WO2018113431A1 - 一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多功能酿酒酵母 - Google Patents

一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多功能酿酒酵母 Download PDF

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WO2018113431A1
WO2018113431A1 PCT/CN2017/109653 CN2017109653W WO2018113431A1 WO 2018113431 A1 WO2018113431 A1 WO 2018113431A1 CN 2017109653 W CN2017109653 W CN 2017109653W WO 2018113431 A1 WO2018113431 A1 WO 2018113431A1
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gene
bsmbi
saccharomyces cerevisiae
vector
cellulose
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张宏刚
康小龙
李莉
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广州格拉姆生物科技有限公司
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Definitions

  • the invention relates to the fields of genetic engineering and fermentation engineering, and more particularly to a multifunctional Saccharomyces cerevisiae which can degrade cellulose to produce probiotic oligosaccharides and secrete antibacterial peptides.
  • Cellulose is the most abundant renewable resource in the world. The development and utilization of cellulose can effectively promote the sustainable development of animal husbandry in China. Except for the rumen microorganisms of ruminants, which can ferment cellulose, other livestock themselves lack cellulase, which is not effective for cellulose or low utilization rate.
  • cello-oligosaccharide COS
  • COS cello-oligosaccharide
  • Cellooligosaccharides can generally be prepared by degradation of cellulose. The use of agricultural by-products such as straw, bagasse and other cellulose sources to produce cellooligosaccharides will have high environmental and economic benefits.
  • Cellulase and its metabolite cellooligosaccharide have the following effects: 1 supplementing the deficiency of endogenous enzymes in animals, stimulating enzyme secretion, for example, insufficient amount of cellulase in herbivores, which limits the digestion and absorption of crude fiber. It can be improved by supplementing the exogenous cellulase. 2 Synergistically with other enzymes to destroy plant cell walls and promote the absorption and utilization of nutrients. Cellulase can synergize with hemicellulase, pectinase and the like to destroy the cell wall of plants, release intracellular amylase and protease, and facilitate digestion and absorption of animals.
  • antimicrobial peptides have strong alkaline, thermal stability and broad-spectrum antibacterial properties.
  • Antibacterial peptides inhibit the growth of pathogenic bacteria. Different antimicrobial peptides have different killing ability against bacteria, fungi, protozoa and viruses; antibacterial peptides also have selective immune activation and regulation functions.
  • the invention co-expresses the cellulase gene and the antibacterial peptide gene through the Saccharomyces cerevisiae system to realize the secretion expression of the cellulase and the antimicrobial peptide.
  • the endo- ⁇ -1,4-glucanase gene, the exo- ⁇ -1,4-glucanase gene, and the ⁇ -glucanase gene in the cellulose degradation gene are classified as endo- ⁇ -1
  • the 4-glucanase gene and the exo- ⁇ -1,4-glucanase gene or the ⁇ -glucanase gene are combined to achieve the maximization of cellooligosaccharide and reduce the amount of glucose produced by cellulose. .
  • the technical problem to be solved by the present invention is to provide a method that can be applied in order to overcome the above-mentioned deficiencies of the prior art.
  • Feeding, industrial alcohol production, kitchen waste, etc. can secrete cellulase and degrade cellulose to produce probiotic oligosaccharides, and secrete antibacterial peptide multi-functional recombinant Saccharomyces cerevisiae.
  • the present invention is by exo- ⁇ -1,4-glucanase (Exo- ⁇ -1,4-glucanase, CBH) gene, endo- ⁇ -1,4-glucanase (Endo- ⁇ -1) , 4-glucanase, EG) gene, ⁇ -glucosidase ( ⁇ -D-Glucosidase, BGL) and other cellulose degradation related genes, and combined with antibacterial peptide (Antimicrobial peptide) gene, constructed into Saccharomyces cerevisiae integrated expression vector Into, it is transferred into Saccharomyces cerevisiae to achieve secretory expression, so that the synergistic action between cellulases produces more cellooligosaccharides, and the secreted antibacterial peptides have antibacterial activity, thereby obtaining multifunctional probiotic recombinant Saccharomyces cerevisiae.
  • Exo- ⁇ -1,4-glucanase, CBH endo- ⁇ -1,4-glucan
  • the object of the present invention is to provide a Saccharomyces cerevisiae multi-gene co-expression vector capable of degrading cellulose to produce probiotic oligosaccharides and secreting antibacterial peptides, and a method for constructing the same.
  • Another object of the present invention is to provide a multifunctional Saccharomyces cerevisiae which can degrade cellulose to produce probiotic oligosaccharides and secrete antibacterial peptides, and a method for constructing the same.
  • a Saccharomyces cerevisiae multi-gene co-expression vector capable of degrading cellulose to produce probiotic oligosaccharide and secreting antibacterial peptide the vector containing an enzyme gene and an antimicrobial peptide gene degrading cellulose;
  • the base sequence of the antimicrobial peptide gene is as shown in SEQ ID NO: 4;
  • the cellulose-degrading enzyme gene is selected from at least one of an exo- ⁇ -1,4-glucanase gene, an endo- ⁇ -1,4-glucanase gene, and a ⁇ -glucosidase gene.
  • the base sequence of the exo- ⁇ -1,4-glucanase gene is as shown in SEQ ID NO: 1
  • the base sequence of the endo- ⁇ -1,4-glucanase gene is As shown in SEQ ID NO: 2
  • the base sequence of the ⁇ -glucosidase gene is shown in SEQ ID NO: 3.
  • the cellulose-degrading enzyme gene is an exo- ⁇ -1,4-glucanase gene and an endo- ⁇ -1,4-glucanase gene.
  • an ⁇ -signal peptide gene sequence is present upstream of the antimicrobial peptide gene, and the base sequence of the ⁇ -signal peptide gene is as shown in SEQ ID NO: 5.
  • the promoter of the cellulose-degrading enzyme gene is selected from the group consisting of pgk1-1 and pgk1-2
  • the terminator is selected from the group consisting of pgkt1-1 and pgkt1-2
  • the promoter of the antimicrobial peptide gene is pgk1-3, terminating The child is pgkt1-3;
  • the base sequence of the pgk1-1 is as shown in SEQ ID NO: 6.
  • the base sequence of the pgkt1-1 is as shown in SEQ ID NO: 7.
  • the base sequence of the pgk1-2 is as shown in SEQ ID NO: 8.
  • the base sequence of the pgkt1-2 is as shown in SEQ ID NO:9;
  • the base sequence of the pgkt1-3 is shown in SEQ ID NO:11.
  • the backbone of the vector is a pGAPZaA plasmid.
  • the above vector contains a 25s rDNA gene fragment of Saccharomyces cerevisiae, and the nucleotide sequence thereof is shown in SEQ ID NO: 13.
  • a method for constructing a Saccharomyces cerevisiae multi-gene co-expression vector capable of degrading cellulose to produce probiotic oligosaccharides and secreting antibacterial peptides comprising the steps of:
  • the S1 integrated expression vector pTEGC-BsmBI was constructed:
  • the base sequence is ligated into the vector pGAPZaA-G418 multiple cloning site BamHI and EcoRI, and the vector pGAPZaA-G418-rDNA is obtained;
  • the vector pGAPZaA-G418-rDNA was double 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: 15 was linear.
  • the vector pTEGC was ligated to obtain the integrated expression vector pTEGC-BsmBI;
  • 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.
  • primers were used to amplify pgk1- by PGK1F1-BsmBI and PGK1R1-BsmBI, PGK1F2-BsmBI and PGK1R2-BsmBI, PGK1F3-BsmBI and PGK1R3-BsmBI, respectively.
  • 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.
  • S3.2 Endo- ⁇ -1,4-glucanase gene fragment containing BsmBI cleavage site egII using T vector containing endo- ⁇ -1,4-glucanase gene fragment egII as template
  • the specific primers egF-BsmBI and egR-BsmBI are amplified, and an endo- ⁇ -1,4-glucanase gene fragment egII containing a cleavage site of BsmBI is obtained;
  • S3.3 ⁇ -signal peptide-antibacterial peptide gene was obtained by using T-vector containing ⁇ -signal peptide gene sequence and T-vector containing antibacterial peptide as template, through primers MfaF4-BsmBI, Mfa-ampR, Mfa-ampF and Mfa -ampR-BsmBI for overlap extension PCR, the ⁇ -signal peptide sequence was ligated into the 5' end of the antibacterial peptide gene without signal peptide, and the mfa-amp gene fragment was amplified, ie, the ⁇ -signal peptide gene sequence and the antimicrobial peptide gene were contained. a fragment of a sequence;
  • exo- ⁇ -1,4-glucanase gene expression cassette element pgk1-1, cbh2, pgkt1-1 obtained above; endo- ⁇ -1,4-glucanase gene expression cassette element pgk1-2, EgII, 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, the type IIs restriction endonuclease BsmBI was utilized.
  • the above integrated expression vector pTEGC-BsmBI was cleaved and linearized; the fragments used were ligated into the linearized integrated expression vector pTEGC-BsmBI by one-step method to obtain a Saccharomyces cerevisiae multi-gene co-expression vector;
  • PGK1R1-BsmBI CGTCTCGGctaTATATTTGTTGTAAA
  • PGK1F2-BsmBI CGTCTCAgtcaGAAGTACCTTCAAAG
  • PGK1R2-BsmBI CGTCTCGGcatTATATTTGTTGTAAA
  • PGK1F3-BsmBI CGTCTCAtgcaGAAGTACCTTCAAAG
  • PGK1R3-BsmBI CGTCTCGtcgaTATATTTGTTGTAAA
  • PGKT1F1-BsmBI CGTCTCAtgtacGATCTCCCATCGTCTCTACT
  • PGKT1R1-BsmBI CGTCTCGGgtcaAAGCTTTTTCGAAACGCAG
  • PGKT1R2-BsmBI CGTCTCGtgcaAAGCTTTTTCGAAACGCAG
  • PGKT1R3-BsmBI CGTCTCGagtcAAGCTTTTTCGAAACGCAG
  • cbhR-BsmBI CGTCTCAgtacTTACAAGAAAGATGGGTTAGCGT
  • egR-BsmBI CGTCTCAtacg TTAGTTCTTCCACTTTGGAACGA
  • Mfa-ampR TGGACAACCGAAACCTCTTTTCTCGAGA
  • Mfa-ampF TCTCGAGAAAAGAGGTTTCGGTTGTCCA
  • Mfa-ampR-BsmBI CGTCTCAtagcTTACTTCTTACCAGAACA.
