation of xylan-containing raw materials

experimental method:

Recombinant Saccharomyces cerevisiae (Glam-x1, Glam-x2, Glam-x3, Glam-x4) constructed in the same manner as in Examples 2, 6, 7, and 8 and the unmodified original host Saccharomyces cerevisiae were taken separately. Fermentation of xylan-containing raw materials is as follows:

1) Inclined resuscitation: Different recombinant Saccharomyces cerevisiae were inoculated on the YPD agar slant, cultured at 30 ° C for 2 d, and resuscitated and activated.

2) Seed liquid activation: Single colonies were picked from the inclined surface, inoculated into a whole nutrient 50 ml of YPD liquid medium, and shake cultured at 30 ° C and 220 rpm.

3) Three-stage amplification liquid fermentation. According to the 500mL to 5L, and then to the 30L three-stage amplification, the inoculation amount is 10% (v / v), 10% (v / v), 16.7% (v / v), inoculation to ensure the amount of each group of Saccharomyces The same, so that a large number of bacteria are quickly obtained. The liquid fermentation medium is as follows: molasses is a carbon source, the three-stage amplification concentration is 15g/l→50g/l→100g/l; the corn slurry is nitrogen source, and the three-stage amplification concentration is 0.4% (v/v)→0.6 %(v/v)→0.8%(v/v); inorganic salts include 0.1% (w/v) magnesium sulfate, 0.05% (w/v) calcium chloride, 0.1% (w/v) potassium dihydrogen phosphate 0.1% (w/v) disodium hydrogen phosphate; The initial pH was adjusted at 6.0.

4) High density aerobic solid state fermentation. According to the following formula: according to the bagasse: bran: corn flour: soybean meal: xylan ratio is 41.9:25:18:15:0.1, the ratio of water to water is 1:1.44. The water includes liquid fermentation broth and partially supplemented distilled water (ratio 0.44:1), and the seed liquid inoculum is 44% (v/m, L/kg). Turn the material every 8 hours, increase the ventilation within 2 hours after the material is turned over, sprinkle the water or buffer to replenish the humidity, and adjust the pH to 6.0. The time of the high-density solid state fermentation was 3d.

5) Deep anaerobic fermentation breaks the wall. Stacking high-density solid fermented material, increasing the thickness of the layer, adding Hcl diluent to reduce the pH of the fermented material to 5.5, the temperature to 28 ° C, deep fermentation anaerobic 48 h; then, adding Hcl dilution to reduce the pH of the fermented material to 4.0, while the fermentation temperature was raised to 55 ° C for 20 h. Yeast break rate can reach 95%, no live yeast.

6) Refer to GB/T 6432-1994 "Determination of crude protein in feed" by Kjeldahl method to determine crude protein content; refer to national standard GB/T 6434-2006 "Determination of crude fiber content in feed"; reference GB/T 22492-2008 "Soybean Peptide Powder" to extract and determine the acid-soluble protein content; refer to GB/T 13093-2006 "Determination of the total number of bacteria in the feed" to detect the number of bacteria, HPLC analysis of xylooligosaccharides, yeast culture of recombinant bacteria The crude protein, acid-soluble protein, crude fiber, xylobiose, xylotriose, bacteria number (number of bacteria) and other indicators are compared with commercially available products.

Experimental results:

The test results are shown in Table 11, and it was found that the yeast cultures of the present invention are superior to the commercially available products, and provide sufficient amount of xylooligosaccharides (for example, xylobiose and xylotriose). The yeast culture of the present invention has significantly fewer bacteria than yeast and is smaller than the commercially available product and the control group. Among them, the recombinant Saccharomyces cerevisiae (Glam-x1) index of Example 2 is superior to the yeast culture of the recombinant Saccharomyces cerevisiae of the commercial product and other examples.

Table 11 Detection of product components for fermentation of xylan-containing raw materials by different recombinant Saccharomyces cerevisiae

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that are made without departing from the spirit and scope of the invention are intended to be equivalents and are included in the scope of the invention.

Claims (10)

1. A Saccharomyces cerevisiae multi-gene co-expression vector capable of producing xylooligosaccharides and antimicrobial peptides, characterized in that the carrier contains β-1,4-endo-xylanase gene and β-1,4-xyloside Enzyme gene, antimicrobial peptide gene;

   The base sequence of the β-1,4-endo-xylanase gene is shown in SEQ ID NO: 1.