  • a method for constructing a multifunctional Saccharomyces cerevisiae capable of degrading cellulose to produce probiotic oligosaccharides and secreting antibacterial peptides transforming the Saccharomyces cerevisiae multi-gene co-expression vector constructed above into a Saccharomyces cerevisiae host, screening positive monoclonal colonies, and verifying by sequencing Correct, that is, multi-functional Saccharomyces cerevisiae that can degrade cellulose to produce probiotic oligosaccharides and secrete antimicrobial peptides.
  • Recombinant Saccharomyces cerevisiae can simultaneously secrete related enzymes and antibacterial peptides for fiber degradation.
  • Cellulose-degrading enzymes can supplement the deficiency or deficiency of endogenous cellulase, and can be adapted to the conditions of enzymes through the organic combination of cellulose degrading enzyme genes. Degradation of natural cellulose to produce cellooligosaccharides, thereby promoting the growth of probiotics.
  • the antibacterial peptide is directed against bacteria or pathogenic bacteria, and the antibacterial peptide can promote the immunity of the body and promote the growth of the body. This will play a role in the application of feed, kitchen waste and other applications.
  • 1 is a Congo red staining method to verify the cellulase activity of the recombinant Saccharomyces cerevisiae constructed in Example 2; 1 to 6 are the recombinant Saccharomyces cerevisiae constructed in Example 2, and SV is the host Saccharomyces cerevisiae as a negative control;
  • Example 2 is a test result of the antibacterial activity of the recombinant Saccharomyces cerevisiae against Staphylococcus aureus ATCC22023 in Example 2; in the figure, both are the fermentation broth of the recombinant Saccharomyces cerevisiae of the present invention, and "+" is ampicillin as a positive control; -" is H 2 O, as a negative control;
  • Figure 4 is a graph showing the effect of different recombinant Saccharomyces cerevisiae on the production of ethanol using natural cellulose;
  • Figure 5 is a graph showing the effect of different recombinant Saccharomyces cerevisiae on the production of cellobiose using natural cellulose;
  • Figure 6 is a graph showing the effect of different recombinant Saccharomyces cerevisiae on the production of cellotriose from natural cellulose.
  • Example 1 Construction of a Saccharomyces cerevisiae multi-gene co-expression vector capable of degrading cellulose to produce probiotic oligosaccharides and secreting antibacterial peptides
  • 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 DH5 ⁇ 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 the T-load without mutation.
  • the base sequence of the G418 resistance gene is shown in SEQ ID NO: 12.
  • 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 and the G418 resistance gene in the above-mentioned digested product were recovered and purified, and the G418 resistance gene was ligated into the vector pGAPZaA by T4 ligase to obtain the vector pGAPZaA-G418.
  • 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 DH5 ⁇ 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: 13.
  • 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.
  • 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 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 The restriction endonuclease cleaves Bgl II and EcoRI to cleave the plasmid pGAPZaA-G418-rDNA, and excises the GAP promoter and a-signal
  • the primers PMDF-BsmBI and PMDR-BsmBI 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 Infineon sequencing, and the sequencing was correct without mutation.
  • the base sequence of BsmBI-2 is shown in SEQ ID NO: 14.
  • 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.
  • 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.
  • the pgk1-1 promoter fragment (the base sequence is shown in SEQ ID NO: 6) was amplified using PGK1F1-BsmBI and PGK1R1-BsmBI primers (see Table 4) for expression. Cut ⁇ -1,4-gluco The promoter of the glycanase gene.
  • the S. cerevisiae gene DNA was used as a template, and the pgk1-2 promoter fragment (the base sequence is shown in SEQ ID NO: 8) was amplified using PGK1F2-BsmBI and PGK1R2-BsmBI primers (see Table 4).
  • a promoter expressing an endo- ⁇ -1,4-glucanase gene was amplified using PGK1F2-BsmBI and PGK1R2-BsmBI primers (see Table 4).
  • the pgk1-3 promoter fragment (the base sequence is shown in SEQ ID NO: 10) was amplified using PGK1F3-BsmBI and PGK1R3-BsmBI primers (see Table 4).
  • a promoter that expresses the antimicrobial peptide gene was amplified using PGK1F3-BsmBI and PGK1R3-BsmBI primers (see Table 4).
  • 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.
  • the pgkt1-1 terminator (the base sequence is shown in SEQ ID NO: 7) was amplified using the primers PGKT1F1-BsmBI and PGKT1R1-BsmBI (see Table 4) for expression of exo- ⁇ a terminator of the -1,4-glucanase gene.
  • the pgkt1-2 terminator (the base sequence is shown in SEQ ID NO: 9) was amplified using primers PGKT1F2-BsmBI and PGKT1R2-BsmBI (see Table 4) for expression of endo- ⁇ a terminator of the -1,4-glucanase 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: 11) was amplified using the primers PGKT1F3-BsmBI and PGKT1R3-BsmBI (see Table 4) for expression of the antimicrobial peptide gene. Terminator.
  • 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.
  • Mfa-BsmBI ie, ⁇ -signal containing Iis-type restriction endonuclease BsmBI sequence
  • MfaF and MfaR primers see Table 5
  • Fragment of the peptide gene sequence is as follows: 98 ° C for 10 s, 55 ° C for 15 s, 72 ° C for 30 s, 30 cycles, 72 ° C for 10 min; connect to the T vector, send samples for sequencing, select the correct positive clone, and thus
  • the ⁇ -signal peptide gene (the base sequence thereof is shown as SEQ ID NO: 5) is stored in the T vector.
  • 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.
  • exo- ⁇ -1,4-glucanase gene cbh2 refer to the exo- ⁇ -1,4-glucanase gene cbh2 of Trichoderma reesei published by NCBI (GenBank: KF992142) .1)
  • the base sequence of the exo- ⁇ -1,4-glucanase gene cbh2 optimized by artificial synthesis is shown in SEQ ID NO: 1.
  • endo- ⁇ -1,4-glucanase gene egII refer to the endo- ⁇ -1,4-glucanase gene egII of Penicillium decumbens published by NCBI (GenBank: EU315320) .1)
  • the base sequence of the endo- ⁇ -1,4-glucanase gene egII optimized by artificial synthesis is shown in SEQ ID NO: 2.
  • antibacterial peptide gene the antibacterial peptide Cg-Defh2 mutant Cg-Defh2-T of the long oyster Crassostrea gigas optimized by artificial synthesis, the base sequence of which is shown in SEQ ID NO: 4, and its amino acid sequence is GFCGCGDQYQCNRHCKSIGCRAGYCDAVTLWLPCTCTRCSGKK (SEQ ID NO: 43).
  • exo- ⁇ -1,4-glucanase gene cbh2 the endo- ⁇ -1,4-glucanase gene egII, and the antibacterial peptide Cg-Defh2 mutant Cg-Defh2-T gene sequence obtained above are respectively stored in In the pMD19-T Simple plasmid, spare.
  • Exo- ⁇ -1,4-glucanase gene fragment containing BsmBI cleavage site cbh2 using a T vector containing exo- ⁇ -1,4-glucanase gene fragment cbh2 as a template, with specificity
  • the primers cbhF-BsmBI and cbhR-BsmBI were amplified to obtain an exo- ⁇ -1,4-glucanase gene fragment cbh2 containing a cleavage site of BsmBI.
  • Endo- ⁇ -1,4-glucanase gene fragment containing BsmBI cleavage site egII using T-vector containing endo- ⁇ -1,4-glucanase gene fragment egII as template
  • the primers egF-BsmBI and egR-BsmBI were amplified to obtain an endo- ⁇ -1,4-glucanase gene fragment egII containing a cleavage site of BsmBI.
  • T-vector containing ⁇ -signal peptide gene sequence, T vector containing antibacterial peptide Cg-Defh2 mutant Cg-Defh2-T gene as template, and primer MfaF4- BsmBI, Mfa-ampR, Mfa-ampF, and Mfa-ampR-BsmBI (see Table 5) were ligated to the 5' end of the anti-peptide gene of the signal-free peptide by overlap extension PCR (SOE-PCR).
  • SOE-PCR overlap extension PCR
  • the above amplified gene fragments were ligated into the pMD19-Simple vector, and verified by sequencing, and the correct positive clones were retained.
  • the expression vector pTEGC-BsmBI was integrated and 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-cbh2-egII-amp was obtained, transformed into E. coli DH5a, and the transformants were selected and verified by sequencing.
  • the positive transformants were ligated, and the plasmid was extracted to obtain a multi-gene co-expression vector pTEGC-cbh2-egII-amp capable of degrading cellulose to produce probiotic oligosaccharides and secreting antibacterial peptides.
  • Example 2 Construction of a multifunctional Saccharomyces cerevisiae capable of degrading cellulose to produce probiotic oligosaccharides and secreting antimicrobial peptides
  • the multi-gene co-expression vector pTEGC-cbh2-egII-amp constructed in Example 1 was transformed into E. coli DH5a for activation and cultured in liquid LB medium overnight, and the plasmid was extracted and purified.
  • the restriction endonuclease HpaI was used for linearization digestion, recovered and purified, and used.
  • the multi-gene co-expression vector pTEGC-cbh2-egII-amp was linearized and transferred to S. cerevisiae using the electroporation transformation method with reference to the instructions of the Bio-Rad clicker (resistance screening has been performed, and the G418 tolerance concentration is up to 200 ⁇ g.
  • the G418 tolerance concentration is up to 200 ⁇ g.
  • the verified transformants were subjected to high-resistance screening in YPD liquid medium containing different concentrations of G418, and the obtained monoclonal colonies were verified by sequencing to obtain correctly linked positive recombinant yeast transformants, which can degrade cellulose production probiotics.
  • the recombinant Saccharomyces cerevisiae which degraded cellulose to produce probiotic oligosaccharides and secreted antibacterial peptides constructed in this example was transferred to YP agar plates containing 1% CMC-Na (recipe as follows: 15 g/l agar, 10 g/l CMC- In Na), culture 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.
  • Fig. 1 to 6 are the recombinant Saccharomyces cerevisiae constructed in this example, and the SV is the host Saccharomyces cerevisiae as a negative control. It can be seen that the different monoclonal clones of the recombinant Saccharomyces cerevisiae constructed in this example There are obvious hydrolyzed circles around, indicating that the recombinant Saccharomyces cerevisiae constructed in this example has good cellulase activity.
  • the recombinant Saccharomyces cerevisiae which can degrade cellulose and produce probiotic oligosaccharide and secrete antibacterial peptide constructed in this example was transferred to YPD liquid medium, cultured for 60 hours, the culture solution was aspirated, and the supernatant was collected by centrifugation, and the national standard GB/T was referred to. 23881-2009 Determination of feed cellulase activity (filter paper method) Determination of total cellulase activity.
  • the fermentation broth of the recombinant Saccharomyces cerevisiae obtained in the present example was added to an Oxford cup, the sterilized water was used as a negative control, and ampicillin (1.5 ⁇ g) was used as a positive control, and cultured at 37 ° C for 16-18 hours to observe the inhibition zone.
  • Example 3 Construction of a multi-gene co-expression vector capable of degrading cellulose to produce probiotic oligosaccharides and secreting antimicrobial peptides
  • the method for constructing the S. cerevisiae multi-gene co-expression vector in this example is the same as in Example 1, except that the exo- ⁇ -1,4-glucanase gene ligated into the vector is replaced with the ⁇ -glucosidase gene (bgl) ( The base sequence is as shown in SEQ ID NO: 3) Further, the ⁇ -glucosidase gene was obtained by codon-optimized and chemically synthesized with reference to the NCBI-expressed ⁇ -glucosidase gene bgl (GenBank: D64088.1) of Aspergillus aculeatus, and the others were the same as in Example 1.
  • the Saccharomyces cerevisiae multi-gene co-expression vector constructed in this example was named pTEGC-bgl-egII-amp.
  • Example 4 Construction of a multi-gene co-expression vector capable of degrading cellulose to produce probiotic oligosaccharides and secreting antimicrobial peptides
  • the method for constructing the S. cerevisiae multi-gene co-expression vector is the same as in Example 1, except that the endo- ⁇ -1,4-glucanase gene ligated into the vector is replaced with the ⁇ -glucosidase gene (base sequence).
  • the ⁇ -glucosidase gene is codon-optimized, chemically synthesized with reference to NCBI
  • the ⁇ -glucosidase gene bgl (GenBank: D64088.1) of Aspergillus aculeatus.
  • the Saccharomyces cerevisiae multi-gene co-expression vector constructed in this example was named pTEGC-cbh2-bgl-amp.
  • Example 5 Construction of a multifunctional Saccharomyces cerevisiae capable of degrading cellulose to produce probiotic oligosaccharides and secreting antimicrobial peptides
  • the multi-gene co-expression vector pTEGC-bgl-egII-amp constructed in Example 3 was transformed into E. coli DH5a for activation and cultured in liquid LB medium overnight, and the plasmid was extracted and purified.
  • the restriction endonuclease HpaI was used for linearization, purification and purification.
  • the pTEGC-bgl-egII-amp linearized by electroporation was transferred into Saccharomyces cerevisiae (reacted with reference to the Bio-Rad clicker).
  • the G418 tolerance was up to 200 ⁇ g/ml medium, and cultured on a YPD plate with a G418 concentration of 300 ⁇ g/ml for more than 48 hours, and the single colony grown was selected as a transformant.
  • the verified transformants were subjected to high-resistance screening in YPD liquid medium containing different concentrations of G418, and the obtained monoclonal colonies were verified by sequencing to obtain correctly linked positive recombinant yeast transformants, which can degrade cellulose production probiotics.
  • a multifunctional Saccharomyces cerevisiae which is a cellooligosaccharide and secretes an antimicrobial peptide, referred to as Glam-c2.
  • Example 6 Construction of a multifunctional Saccharomyces cerevisiae capable of degrading cellulose to produce probiotic oligosaccharides and secreting antimicrobial peptides
  • the multi-gene co-expression vector pTEGC-cbh2-bgl-amp constructed in Example 4 was transformed into E. coli DH5a for activation and cultured in liquid LB medium overnight, and the plasmid was extracted and purified.
  • the restriction endonuclease HpaI was used for linearization digestion, purification and purification.
  • the pTEGC-cbh2-bgl-amp linearized by electroporation was transferred into Saccharomyces cerevisiae (with resistance) according to the instructions of Bio-Rad clicker.
  • the G418 tolerance was up to 200 ⁇ g/ml medium, and cultured on a YPD plate with a G418 concentration of 300 ⁇ g/ml for more than 48 hours, and the single colony grown was selected as a transformant.
  • the verified transformants were subjected to high-resistance screening in YPD liquid medium containing different concentrations of G418, and the obtained monoclonal colonies were verified by sequencing to obtain correctly linked positive recombinant yeast transformants, which can degrade cellulose production probiotics.
  • a multifunctional Saccharomyces cerevisiae that is a cellooligosaccharide and secretes an antimicrobial peptide referred to as Glam-c3.
  • the antibacterial peptide Cg-Defh2 of the long oyster Crassostrea gigas and its mutant Cg-Defh2-T (the base sequence of which is shown in SEQ ID NO: 4) were synthesized by the biotechnology company.
  • Escherichia coli CICC10899 and Staphylococcus aureus As an indicator strain, ATCC22023 was used to detect the size of the inhibition zone of different types of antimicrobial peptides by the Oxford Cup method (repeated 3 times), and compared the size of the inhibition zone of the antimicrobial peptide before and after the mutation.
  • the outer diameter of the Oxford Cup is 7.8mm.
  • 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).
  • Example 7 The test results are shown in Table 7. It can be seen that the fermentation broth of the recombinant Saccharomyces cerevisiae constructed in Example 2 has the most obvious inhibition zone against Staphylococcus aureus ATCC22023 and Bacillus subtilis, and the antibacterial activity is the strongest; Example 6 The recombinant Saccharomyces cerevisiae constructed by -8 had antibacterial effects (inhibition zone size) against Staphylococcus aureus ATCC22023 and Escherichia coli CICC10899, but slightly lower than that of Example 2.
  • the outer diameter of the Oxford Cup is 7.8mm.
  • the strains identified by PCR and identified by Congo red staining were selected to obtain the same amount of activated recombinant Saccharomyces cerevisiae constructed in Examples 2, 5 and 6 and the unmodified original host Saccharomyces cerevisiae, respectively.
  • YPD plus 0.5% of the 200-grafted bagasse cellulosic material as the inducing substrate
  • the cellulase activity of the feed GB/T 23881-2009 was referred to.
  • the measurement measures the total cellulase activity.
  • the "one" fermentation supernatant was collected by centrifugation, and the fermentation supernatant was subjected to ultrafiltration concentration using an appropriate size ultrafiltration tube to obtain the recombinant bacteria Glam-c1, Glam-c2, Glam- constructed in Examples 2, 5 and 6.
  • the concentrated cellulase concentrate secreted by c3 and the concentrated solution of the fermentation broth of the host recombinant bacteria were subjected to filtration sterilization treatment using a 0.2 um filter.
  • the recombinant bacteria group was added to the cellulose substrate of 200-graft bagasse according to the total cellulase activity of 20 U/g.
  • the pH was controlled at 5.5 with buffer and enzymatically hydrolyzed at 50 °C for 48 h. Cellobiose and cellotriose and glucose content were measured.
  • the results of the total cellulase enzyme activity assay are shown in Table 8.
  • the total cellulase activity of Glam-c1, Glam-c2, and Glam-c3 was about 1.0 U/ml or higher, which was higher than that of the control host brewing yeast.
  • Example 2 constructed recombinant S. cerevisiae Glam-c1 highest.
  • the test results of the ability of each recombinant Saccharomyces cerevisiae to degrade fibers were as shown in Table 9, and the weights of Examples 2, 5, and 6 were constructed.
  • the concentrations of cellobiose, cellotriose and glucose obtained by the group of Saccharomyces cerevisiae Glam-c1, Glam-c2 and Glam-c3 after 48h digestion were significantly higher than those of the host Saccharomyces cerevisiae, and the recombinant bacteria constructed in Example 2
  • the cellobiose, cellotriose and glucose obtained after Glam-c1 digestion have the highest concentration.
  • Example 2 The above results indicate that the construction of recombinant S. cerevisiae in Example 2 has a good activity for degrading cellulose to produce probiotic oligosaccharides, that is, exo- ⁇ -1,4-glucanase, endo- ⁇ in recombinant Saccharomyces cerevisiae
  • probiotic oligosaccharides that is, exo- ⁇ -1,4-glucanase, endo- ⁇ in recombinant Saccharomyces cerevisiae
  • the recombinant Saccharomyces cerevisiae has the best cellulase activity and is capable of obtaining the highest cellooligosaccharide.
  • the straw slag is pretreated as follows: washed, dried, pulverized, passed through a standard 250 mesh sieve, and used.
  • Activated S. cerevisiae was grown by seed medium to an OD 600 to 6.0 with seed medium as follows: 2% (w/w) corn syrup, 5% (w/w) glucose.
  • the recombinant Saccharomyces cerevisiae was added to the fermentation medium (the medium formula was as follows: 15% (w/w) straw slag, 3% (w/w) corn syrup, 1 % ammonium sulfate), cultured for more than 96h.
  • the medium formula was as follows: 15% (w/w) straw slag, 3% (w/w) corn syrup, 1 % ammonium sulfate), cultured for more than 96h.
  • a standard curve was prepared by HPLC to measure the ethanol content, cellooligosaccharides such as cellobiose and cellotriose.
  • the detection results are shown in 4 to 6. It can be seen that the recombinant bacteria Glam-c1, Glam-c2 and Glam-c3 can produce ethanol using natural cellulose, wherein the recombinant Saccharomyces cerevisiae Glam-c1 constructed in Example 2 utilizes fibers.
  • the enzyme degrades the glucose produced by the cellulose substrate and the residual sugar of the substrate material to produce ethanol concentration, and the cellulase enzymatic substrate produces the highest concentration of cellobiose and cellotriose; the recombinant Saccharomyces cerevisiae Glam- constructed in Example 6
  • the recombinant Saccharomyces cerevisiae Glam-c2 constructed in Example 5 is the lowest; the host Saccharomyces cerevisiae can utilize the residual sugar in the raw material to produce ethanol, and the substrate raw material is removed.
  • the residual cellooligosaccharide does not substantially form cellooligosaccharides (cellobiose and cellotriose). It can be seen that the recombinant Saccharomyces cerevisiae Glam-c1 constructed in Example 2 has the best cellulose degradation ability, and the expression of the related enzyme gene is the best.
  • Recombinant Saccharomyces cerevisiae ie, Glam-c1, Glam-c2, and Glam-c3 constructed in the same manner as in Examples 2, 5, and 6 and unmodified original host Saccharomyces cerevisiae for high-fiber solid-state fermentation, respectively.
  • the medium is fermented, and the specific method is as follows:
  • the solid fermentation medium is: bagasse (pulverized over 200 mesh): bran: corn flour: the ratio of soybean meal is 10:30:20:40, and the ratio of feed to water is 1:1.4.