   The base sequence of the β-1,4-xylosidase gene is shown in SEQ ID NO: 2.
2. The Saccharomyces cerevisiae multi-gene co-expression vector capable of producing xylooligosaccharide and antibacterial peptide according to claim 1, wherein the antibacterial peptide gene is selected from the group consisting of an oriental bell antibacterial peptide BLP-2 mutant BLP- 2-T, heterochromatic ladybug antibacterial peptide Haxy-Col1 mutant Haxy-Col1-T, salmon antibacterial peptide mutant, crispy frog antibacterial peptide mutant Lf-cath-T;

   The base sequence of the oriental bell antibacterial peptide BLP-2 mutant BLP-2-T is shown in SEQ ID NO: 3;

   The base sequence of the Hazel-Col1 mutant Haxy-Col1-T of the S. cerevisiae antibacterial peptide is shown in SEQ ID NO: 4;

   The base sequence of the salmon antibacterial peptide mutant is as shown in SEQ ID NO: 5;

   The base sequence of the crispy frog antibacterial peptide mutant Lf-cath-T is shown in SEQ ID NO: 6.
3. The Saccharomyces cerevisiae multi-gene co-expression vector capable of producing xylooligosaccharides and antimicrobial peptides according to claim 1, wherein an α-signal peptide gene sequence and an α-signal peptide gene are present upstream of the antimicrobial peptide gene. The base sequence is shown in SEQ ID NO: 7.
4. A Saccharomyces cerevisiae multi-gene co-expression vector capable of producing xylooligosaccharides and antimicrobial peptides according to claim 1, wherein the promoter of the β-1,4-endo-xylanase gene is Pgk1-1, the base sequence thereof is shown in SEQ ID NO: 8, the terminator is pgkt1-1, and the base sequence thereof is shown in SEQ ID NO: 9.

   The promoter of the β-1,4-xylosidase gene is pgk1-2, the base sequence thereof is shown in SEQ ID NO: 10, the terminator is pgkt1-2, and the base sequence thereof is SEQ ID NO: 11. Shown

   The promoter of the antimicrobial peptide gene is pgk1-3, the base sequence thereof is shown in SEQ ID NO: 12, the terminator is pgkt1-3, and the base sequence thereof is shown in SEQ ID NO: 13.
5. The Saccharomyces cerevisiae multi-gene co-expression vector capable of producing xylooligosaccharides and antimicrobial peptides according to claim 1, wherein the vector has a G418 resistance gene in the selection gene.
6. A Saccharomyces cerevisiae multi-gene co-expression vector capable of producing xylooligosaccharides and antimicrobial peptides according to claim 1, wherein the backbone of the vector is a pGAPZaA plasmid.
7. The Saccharomyces cerevisiae multi-gene co-expression vector capable of producing xylooligosaccharides and antimicrobial peptides according to claim 1, wherein the vector comprises a 25s rDNA gene fragment of Saccharomyces cerevisiae, the base sequence of which is SEQ. ID NO: 15 is shown.
8. A Saccharomyces cerevisiae capable of producing xylooligosaccharides and antimicrobial peptides, wherein the recombinant Saccharomyces cerevisiae genome is inserted with the multi-gene co-expression vector of any one of claims 1 to 7.
9. The method for constructing a Saccharomyces cerevisiae multi-gene co-expression vector capable of producing xylooligosaccharides and antimicrobial peptides according to any one of claims 1 to 6, comprising the steps of:

   The S1 integrated expression vector pTEGC-BsmBI was constructed:

   S1.1 ligated the G418 resistance gene between the multiple cloning sites Msc I and EcoR V of the pGAPZaA plasmid vector to obtain the vector pGAPZaA-G418;

   S1.2, the base sequence is ligated into the vector pGAPZaA-G418 multiple cloning site BamHI and EcoRI, and the vector pGAPZaA-G418-rDNA is obtained;

   S1.3 The vector pGAPZaA-G418-rDNA was digested with Bgl II and EcoRI, and the large fragment product was recovered to obtain a linearized vector pTEGC, and the BsmBI-2 fragment represented by SEQ ID NO: 16 was linear. The vector pTEGC was ligated to obtain the integrated expression vector pTEGC-BsmBI;

   S2 promoter, terminator amplification

   Amplification of S2.1 promoter: using S. cerevisiae genomic DNA as a template, primers were used to amplify pgk1- by PGK1F1-BsmBI and PGK1R1-BsmBI, PGK1F2-BsmBI and PGK1R2-BsmBI, PGK1F3-BsmBI and PGK1R3-BsmBI, respectively. 1. pgk1-2, pgk1-3 promoter fragment;