  • the inoculum of seed solution was 40% (v/m, L/kg). The air was passed through the shallow pan, and the material was periodically turned and cultured at 28 °C for 48 h. The thickness of the layer was further increased and deep anaerobic fermentation was carried out for 48 h.
  • the material is dried at low temperature, pulverized, and packaged to obtain a yeast culture.
  • a total of 180 weaned piglets of 35 days old were randomly divided into 6 treatment groups with 3 replicates in each group. During the test period of 40 days, the six treatment groups were:
  • Basal diet (blank control group);
  • the results of the yeast culture component detection are shown in Table 10. As can be seen from the above, the recombinant brewing yeast (Glam-C1) of Example 2 fermented the medium containing the high fiber material, and the cellooligosaccharide content was the highest, and the number of bacteria was the least.
  • Table 10 Composition test table of yeast culture after fermentation of different recombinant Saccharomyces cerevisiae
  • Cellooligosaccharides mainly detect cellobiose and cellotriose.
  • the results of the feeding animal effect test are shown in Table 11, and the diarrhea rate of the yeast culture (Group A) using the recombinant Saccharomyces cerevisiae (Glam-C1) of Example 2 was lower than that of the control group and the commercially available yeast culture group D.
  • Other indicators are also better than other groups. Among them, the indicators of the E group of the commercially available yeast culture supplemented with cellooligosaccharide were close to the group A.
  • the genetically modified Saccharomyces cerevisiae of Examples 5 and 6 had higher index of Group B (Glam-C2) and Group C (Glam-C3) than Group D of the commercially available yeast culture, and was close to Group A.
  • the yeast culture of the recombinant Saccharomyces cerevisiae of Example 2 can exhibit the advantages of the yeast culture, and at the same time, it can supplement the cellooligosaccharide and exert its probiotic effect.

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Abstract

提供一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多功能酿酒酵母，该重组酿酒酵母中含有能利用能降解纤维素生产益生纤维寡糖并分泌抗菌肽的酿酒酵母多基因共表达载体，该载体中含有降解纤维素的酶基因和抗菌肽基因。

Description

一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多功能酿酒酵母 技术领域
本发明涉及基因工程和发酵工程等领域，更具体地，涉及一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多功能酿酒酵母。
背景技术
纤维素是世界上最丰富的可再生资源，开发和利用纤维素能够有效地促进我国畜牧业可持续发展的。除反刍动物瘤胃微生物可发酵利用纤维素外，其他家畜本身缺乏纤维素酶(cellulase)，对纤维素不能有效利用或利用率低。此外，纤维素降解产物之一纤维寡糖(cello-oligosaccharide，COS)，是一种新型的功能性寡糖，由2～7个吡喃型葡萄糖单元以β-1，4-糖苷键连接而成。纤维寡糖一般可以通过纤维素降解而制备。利用农业副产物例如秸秆、甘蔗渣等纤维素来源来生产纤维寡糖将具有较高的环境效益和经济效益。
纤维素酶及其代谢产物纤维寡糖等具有以下作用：①补充动物内源酶的不足，刺激酶分泌，例如在草食动物中的纤维素酶量不足，会使粗纤维的消化吸收受到限制，补充外源纤维素酶后可以得到改善。②与其他酶类协同作用，破坏植物细胞壁，促进营养物质吸收利用。纤维素酶可与半纤维素酶、果胶酶等协同作用，破坏植物的细胞壁，使胞内的淀粉酶和蛋白酶释放，利于动物消化吸收。③消除非淀粉多糖类抗营养因子，提高营养价值；④提高机体的代谢水平，增强免疫力，例如维持小肠绒毛形态完整性⑤改善动物微生态环境。纤维寡糖可促进有益微生物的生长，增加单细胞蛋白的含量。因而，通过对纤维素酶的研究是纤维素作为饲料开发和利用的关键。
抗菌肽(antimicrobial peptide)这类活性多肽多数具有强碱性、热稳定性以及广谱抗菌等特点。抗菌肽抑制致病菌生长，不同抗菌肽对细菌、真菌、原虫以及病毒等具有不同杀伤能力；抗菌肽还具有选择性免疫激活和调节功能。
本发明通过酿酒酵母系统将纤维素酶基因和抗菌肽基因共表达，实现纤维素酶和抗菌肽分泌表达。其中，将纤维素降解基因中内切β-1,4-葡聚糖酶基因、外切β-1,4-葡聚糖酶基因、β-葡聚糖酶基因，按照以内切β-1,4-葡聚糖酶基因和外切β-1,4-葡聚糖酶基因或β-葡聚糖酶基因的俩俩搭配，实现纤维寡糖的最大化，减少纤维素生产葡萄糖的量。将重组酿酒酵母应用到饲料添加、动物养殖以及餐厨废弃物降解中，可以有效发挥其协同作用，发挥益生作用，既促进益生菌的生长，又同时抑制致病菌。
发明内容
本发明所要解决的技术问题是，为了克服现有技术的上述不足，提供一种能够应用在 饲料添加、工业酒精生产、餐厨废弃物等中能分泌纤维素酶并降解纤维素生产益生纤维寡糖的，且分泌抗菌肽多功能重组酿酒酵母。
本发明是通过将外切β-1,4-葡聚糖酶(Exo-β-1,4-glucanase，CBH)基因、内切β-1,4-葡聚糖酶(Endo-β-1,4-glucanase，EG)基因、β-葡萄糖苷酶(β-D-Glucosidase，BGL)等纤维素降解相关基因的合理搭配，再结合抗菌肽(Antimicrobial peptide)基因，构建到酿酒酵母整合表达载体中，转入到酿酒酵母中，实现分泌表达，从而纤维素酶间的协同作用产生更多的纤维寡糖，分泌出的抗菌肽类具有抗菌活性，从而得到具有多功能益生重组酿酒酵母。
本发明的目的在于提供一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的酿酒酵母多基因共表达载体及其构建方法。
本发明的另一目的在于提供一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多功能酿酒酵母及其构建方法。
本发明所采取的技术方案是：
一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的酿酒酵母多基因共表达载体，该载体中含有降解纤维素的酶基因、抗菌肽基因；
所述抗菌肽基因的碱基序列如SEQ ID NO：4所示；
所述降解纤维素的酶基因选自外切β-1,4-葡聚糖酶基因、内切β-1,4-葡聚糖酶基因、β-葡萄糖苷酶基因中的至少一种。
进一步的，所述外切β-1,4-葡聚糖酶基因的碱基序列如SEQ ID NO：1所示，所述内切β-1,4-葡聚糖酶基因的碱基序列如SEQ ID NO：2所示，所述β-葡萄糖苷酶基因的碱基序列如SEQ ID NO：3所示。
进一步的，所述降解纤维素的酶基因为外切β-1,4-葡聚糖酶基因和内切β-1,4-葡聚糖酶基因。
进一步的，上述抗菌肽基因上游存在α-信号肽基因序列，α-信号肽基因的碱基序列如SEQ ID NO：5所示。
进一步的，所述降解纤维素的酶基因的启动子选自pgk1-1、pgk1-2，终止子选自pgkt1-1、pgkt1-2；所述抗菌肽基因的启动子为pgk1-3，终止子为pgkt1-3；
所述pgk1-1的碱基序列如SEQ ID NO：6所示；
所述pgkt1-1的碱基序列如SEQ ID NO：7所示；
所述pgk1-2的碱基序列如SEQ ID NO：8所示；
所述pgkt1-2的碱基序列如SEQ ID NO：9所示；
所述pgk1-3，的碱基序列如SEQ ID NO：10所示；
所述pgkt1-3的碱基序列如SEQ ID NO：11所示。
进一步的，所述载体的骨架为pGAPZaA质粒。
进一步的，上述载体中含有酿酒酵母菌的25s rDNA基因片段，其碱基序列如SEQ ID NO：13所示。
一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多功能酿酒酵母，该重组酿酒酵母基因组中插入有上述任一所述的多基因共表达载体。
一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的酿酒酵母多基因共表达载体的构建方法，其特征在于：包括以下步骤：
S1整合表达载体pTEGC-BsmBI构建：
S1.1将G418抗性基因连入pGAPZaA质粒载体的多克隆位点Msc I和EcoR V之间，获得载体pGAPZaA-G418；
S1.2将碱基序列如SEQ ID NO：13所示的rDNA基因序列的连入载体pGAPZaA-G418多克隆位点BamHI和EcoRI之间，获得载体pGAPZaA-G418-rDNA；
S1.3将载体pGAPZaA-G418-rDNA经Bgl II和EcoRI双酶切后，回收大片段产物，得到线性化载体pTEGC，将碱基序列如SEQ ID NO：15所示的BsmBI-2片段与线性化载体pTEGC连接，得整合表达载体pTEGC-BsmBI；
S2启动子、终止子的扩增
S2.1启动子的扩增：以酿酒酵母基因组DNA为模板，分别用引物对PGK1F1-BsmBI和PGK1R1-BsmBI、PGK1F2-BsmBI和PGK1R2-BsmBI、PGK1F3-BsmBI和PGK1R3-BsmBI分别扩增出pgk1-1、pgk1-2、pgk1-3启动子片段；
S2.2终止子的扩增：以酿酒酵母基因组DNA为模板，分别用引物对PGKT1F1-BsmBI和PGKT1R1-BsmBI、PGKT1F2-BsmBI和PGKT1R2-BsmBI、PGKT1F3-BsmBI和PGKT1R3-BsmBI分别扩增出pgkt1-1、pgkt1-2、pgkt1-3终止子片段；
S3α-信号肽基因、外切β-1,4-葡聚糖酶基因、内切β-1,4-葡聚糖酶基因、抗菌肽基因的获得
S3.1含BsmBI酶切位点的外切β-1,4-葡聚糖酶基因的获得：以含外切β-1,4-葡聚糖酶基因序列的T载体为模板，通过引物cbhF-BsmBI以及cbhR-BsmBI进行扩增，获得含有BsmBI的切割位点的外切β-1,4-葡聚糖酶基因片段cbh2；
S3.2含BsmBI酶切位点的内切β-1,4-葡聚糖酶基因片段egII：以含内切β-1,4-葡聚糖酶基因片段egII的T载体为模板，用特异引物egF-BsmBI以及egR-BsmBI进行扩增，获得含有BsmBI的切割位点的内切β-1,4-葡聚糖酶基因片段egII；
S3.3α-信号肽-抗菌肽基因的获得：分别以含α-信号肽基因序列的T载体、含抗菌肽的T载体为模板，通过引物MfaF4-BsmBI、Mfa-ampR、Mfa-ampF以及Mfa-ampR-BsmBI进行重叠延伸PCR将α-信号肽序列定向连入无信号肽的抗菌肽基因的5’端，扩增出mfa-amp基因片段，即含有α-信号肽基因序列和抗菌肽基因序列的片段；
S4酿酒酵母多基因共表达载体的构建
将上述获得的外切β-1,4-葡聚糖酶基因表达盒元件pgk1-1、cbh2、pgkt1-1；内切β-1,4-葡聚糖酶基因表达盒元件pgk1-2、egII、pgkt1-2；抗菌肽基因表达盒元件pgk1-3、mfa-amp、pgkt1-3利用IIs型限制性内切酶BsmBI进行酶切，纯化回收；同时，利用IIs型限制性内切酶BsmBI切割上述整合表达载体pTEGC-BsmBI，将其线性化；将所用这些片段通过一步法定向连入线性化的整合表达载体pTEGC-BsmBI中，即得酿酒酵母多基因共表达载体；
上述所述引物的碱基序列如下：
PGK1F1-BsmBI：CGTCTCAgatc GAAGTACCTTCAAAG
PGK1R1-BsmBI：CGTCTCGgctaTATATTTGTTGTAAA
PGK1F2-BsmBI：CGTCTCAgtcaGAAGTACCTTCAAAG
PGK1R2-BsmBI：CGTCTCGgcatTATATTTGTTGTAAA
PGK1F3-BsmBI：CGTCTCAtgcaGAAGTACCTTCAAAG
PGK1R3-BsmBI：CGTCTCGtcgaTATATTTGTTGTAAA
PGKT1F1-BsmBI：CGTCTCAtgtacGATCTCCCATCGTCTCTACT
PGKT1R1-BsmBI：CGTCTCGgtcaAAGCTTTTTCGAAACGCAG
PGKT1F2-BsmBI：CGTCTCAtacgGATCTCCCATCGTCTCTACT
PGKT1R2-BsmBI：CGTCTCGtgcaAAGCTTTTTCGAAACGCAG
PGKT1F3-BsmBI：CGTCTCAatcgGATCTCCCATCGTCTCTACT
PGKT1R3-BsmBI：CGTCTCGagtcAAGCTTTTTCGAAACGCAG
cbhF-BsmBI：CGTCTCAgcta ATGATCGTTGGTATCTTGACTACT
cbhR-BsmBI：CGTCTCAgtacTTACAAGAAAGATGGGTTAGCGT
egF-BsmBI：CGTCTCAgcatATGAGATCTTCTCACAAGAGAAACT
egR-BsmBI：CGTCTCAtacg TTAGTTCTTCCACTTTGGAACGA
MfaF4-BsmBI：CGTCTCAtcga ATGAGATTTCCTTCAATTTTTAC
Mfa-ampR：TGGACAACCGAAACCTCTTTTCTCGAGA
Mfa-ampF：TCTCGAGAAAAGAGGTTTCGGTTGTCCA
Mfa-ampR-BsmBI：CGTCTCAtagcTTACTTCTTACCAGAACA。
一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多功能酿酒酵母的构建方法，将上述构建的酿酒酵母多基因共表达载体转化酿酒酵母宿主，筛选出阳性单克隆菌落，并测序验证正确，即得能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多功能酿酒酵母。
本发明的有益效果是：
基因重组酿酒酵母能够同时分泌纤维降解的相关酶以及抗菌肽，纤维素降解酶可以补充个内源性纤维素酶的缺乏或不足，通过纤维素降解酶基因间的有机搭配，能够在酶适宜条件下对天然纤维素进行降解生产纤维寡糖，从而能促进益生菌的生长等。抗菌肽针对杂菌或致病菌，同时抗菌肽能够促进机体的免疫力提高，促进机体的生长。从而在饲料添加、餐厨废弃物等等应用领域发挥作用。
附图说明
图1为刚果红染色法验证实施例2构建的重组酿酒酵母的纤维素酶活；1～6均为实施例2构建的重组酿酒酵母，SV为宿主酿酒酵母作为阴性对照；
图2为实施例2重组酿酒酵母对金色葡萄球菌ATCC22023的抑菌活性检测结果；图中①、②均为本发明重组酿酒酵母菌的发酵液，“+”为氨苄青霉素，作为阳性对照；“-”为H2O，作为阴性对照；
图3为实施例2重组酿酒酵母对枯草芽孢杆菌的抑菌活性检测结果；图中①、②均为本发明重组酿酒酵母菌的发酵液，“+”为氨苄青霉素，作为阳性对照；“-”为H2O，作为阴性对照；
图4为不同重组酿酒酵母利用天然纤维素生产乙醇的效果检测；
图5为不同重组酿酒酵母利用天然纤维素生产纤维二糖的效果检测；
图6为不同重组酿酒酵母利用天然纤维素生产纤维三糖的效果检测。
具体实施方式
下面结合具体实施例对本发明作进一步的说明，但并不局限于此。
实施例1一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的酿酒酵母多基因共表达载体的构建
一、整合表达载体pTEGC-BsmBI构建
1)G418抗性基因的获得
PCR扩增目的基因，以载体pPIC9k为模板，利用G418F-MscI和G418R-EcoRV引物(表1)扩增G418抗性基因。PCR反应条件：98℃10s，55℃15s，72℃50s，30个循环，72℃10min。经2％琼脂糖凝胶电泳验证。
目的基因回收、纯化、转化大肠杆菌、验证、送样测序。回收纯化目的片段，保存于-20℃备用。将得到的G418抗性基因与T载体连接，转化大肠杆菌DH5α菌株，37℃培养，提取其质粒DNA，利用G418F-MscI和G418R-EcoRV引物进行菌落PCR筛选阳性菌株，将阳性克隆送至英潍捷基测序验证基因的正确性。测序结果表明：G418抗性基因及其酶切位点正确连入T载，未发生突变，G418抗性基因的碱基序列如SEQ ID NO：12所示。
表1 扩增G418抗性基因引物
Figure PCTCN2017109653-appb-000001
注：下划线处字母为限制性内切酶的识别/切割序列。
2)载体pGAPZaA-G418的构建
在37℃下，利用限制性内切酶MscI和EcoRV切割pGAPZaA质粒，并在1.5％的琼脂糖凝胶电泳验证；利用限制性内切酶切割MscI和EcoRV切割pMD-G418载体，获得G418抗性基因，在1.5％的琼脂糖凝胶电泳验证；回收纯化上述酶切产物中的pGAPZaA载体、G418抗性基因，利用T4连接酶将G418抗性基因连入载体pGAPZaA，获得载体pGAPZaA-G418。
3)rDNA基因扩增
以酿酒酵母基因组DNA为模板，采用引物rDNAF和rDNAR引物(见表2)PCR扩增rDNA基因；PCR扩增条件为：98℃10s，55℃15s，72℃60s，30个循环，72℃10min；在1％琼脂糖凝胶电泳验证，并在上下游分别引入EcoRI和BamHI酶切位点。
将得到的rDNA基因与T载体连接，转化大肠杆菌DH5α菌株，37℃培养，提取其质粒DNA，利用rDNAF和rDNAR引物进行菌落PCR筛选阳性菌株。测序结果表明：rDNA基因及其酶切位点正确连入T载，未发生突变，rDNA基因的碱基序列如SEQ ID NO：13所示。
表2 扩增rDNA基因引物
Figure PCTCN2017109653-appb-000002
Figure PCTCN2017109653-appb-000003
注：下划线处字母为限制性内切酶的识别/切割序列。
4)载体pGAPZaA-G418-rDNA构建
利用限制性内切酶BamHI和EcoRI分切下上述T载体上的rDNA片段、切割质粒pGAPZaA-G418，回收纯化pGAPZaA-G418载体骨架，利用T4连接酶将rDNA连入线性化后的载体pGAPZaA-G418，获得重组载体pGAPZaA-G418-rDNA。
5)整合表达载体pTEGC-BsmBI构建
限制性内切酶切割Bgl II和EcoRI切割质粒pGAPZaA-G418-rDNA，切除该载体上BglII到EcoRI酶切位点间的GAP启动子、a-信号肽等序列，回收大片段产物，得到线性化载体pTEGC。
以pMD19-T simple载体为模板，通过引物PMDF-BsmBI和PMDR-BsmBI(见表3)扩增出含有2个BsmBI酶切位点识别序列，约233bp的片段BsmBI-2，连入T载体，送至英潍捷基测序，测序正确，未发生突变，BsmBI-2的碱基序列如SEQ ID NO：14所示。