   Amplification of S2.2 terminator: using S. cerevisiae genomic DNA as a template, primers were used to amplify pgkt1-, PGKT1F1-BsmBI and PGKT1R1-BsmBI, PGKT1F2-BsmBI and PGKT1R2-BsmBI, PGKT1F3-BsmBI and PGKT1R3-BsmBI, respectively. 1. pgkt1-2, pgkt1-3 terminator fragment;

   Acquisition of S3α-signal peptide gene, α-amylase gene, glucoamylase gene and antimicrobial peptide gene

   S3.1 Acquisition of β-1,4-endo-xylanase gene containing BsmBI cleavage site: using T-vector containing β-1,4- endoxylanase gene sequence as template, by primer XynF-BsmBI and xynR-BsmBI are amplified to obtain a fragment of xynA1 gene, ie, a fragment containing a β-1,4-endo-xylanase gene;

   S3.2 Acquisition of β-1,4-xylosidase gene containing BsmBI cleavage site: T-vector containing β-1,4-xylosidase gene sequence as template, by primers xylF-BsmBI and xylR- BsmBI is amplified to obtain a fragment of xyl-1 gene, that is, a fragment containing a β-1,4-xylosidase gene;

   Obtainment of S3.3α-signal peptide-antibacterial peptide gene: The T-vector containing the α-signal peptide gene sequence and the T-vector containing the antimicrobial peptide were used as templates, and the α-signal peptide sequence was directionally linked to no signal by overlap extension PCR. The 5' end of the antibacterial peptide gene of the peptide amplifies the mfa-amp gene fragment, ie, the fragment containing the α-signal peptide gene sequence and the antibacterial peptide gene sequence; during the overlap extension PCR, the amplification product mfa is amplified by the primer - Both ends of the amp are introduced in opposite directions of cutting Cutting sequence of BsmBI;

   Construction of S4 Saccharomyces Cerevisiae Multi-gene Co-expression Vector

   The β-1,4-endoxylanase gene expression cassette element pgk1-1, xynA1, pgkt1-1 obtained above; β-1,4-xylosidase gene expression cassette element pgk1-2, xyl-1 , pgkt1-2; antibacterial peptide gene expression cassette elements pgk1-3, mfa-amp, pgkt1-3 were digested with the type IIs restriction endonuclease BsmBI, purified and recovered; meanwhile, cut with the type IIs restriction endonuclease BsmBI The above integrated expression vector pTEGC-BsmBI is linearized; the fragments used are ligated into the linearized integrated expression vector pTEGC-BsmBI by a one-step method to obtain a Saccharomyces cerevisiae multi-gene co-expression vector;

   The base sequences of the above primers are as follows:

   PGK1F1-BsmBI: CGTTCCAPidc GAAGTACCTTCAAAG

   PGK1R1-BsmBI: CGTCTCGGctaTATATTTGTTGTAAA

   PGK1F2-BsmBI: CGTCTCAgtcaGAAGTACCTTCAAAG

   PGK1R2-BsmBI: CGTCTCGGcatTATATTTGTTGTAAA

   PGK1F3-BsmBI: CGTCTCAtgcaGAAGTACCTTCAAAG

   PGK1R3-BsmBI: CGTCTCGtcgaTATATTTGTTGTAAA

   PGKT1F1-BsmBI: CGTCTCAtgtacGATCTCCCATCGTCTCTACT

   PGKT1R1-BsmBI: CGTCTCGGgtcaAAGCTTTTTCGAAACGCAG

   PGKT1F2-BsmBI: CGTCTCAtacgGATCTCCCATCGTCTCTACT

   PGKT1R2-BsmBI: CGTCTCGtgcaAAGCTTTTTCGAAACGCAG

   PGKT1F3-BsmBI: CGTCTCAatcgGATCTCCCATCGTCTCTACT

   PGKT1R3-BsmBI: CGTCTCGagtcAAGCTTTTTCGAAACGCAG

   xynF-BsmBI: CCTTCCAgcta ATGAAGGTTACTGCTGCT

   xynR-BsmBI: CGTCTCAgtac TTAAGAAGAGATAGTAACA

   xylF-BsmBI: CGTCTCAgcatATGCCAGGTGCTGCTTCTATCGTTGCT

   xylR-BsmBI: CGTCTCAtacg TTATTGTGGAGCGATCAATTGTTCT.
10. A method for constructing recombinant Saccharomyces cerevisiae capable of producing xylooligosaccharides and antimicrobial peptides, characterized in that the Saccharomyces cerevisiae multi-gene co-expression vector constructed according to claim 9 is transformed into a Saccharomyces cerevisiae host, positive monoclonal colonies are screened, and sequenced Properly verified, it is a recombinant Saccharomyces cerevisiae that produces xylooligosaccharides and antimicrobial peptides.

Images