利用限制性内切酶切割Bgl II和EcoR I切下重组后的T载体，回收约233bp的DNA片段BsmBI-2，然后利用T4连接酶将其正确连入线性化载体pTEGC，获得整合表达载体pTEGC-BsmBI。
表3 扩增含BsmBI骨架DNA引物
Figure PCTCN2017109653-appb-000004
注：下划线处的大写字母为BglII或EcoRI酶切位点；小写字母为IIs型限制性内切酶BsmBI酶的识别序列。
二、启动子、终止子的扩增
1)启动子的扩增：
以酿酒酵母基因组DNA为模板，利用PGK1F1-BsmBI和PGK1R1-BsmBI引物(见表4)扩增出pgk1-1启动子片段(其碱基序列如SEQ ID NO：6所示)，用作表达外切β-1,4-葡 聚糖酶基因的启动子。
同理，以酿酒酵母基因DNA为模板，利用PGK1F2-BsmBI和PGK1R2-BsmBI引物(见表4)扩增出pgk1-2启动子片段(其碱基序列如SEQ ID NO：8所示)，用作表达内切β-1,4-葡聚糖酶基因的启动子。
同理，以酿酒酵母基因DNA为模板，利用PGK1F3-BsmBI和PGK1R3-BsmBI引物(见表4)扩增出pgk1-3启动子片段(其碱基序列如SEQ ID NO：10所示)，用作表达抗菌肽基因的启动子。
上述扩增所得启动子基因片段均分别连入到pMD19-T Simple载体中，测序验证，保留正确的阳性克隆。
2)终止子的扩增：
以酿酒酵母基因组DNA为模板，利用引物PGKT1F1-BsmBI和PGKT1R1-BsmBI(见表4)扩增pgkt1-1终止子(其碱基序列如SEQ ID NO：7所示)，用于表达外切β-1,4-葡聚糖酶基因的终止子。
以酿酒酵母基因组DNA为模板，利用引物PGKT1F2-BsmBI和PGKT1R2-BsmBI(见表4)扩增pgkt1-2终止子(其碱基序列如SEQ ID NO：9所示)，用于表达内切β-1,4-葡聚糖酶基因的终止子。
以酿酒酵母基因组DNA为模板，利用引物PGKT1F3-BsmBI和PGKT1R3-BsmBI(见表4)扩增pgkt1-3终止子(其碱基序列如SEQ ID NO：11所示)，用于表达抗菌肽基因的终止子。
上述扩增得到终止子基因片段均连入到pMD19-T Simple载体中，测序验证，保留正确的阳性克隆。
表4 扩增酿酒酵母启动子、终止子的引物
Figure PCTCN2017109653-appb-000005
Figure PCTCN2017109653-appb-000006
注：下划线处大写字母为IIs型限制性内切酶BsmBI的识别序列，下划线小写粗体字母为IIs型限制性内切酶BsmBI的切割序列。
三、α-信号肽基因、外切β-1,4-葡聚糖酶基因cbh2、内切β-1,4-葡聚糖酶基因egII、抗菌肽基因amp的扩增
1)α-信号肽基因的获得：以酿酒酵母基因组DNA为模板，利用MfaF和MfaR引物(见表5)扩增得到Mfa-BsmBI(即含有Iis型限制性内切酶BsmBI序列的α-信号肽基因序列的片段)片段，扩增程序如下：98℃10s，55℃15s，72℃30s，30个循环，72℃10min；连入T载体，送样测序，挑选正确的阳性克隆，从而将α-信号肽基因(其碱基序列如SEQ ID NO：5所示)保存至T载体中。
表5扩增α-信号肽基因、外切β-1,4-葡聚糖酶基因cbh2、内切β-1,4-葡聚糖酶
基因egII、抗菌肽基因amp引物
Figure PCTCN2017109653-appb-000007
Figure PCTCN2017109653-appb-000008
注：下划线处大写字母为IIs型限制性内切酶BsmBI的识别序列，下划线处小写粗体字母为IIs型限制性内切酶BsmBI的切割序列。
2)外切β-1,4-葡聚糖酶基因cbh2的获得：参照NCBI公布的里氏木霉(Trichoderma reesei)的外切β-1,4-葡聚糖酶基因cbh2(GenBank:KF992142.1)，通过人工合成优化后的外切β-1,4-葡聚糖酶基因cbh2的碱基序列如SEQ ID NO：1所示。
3)内切β-1,4-葡聚糖酶基因egII的获得：参照NCBI公布的斜卧青霉(Penicillium decumbens)的内切β-1,4-葡聚糖酶基因egII(GenBank:EU315320.1)，通过人工合成优化后的内切β-1,4-葡聚糖酶基因egII的碱基序列如SEQ ID NO：2所示。
4)抗菌肽基因的获得：通过人工合成优化后的长牡蛎Crassostrea gigas的抗菌肽Cg-Defh2突变体Cg-Defh2-T，其碱基序列如SEQ ID NO：4所示，其氨基酸序列为GFGCPGDQYQCNRHCKSIGCRAGYCDAVTLWLPCTCTRCSGKK(SEQ ID NO:43)。
将上述所得外切β-1,4-葡聚糖酶基因cbh2、内切β-1,4-葡聚糖酶基因egII、抗菌肽Cg-Defh2突变体Cg-Defh2-T基因序列分别保存于pMD19-T Simple质粒中，备用。
5)含BsmBI酶切位点的外切β-1,4-葡聚糖酶基因片段cbh2：以含外切β-1,4-葡聚糖酶基因片段cbh2的T载体为模板，用特异引物cbhF-BsmBI以及cbhR-BsmBI(见表5)进行扩增，获得含有BsmBI的切割位点的外切β-1,4-葡聚糖酶基因片段cbh2。
6)含BsmBI酶切位点的内切β-1,4-葡聚糖酶基因片段egII：以含内切β-1,4-葡聚糖酶基因片段egII的T载体为模板，用特异引物egF-BsmBI以及egR-BsmBI(见表5)进行扩增，获得含有BsmBI的切割位点的内切β-1,4-葡聚糖酶基因片段egII。
7)α-信号肽-抗菌肽基因的获得：分别以含α-信号肽基因序列的T载体、含抗菌肽Cg-Defh2突变体Cg-Defh2-T基因的T载体为模板，用引物MfaF4-BsmBI、Mfa-ampR、Mfa-ampF以及Mfa-ampR-BsmBI(见表5)通过重叠延伸PCR(SOE-PCR)将α-信号肽序列定向连入无信号肽的抗菌肽基因的5’端，扩增出mfa-amp基因片段(含有α-信号肽基因序列和抗菌肽Cg-Defh2突变体Cg-Defh2-T基因)。
上述扩增得到基因片段均连入到pMD19-Simple载体中，测序验证，保留正确的阳性克隆。
四、能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多基因共表达载体的构建
将上述“二”和“三”中获得的外切β-1,4-葡聚糖酶基因表达盒元件pgk1-1(启动子)、cbh2(含有外切β-1,4-葡聚糖酶基因cbh2)、pgkt1-1(终止子)；内切β-1,4-葡聚糖酶基因表达盒元件pgk1-2(启动子)、egII(含有内切β-1,4-葡聚糖酶基因egII)、pgkt1-2(终止子)；抗菌肽基因表达盒元件pgk1-3(启动子)、mfa-amp(含有α-信号肽基因序列和抗菌肽Cg-Defh2突变体Cg-Defh2-T基因)、pgkt1-3(终止子)利用IIs型限制性内切酶BsmBI分别从T载体切下，纯化回收；同时，利用IIs型限制性内切酶BsmBI切割上述“一”中构建的整合表达载体pTEGC-BsmBI，将其线性化。利用T4连接酶将上述片段一次连接反应定向连入整合表达载体pTEGC-BsmBI，获得酿酒酵母多基因共表达载体pTEGC-cbh2-egII-amp，转化大肠杆菌DH5a，挑选转化子，测序验证，获得正确连接的阳性转化子，提取质粒，即得能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多基因共表达载体pTEGC-cbh2-egII-amp。
实施例2能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多功能酿酒酵母的构建
一、多基因共表达载体pTEGC-cbh2-egI I-amp的预处理
将实施例1构建好的多基因共表达载体pTEGC-cbh2-egII–amp转化大肠杆菌DH5a进行活化并在液体LB培养基中培养过夜，提取其质粒，并进行纯化回收。利用限制性内切酶HpaI进行线性化酶切，回收纯化，备用。
二、重组酵母的筛选与验证
多基因共表达载体pTEGC-cbh2-egII–amp线性化后，参考Bio-Rad点击仪的使用说明，采用电穿孔转化法转入酿酒酵母(已做抗性筛选，其G418耐受浓度最高为200μg/ml培养基)中，在G418浓度为300μg/ml的YPD平板上培养48h以上，挑取长出的单菌落为转化子。验证后的转化子逐步在含不同浓度的G418的YPD液体培养基中进行高抗筛选，获得的单克隆菌落，测序验证，获得正确连接的阳性重组酵母转化子，即得能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多功能酿酒酵母，简称为Glam-c1。
三、重组酿酒酵母的纤维素酶的活性检测
1)刚果红染色法鉴定重组酿酒酵母的纤维素酶活性
实验方法：
将本实施例构建的能降解纤维素生产益生纤维寡糖并分泌抗菌肽的重组酿酒酵母转接到含1％CMC-Na的YP琼脂平板(配方如下：15g/l琼脂、10g/l CMC-Na)中，培养72h以上。然后，加入10mL 0.1％刚果红染色液，常温染色40min，再用1M NaCl溶液脱色30min，观察水解圈。
实验结果：
观察结果如图1所示，图中1～6均为本实施例构建的重组酿酒酵母，SV为宿主酿酒酵母作为阴性对照，从中可以看出，本实施例构建的重组酿酒酵母的不同单克隆周围均有明显的水解圈出现，说明本实施例构建的重组酿酒酵母具有很好的纤维素酶活性。
2)纤维素酶酶活的测定
实验方法：
将本实施例构建的能降解纤维素生产益生纤维寡糖并分泌抗菌肽的重组酿酒酵母转接到YPD液体培养基中，培养60h，吸取培养液，离心收集上清液，参考国标GB/T 23881-2009饲用纤维素酶活性的测定(滤纸法)测定总纤维素酶酶活。
实验结果：
总纤维素酶活性检测结果如表6所示，从中可以看出，实施例2重组酿酒酵母Glam-c1的总纤维素酶酶活约在1.0U/ml以上，显著高于对照组宿主酿酒酵母。
表6 实施例2构建的重组酿酒酵母的纤维素酶活性
组别 纤维素酶酶活(U/ml)
宿主酿酒酵母 0.01
实施例2重组酿酒酵母Glam-c1 1.12
四、重组酿酒酵母的抑菌效果检测
实验方法：
将革兰氏阳性菌金色葡萄球菌ATCC22023、枯草芽孢杆菌作为受试菌，经液体培养基中培养至在OD600nm＝0.4，适当稀释、混匀、均匀涂布至MH培养基平板上(培养基配方为：5g/l牛肉膏浸粉、17.5g/l酪素水解物、1.5g/l淀粉、琼脂粉20g/l)。将本实施例所得重组酿酒酵母菌的发酵液加入牛津杯中，以灭菌水为阴性对照，以氨苄青霉素(1.5μg)为阳性对照，在37℃培养16-18h，观察抑菌圈情况。
实验结果：
实验结果如图2和图3所示，从中可以看出，本实施例构建的重组酿酒酵母菌的发酵液对金色葡萄球菌ATCC22023和枯草芽孢杆菌均有明显的抑菌圈，说明所得重组酿酒酵母菌成功分泌出抗菌肽。
实施例3能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多基因共表达载体的构建
本实施例构建酿酒酵母多基因共表达载体的方法同实施例1，除了将连入载体中的外切β-1,4-葡聚糖酶基因替换为β-葡萄糖苷酶基因(bgl)(碱基序列如SEQ ID NO：3所示) 外，其中β-葡萄糖苷酶基因参照NCBI公布棘孢曲霉(Aspergillus aculeatus)的β-葡萄糖苷酶基因bgl(GenBank:D64088.1)经密码子优化，化学合成获得，其他均与实施例1相同，本实施例构建的酿酒酵母多基因共表达载体命名为pTEGC-bgl-egII-amp。
实施例4能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多基因共表达载体的构建
本实施例构建酿酒酵母多基因共表达载体的方法同实施例1，除了将连入载体中的内切β-1,4-葡聚糖酶基因替换为β-葡萄糖苷酶基因(碱基序列如SEQ ID NO：3所示)外，其中β-葡萄糖苷酶基因参照NCBI公布棘孢曲霉(Aspergillus aculeatus)的β-葡萄糖苷酶基因bgl(GenBank:D64088.1)经密码子优化，化学合成获得，，其他均与实施例1相同，本实施例构建的酿酒酵母多基因共表达载体命名为pTEGC-cbh2-bgl-amp。
实施例5能降解纤维素生产益生纤维寡糖并分泌抗菌肽的的多功能酿酒酵母的构建
将实施例3构建好的多基因共表达载体pTEGC-bgl-egII-amp转化大肠杆菌DH5a进行活化并在液体LB培养基中培养过夜，提取其质粒，并进行纯化回收。利用限制性内切酶HpaI进行线性化酶切，回收纯化，参考Bio-Rad点击仪的使用说明，采用电穿孔转化法线性化的pTEGC-bgl-egII-amp转入酿酒酵母(已做抗性筛选，其G418耐受浓度最高为200μg/ml培养基)中，在G418浓度为300μg/ml的YPD平板上培养48h以上，挑取长出的单菌落为转化子。验证后的转化子逐步在含不同浓度的G418的YPD液体培养基中进行高抗筛选，获得的单克隆菌落，测序验证，获得正确连接的阳性重组酵母转化子，即得能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多功能酿酒酵母，简称为Glam-c2。
实施例6能降解纤维素生产益生纤维寡糖并分泌抗菌肽的的多功能酿酒酵母的构建
将实施例4构建好的多基因共表达载体pTEGC-cbh2-bgl-amp转化大肠杆菌DH5a进行活化并在液体LB培养基中培养过夜，提取其质粒，并进行纯化回收。利用限制性内切酶HpaI进行线性化酶切，回收纯化，参考Bio-Rad点击仪的使用说明，采用电穿孔转化法线性化的pTEGC-cbh2-bgl-amp转入酿酒酵母(已做抗性筛选，其G418耐受浓度最高为200μg/ml培养基)中，在G418浓度为300μg/ml的YPD平板上培养48h以上，挑取长出的单菌落为转化子。验证后的转化子逐步在含不同浓度的G418的YPD液体培养基中进行高抗筛选，获得的单克隆菌落，测序验证，获得正确连接的阳性重组酵母转化子，即得能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多功能酿酒酵母，简称为Glam-c3。
实施例7抗菌肽突变体与原抗菌肽的抗菌性能对比
通过生物公司合成长牡蛎Crassostrea gigas的抗菌肽Cg-Defh2及其突变体Cg-Defh2-T(其碱基序列如SEQ ID NO：4所示)。以大肠杆菌CICC10899和金色葡萄球菌 ATCC22023作为指示菌，用牛津杯法检测不同类别抗菌肽的抑菌圈的大小(重复3次)，比较抗菌肽在突变前后抑菌圈的大小。
具体实验结果如表6所示，长牡蛎的抗菌肽突变体Cg-Defh2-T的抗菌性能(抑菌圈大小)优于未突变的抗菌肽Cg-Defh2。
表6 抗菌肽抗菌性能表
Figure PCTCN2017109653-appb-000009
注：牛津杯的外径为7.8mm。
下面对上述不同实施例制备的重组酿酒酵母作进一步的性能检测
一、重组酿酒酵母抑菌活性的检测
实验方法：
将革兰氏阳性菌金色葡萄球菌ATCC22023、枯草芽孢杆菌作为受试菌，经液体培养基中培养至在OD600nm＝0.4-1，适当稀释、混匀、均匀涂布至MH培养基平板中(培养基配方为：5g/l牛肉膏浸粉、17.5g/l酪素水解物、1.5g/l淀粉、琼脂粉20g/l)。分别取等量的活化后的实施例2、5、6构建的重组酿酒酵母菌和未经改造的原始宿主酿酒酵母菌的发酵液上清，牛津杯中，以灭菌水为阴性对照，以氨苄青霉素(1.5μg)为阳性对照，在37℃培养16-18h，观察抑菌圈情况。
实验结果：
检测结果如表7所示，从中可以看出，实施例2构建的重组酿酒酵母菌的发酵液对金色葡萄球菌ATCC22023和枯草芽孢杆菌的抑菌圈最为明显，抑菌活性最强；实施例6-8构建的重组酿酒酵母对金黄色葡萄球菌ATCC22023和大肠杆菌CICC10899均有抑菌效果(抑菌圈大小)，但略低于实施例2的。
表7 不同实施例的重组酿酒酵母菌抑菌统计表
Figure PCTCN2017109653-appb-000010
Figure PCTCN2017109653-appb-000011
注：牛津杯的外径为7.8mm。
二、重组酿酒酵母纤维素酶活性的检测
实验方法：
一、总纤维素酶活测定
挑选经PCR鉴定后重组酵母，经刚果红染色法鉴定的菌株，分别取等量的活化后的实施例2、5、6构建的重组酿酒酵母菌和未经改造的原始宿主酿酒酵母菌，分别YPD(补加0.5％过200目的甘蔗渣的纤维素材料作为诱导底物)液体培养基培养96h，吸取培养液，离心收集上清液，参考国标GB/T 23881-2009饲用纤维素酶活性的测定(滤纸法)测定总纤维素酶酶活。
二、重组菌的复合纤维素酶降解纤维素底物的能力检测
离心收集“一”发酵上清液，利用适宜大小的超滤管对发酵上清液进行超滤浓缩，获得实施例2、5、6中构建的重组菌Glam-c1、Glam-c2、Glam-c3分泌的复合纤维素酶浓缩液以及宿主重组菌的发酵液的浓缩液，利用0.2um大小的过滤器进行过滤除菌处理。重组菌的组别按照总纤维素酶活为20U/g底物，加入到过200目的甘蔗渣的纤维素底物中，利用缓冲液控制pH在5.5，在50摄氏度下酶解48h，利用HPLC检测纤维二糖和纤维三糖以及葡萄糖含量。
实验结果：
总纤维素酶酶活测定结果如表8所示，Glam-c1、Glam-c2、Glam-c3的总纤维素酶酶活约在1.0U/ml以上，高于对照组宿主酿酵母，其中实施例2构建重组酿酒酵母菌Glam-c1最高。
表8 重组菌总纤维素酶酶活
组别 纤维素酶酶活(U/ml)
宿主酿酒酵母 0.01
Glam-c1 1.32
Glam-c2 1.11
Glam-c3 1.08
各重组酿酒酵母菌菌液降解纤维的能力检测结果如表9所示，实施例2、5、6构建重 组酿酒酵母菌Glam-c1、Glam-c2、Glam-c3经48h酶解后获得的纤维二糖、纤维三糖以及葡糖的浓度均明显高于宿主酿酒酵母，其中实施例2构建的重组菌Glam-c1酶解后获得的纤维二糖、纤维三糖以及葡糖的浓度最高。
表9 重组菌酶液酶解纤维素产纤维寡糖
组别 纤维二糖(g/l) 纤维三糖(g/l) 葡萄糖(g/l)
宿主酿酒酵母 - - -
Glam-c1 0.35 0.51 2.1
Glam-c2 0.09 0.25 0.6
Glam-c3 0.06 0.08 1.9
上述结果说明实施例2构建重组酿酒酵母菌具有很好的降解纤维素生产益生纤维寡糖的活性，即在重组酿酒酵母菌中将外切β-1,4-葡聚糖酶、内切β-1,4-葡聚糖酶和抗菌肽进行共同表达时，重组酿酒酵母菌具有最好的纤维素酶活性，能够获得最高纤维寡糖。
三、不同重组酿酒酵直接发酵利用含天然纤维素原料、生产纤维寡糖的效果检测
实验方法：
分别取等量的活化后的实施例2、5和6构建的重组酿酒酵母菌(即Glam-c1、Glam-c2和Glam-c3)和未经改造的原始宿主酿酒酵母对天然纤维素原料进行利用，如秸秆渣。具体操作如下：
①对秸秆渣进行进行如下预处理：洗净、烘干，粉碎，过标准250目数筛子，备用。
②通过种子培养基活化重组酿酒酵母生长至OD600至6.0，种子培养基如下：2％(w/w)玉米浆，5％(w/w)的葡萄糖。
③按照10％(v/v)的接种量，将重组酿酒酵母接入发酵培养基中(培养基配方如下：15％(w/w)秸秆渣、3％(w/w)玉米浆、1％硫酸铵)，培养96h以上。以纯品为标样，利用HPLC法制备标准曲线，检测乙醇含量、纤维寡糖，如纤维二糖和纤维三糖。
实验结果：
检测结果如4～6所示，从中可以看出，重组菌Glam-c1、Glam-c2和Glam-c3都能利用天然纤维素生产乙醇，其中实施例2构建的重组酿酒酵母Glam-c1利用纤维素酶降解纤维素底物产生的葡萄糖以及底物原料的残糖生成乙醇浓度、以及纤维素酶酶解底物产生纤维二糖和纤维三糖浓度最高；实施例6构建的重组酿酒酵母Glam-c3的次之，实施例5构建的重组酿酒酵母Glam-c2最低；宿主酿酒酵母能够利用原料中残糖生成乙醇，除底物原料 中的残留纤维寡糖，基本未生成纤维寡糖(纤维二糖和纤维三糖)。由此可知，实施例2构建的重组酿酒酵母Glam-c1的纤维素降解能力最佳，相关酶基因的表达效果最好。
四、不同重组酿酒酵母对高纤维含量培养基的发酵及饲喂动物的效果检测
实验方法：
1)发酵实验
分别取等量的活化后的实施例2、5和6构建的重组酿酒酵母菌(即Glam-c1、Glam-c2和Glam-c3)和未经改造的原始宿主酿酒酵母对高纤维含量固态发酵培养基进行发酵，其具体方法如下：
①固态发酵。固态发酵培养基为：甘蔗渣(粉碎过200目)：麸皮：玉米粉：豆粕比例为10：30：20：40加入配比，料水比为1：1.4。种子液接种量为40％(v/m，L/kg)在浅盘中通空气，定期翻料，28摄氏度培养48h；再提高料层厚度，进行深层厌氧发酵48h。
②发酵破壁。将同时发酵温度升至55℃进行破壁36h。酵母破壁率可达92％，无活酵母。
③发酵后物料经低温烘干，粉碎，包装，得到酵母培养物。
④对发酵完成的酵母培养物进行检测分析。检测结果如表10所示。
2)饲喂动物实验：
选取35日龄的断奶仔猪，共180头，随机分为6个处理组，每组3个重复。试验期40d，6个处理组分别为：
基础日粮(空白对照组)；
实验A组，日粮+3‰实施例2酵母培养物(Glam-c1)；
实验B组，日粮+3‰实施例5酵母培养物(Glam-c2)；
实验C组，日粮+3‰实施例6酵母培养物(Glam-c3)；
实验D组，日粮+3‰市售酵母培养物；
实验E组，日粮+3‰市售酵母培养物和0.7‰纤维寡糖。
按照养殖场管理规范进行日常管理，定期喂养，自由采食等。观察、统计仔猪腹泻，头均总增重情况、耗料量等。检测结果如表11所示。
实验结果：
1)酵母培养物成分检测
酵母培养物成分检测结果如表10所示，从中可以看出，实施例2重组酿酒酵母(Glam-C1)发酵含高纤维物料的培养基后，纤维寡糖含量最高，且杂菌数最少。
表10 不同重组酿酒酵母发酵后酵母培养物的成分检测表
Figure PCTCN2017109653-appb-000012
注：纤维寡糖主要检测了纤维二糖和纤维三糖。
2)饲喂动物效果检测
饲喂动物效果检测结果如表11所示，采用实施例2基因重组酿酒酵母(Glam-C1)的酵母培养物(A组)的腹泻率低于对照组和市售酵母培养物的D组，其他指标也优于其他组。其中，补充了纤维寡糖的市售酵母培养物的E组的各项指标接近A组。实施例5、6的基因重组酿酒酵母的B组(Glam-C2)、C组(Glam-C3)的各指标优于市售酵母培养物的D组，接近A组。说明实施例2的基因重组酿酒酵母的酵母培养物能够发挥酵母培养物的优势，同时，能够起到补充纤维寡糖，发挥其益生作用等。
表11 日粮添加不同重组酵母培养物/纤维寡糖对仔猪影响
Figure PCTCN2017109653-appb-000013
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的酿酒酵母多基因共表达载体，其特征在于：该载体中含有降解纤维素的酶基因、抗菌肽基因；
    所述抗菌肽基因的碱基序列如SEQ ID NO：4所示；
    所述降解纤维素的酶基因选自外切β-1,4-葡聚糖酶基因、内切β-1,4-葡聚糖酶基因、β-葡萄糖苷酶基因中的至少一种。
  2. 根据权利要求1所述的一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的酿酒酵母多基因共表达载体，其特征在于：所述外切β-1,4-葡聚糖酶基因的碱基序列如SEQ ID NO：1所示，所述内切β-1,4-葡聚糖酶基因的碱基序列如SEQ ID NO：2所示，所述β-葡萄糖苷酶基因的碱基序列如SEQ ID NO：3所示。
  3. 根据权利要求1所述的一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的酿酒酵母多基因共表达载体，其特征在于：所述降解纤维素的酶基因为外切β-1,4-葡聚糖酶基因和内切β-1,4-葡聚糖酶基因。
  4. 根据权利要求1所述的一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的酿酒酵母多基因共表达载体，其特征在于：抗菌肽基因上游存在α-信号肽基因序列，α-信号肽基因的碱基序列如SEQ ID NO：5所示。
  5. 根据权利要求1所述的一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的酿酒酵母多基因共表达载体，其特征在于：所述降解纤维素的酶基因的启动子选自pgk1-1、pgk1-2，终止子选自pgkt1-1、pgkt1-2；所述抗菌肽基因的启动子为pgk1-3，终止子为pgkt1-3；
    所述pgk1-1的碱基序列如SEQ ID NO：6所示；
    所述pgkt1-1的碱基序列如SEQ ID NO：7所示；
    所述pgk1-2的碱基序列如SEQ ID NO：8所示；
    所述pgkt1-2的碱基序列如SEQ ID NO：9所示；
    所述pgk1-3，的碱基序列如SEQ ID NO：10所示；
    所述pgkt1-3的碱基序列如SEQ ID NO：11所示。
  6. 根据权利要求1所述的一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的酿酒酵母多基因共表达载体，其特征在于：所述载体的骨架为pGAPZaA质粒。
  7. 根据权利要求1所述的一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的酿酒酵母多基因共表达载体，其特征在于：该载体中含有酿酒酵母菌的25s rDNA基因片段，其 碱基序列如SEQ ID NO：13所示。
  8. 一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多功能酿酒酵母，其特征在于：该重组酿酒酵母基因组中插入有权利要求1～7任一所述的多基因共表达载体。
  9. 权利要求1～7任一所述一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的酿酒酵母多基因共表达载体的构建方法，其特征在于：包括以下步骤：
    S1整合表达载体pTEGC-BsmBI构建：
    S1.1将G418抗性基因连入pGAPZaA质粒载体的多克隆位点Msc I和EcoR V之间，
    获得载体pGAPZaA-G418；
    S1.2将碱基序列如SEQ ID NO：13所示的rDNA基因序列的连入载体pGAPZaA-G418多克隆位点BamHI和EcoRI之间，获得载体pGAPZaA-G418-rDNA；
    S1.3将载体pGAPZaA-G418-rDNA经Bgl II和EcoRI双酶切后，回收大片段产物，得到线性化载体pTEGC，将碱基序列如SEQ ID NO：15所示的BsmBI-2片段与线性化载体pTEGC连接，得整合表达载体pTEGC-BsmBI；
    S2启动子、终止子的扩增
    S2.1启动子的扩增：以酿酒酵母基因组DNA为模板，分别用引物对PGK1F1-BsmBI和PGK1R1-BsmBI、PGK1F2-BsmBI和PGK1R2-BsmBI、PGK1F3-BsmBI和PGK1R3-BsmBI分别扩增出pgk1-1、pgk1-2、pgk1-3启动子片段；
    S2.2终止子的扩增：以酿酒酵母基因组DNA为模板，分别用引物对PGKT1F1-BsmBI和PGKT1R1-BsmBI、PGKT1F2-BsmBI和PGKT1R2-BsmBI、PGKT1F3-BsmBI和PGKT1R3-BsmBI分别扩增出pgkt1-1、pgkt1-2、pgkt1-3终止子片段；
    S3 α-信号肽基因、外切β-1,4-葡聚糖酶基因、内切β-1,4-葡聚糖酶基因、抗菌肽基因的获得
    S3.1含BsmBI酶切位点的外切β-1,4-葡聚糖酶基因的获得：以含外切β-1,4-葡聚糖酶基因序列的T载体为模板，通过引物cbhF-BsmBI以及cbhR-BsmBI进行扩增，获得含有BsmBI的切割位点的外切β-1,4-葡聚糖酶基因片段cbh2；
    S3.2含BsmBI酶切位点的内切β-1,4-葡聚糖酶基因片段egII：以含内切β-1,4-葡聚糖酶基因片段egII的T载体为模板，用特异引物egF-BsmBI以及egR-BsmBI进行扩增，获得含有BsmBI的切割位点的内切β-1,4-葡聚糖酶基因片段egII；
    S3.3 α-信号肽-抗菌肽基因的获得：分别以含α-信号肽基因序列的T载体、含抗菌肽的T载体为模板，通过引物MfaF4-BsmBI、Mfa-ampR、Mfa-ampF以及Mfa-ampR-BsmBI 进行重叠延伸PCR将α-信号肽序列定向连入无信号肽的抗菌肽基因的5’端，扩增出mfa-amp基因片段，即含有α-信号肽基因序列和抗菌肽基因序列的片段；
    S4酿酒酵母多基因共表达载体的构建
    将上述获得的外切β-1,4-葡聚糖酶基因表达盒元件pgk1-1、cbh2、pgkt1-1；内切β-1,4-葡聚糖酶基因表达盒元件pgk1-2、egII、pgkt1-2；抗菌肽基因表达盒元件pgk1-3、mfa-amp、pgkt1-3利用IIs型限制性内切酶BsmBI进行酶切，纯化回收；同时，利用IIs型限制性内切酶BsmBI切割上述整合表达载体pTEGC-BsmBI，将其线性化；将所用这些片段通过一步法定向连入线性化的整合表达载体pTEGC-BsmBI中，即得酿酒酵母多基因共表达载体；
    上述所述引物的碱基序列如下：
    PGK1F1-BsmBI：CGTCTCAgatcGAAGTACCTTCAAAG
    PGK1R1-BsmBI：CGTCTCGgctaTATATTTGTTGTAAA
    PGK1F2-BsmBI：CGTCTCAgtcaGAAGTACCTTCAAAG
    PGK1R2-BsmBI：CGTCTCGgcatTATATTTGTTGTAAA
    PGK1F3-BsmBI：CGTCTCAtgcaGAAGTACCTTCAAAG
    PGK1R3-BsmBI：CGTCTCGtcgaTATATTTGTTGTAAA
    PGKT1F1-BsmBI：CGTCTCAtgtacGATCTCCCATCGTCTCTACT
    PGKT1R1-BsmBI：CGTCTCGgtcaAAGCTTTTTCGAAACGCAG
    PGKT1F2-BsmBI：CGTCTCAtacgGATCTCCCATCGTCTCTACT
    PGKT1R2-BsmBI：CGTCTCGtgcaAAGCTTTTTCGAAACGCAG
    PGKT1F3-BsmBI：CGTCTCAatcgGATCTCCCATCGTCTCTACT
    PGKT1R3-BsmBI：CGTCTCGagtcAAGCTTTTTCGAAACGCAG
    cbhF-BsmBI：CGTCTCAgctaATGATCGTTGGTATCTTGACTACT
    cbhR-BsmBI：CGTCTCAgtacTTACAAGAAAGATGGGTTAGCGT
    egF-BsmBI：CGTCTCAgcatATGAGATCTTCTCACAAGAGAAACT
    egR-BsmBI：CGTCTCAtacgTTAGTTCTTCCACTTTGGAACGA
    MfaF4-BsmBI：CGTCTCAtcgaATGAGATTTCCTTCAATTTTTAC
    Mfa-ampR：TGGACAACCGAAACCTCTTTTCTCGAGA
    Mfa-ampF：TCTCGAGAAAAGAGGTTTCGGTTGTCCA
    Mfa-ampR-BsmBI：CGTCTCAtagcTTACTTCTTACCAGAACA。
  10. 一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多功能酿酒酵母的构建方法，其特征在于，将权利要求9构建的酿酒酵母多基因共表达载体转化酿酒酵母宿主，筛选出阳性单克隆菌落，并测序验证正确，即得能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多功能酿酒酵母。
PCT/CN2017/109653 2016-12-20 2017-11-07 一种能降解纤维素生产益生纤维寡糖并分泌抗菌肽的多功能酿酒酵母 WO2018113431A1 (zh)

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