CN113549644A

CN113549644A - Recombinant yeast displaying three NSP enzymes together and construction method and application thereof

Info

Publication number: CN113549644A
Application number: CN202110529058.5A
Authority: CN
Inventors: 张莉; 刘清怡; 邓香连; 熊海容; 郭小华; 王海英
Original assignee: South Central University for Nationalities
Current assignee: South Central Minzu University
Priority date: 2021-05-14
Filing date: 2021-05-14
Publication date: 2021-10-26
Anticipated expiration: 2041-05-14
Also published as: CN113549644B

Abstract

The invention discloses a recombinant yeast for co-displaying three NSP enzymes and a preparation method and application thereof, wherein the recombinant yeast is a pichia pastoris cell wall protein serving as an anchor protein, three NSP enzymes of xylanase DSB, glucanase EG II and pectinase PG5 are co-displayed and expressed on the cell surface of the pichia pastoris to construct three NSP enzyme cell surface co-displayed recombinant yeast engineering bacteria, three NSP enzyme cell surface co-displayed whole cell catalysts are prepared, the highest enzyme activity of the xylanase of the whole cell catalysts is 13236U/g, the highest enzyme activity of the glucanase is 2056U/g, and the highest enzyme activity of the pectinase is 3227U/g during shake flask fermentation. The three NSP enzyme co-display recombinant yeast engineering bacteria can simultaneously and efficiently display and express 3 NSP enzymes, so that one-bacterium multi-enzyme is realized, compared with free enzymes, the three NSP enzyme co-display recombinant yeast engineering bacteria have the advantages of good stability, simple preparation method, easiness in recovery and recycling, reduction in production cost and the like, and when three NSP enzyme co-display whole-cell catalysts degrade and treat NSP in wheat bran serving as a feed raw material, the three enzymes have an obvious synergistic promotion effect.

Description

Recombinant yeast displaying three NSP enzymes together and construction method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to recombinant yeast for displaying three NSP enzymes together and a construction method and application thereof.

Background

Non-starch polysaccharides (NSP) are a general term for most polysaccharide carbohydrates except starch in plant tissues, mainly include cellulose, hemicellulose, pectin and other substances, are main components of plant cell walls, and are abundantly present in various agricultural and sideline products such as corn, wheat, soybean, rice, barley and the like. However, because of the special structure of NSP, there is no endogenous enzyme in animal digestive tract which can hydrolyze the NSP directly, and it can only be fermented and utilized by the microorganism in the organism, so the NSP can cause the anti-nutrition function, increase the animal intestinal cannibalism viscosity, reduce the animal's digestibility of the nutrient substance, thus limiting the wide application of many farm and sideline products in the animal feed.

In order to reduce or eliminate the anti-nutritional effect of NSP in the feed, except for adopting processing methods such as crushing, NaOH treatment, steam tabletting and the like, NSP enzyme preparations such as cellulase, xylanase, mannase, pectinase and the like can be added into the feed, the special structure of NSP can be destroyed by NSP enzyme, so that the utilization rate of the feed is improved, and the effect of reducing the anti-nutritional effect, improving the growth performance of livestock and poultry and improving the intestinal health of livestock and poultry is obviously better than that of adding single enzyme due to the synergistic degradation effect of the single enzymes when the compound enzyme is added, so that the addition of the NSP compound enzyme into the feed is a well-known high-efficiency and safe method for improving the anti-nutritional effect of NSP.

At present, most of NSP complex enzyme is produced in a compounding mode, multiple single enzymes need to be produced at first, the production is complex, the current enzyme preparation products are various in types, the adding modes and the adding levels of the various enzymes are different, and in addition, the temperature in the processes of granulation and the like in the feed production is higher, so that the activity and the stability of the added enzymes are greatly influenced. Therefore, the method has important significance in simplifying the production process of the complex enzyme, improving the stability of the complex enzyme and reducing the industrial production cost.

Disclosure of Invention

In view of the above, the main objective of the present invention is to provide a recombinant yeast displaying three NSP enzymes together, and a construction method and an application thereof, wherein the three NSP enzymes are efficiently displayed and expressed in the same host bacterium, so as to realize "one-bacterium multiple enzymes".

In order to achieve the above object, the present invention provides a method for constructing a recombinant yeast that displays three NSP enzymes in total, comprising the steps of:

1) after codon optimization, the whole gene synthesizes three NSP enzyme genes, including xylanase DSB gene (SEQ ID NO:1), glucanase EG II gene (SEQ ID NO:2) and pectase PG5 gene (SEQ ID NO: 3);

2) the gene of the anchored protein is obtained by PCR amplification from a pichia pastoris X33 genome, and comprises GCW61 shown in SEQ ID NO. 4, GCW51 shown in SEQ ID NO. 5 and GCW21 shown in SEQ ID NO. 6;

3) inserting a nucleotide sequence of xylanase DSB into a polyclonal locus of an expression vector, and then inserting a nucleotide sequence of dockerin GCW61 into the polyclonal locus of the expression vector, so that a gene of xylanase DSB and a gene of dockerin GCW61 form a fusion gene DSB-GCW61, and obtaining xylanase DSB cell surface display expression recombinant plasmid 1 taking GCW61 as dockerin;

4) inserting a nucleotide sequence of glucanase EG II into a polyclonal site of an expression vector, then inserting a nucleotide sequence of dockerin GCW51 into the polyclonal site of the expression vector, so that a gene of the glucanase EG II and a gene of the dockerin GCW51 form a fusion gene EG II-GCW 51, and obtaining a glucanase EG II cell surface display expression recombinant plasmid 2 taking GCW51 as the dockerin;

5) inserting the nucleotide sequence of pectinase PG5 into a polyclonal locus of an expression vector, then inserting the nucleotide sequence of dockerin GCW21 into the polyclonal locus of the expression vector, so that the gene of pectinase PG5 and the gene of dockerin GCW21 form a fusion gene PG5-GCW21, and obtaining pectinase PG5 cell surface display expression recombinant plasmid 3 taking GCW21 as dockerin;

6) the expression vector contains BglII restriction endonuclease sites at the initial position of a promoter, contains BamHI restriction endonuclease sites at the tail end of a terminator, BglII and BamHI are isocaudarner enzymes, recombinant plasmid 2 is subjected to double enzyme digestion by BglII and BamHI to obtain an expression cassette 1, recombinant plasmid 1 is subjected to single enzyme digestion by BamHI, and the expression cassette 1 is inserted into the BamHI sites of the recombinant plasmid 1 to obtain a recombinant plasmid for co-displaying and expressing xylanase DSB and glucanase EG II; carrying out double enzyme digestion on the recombinant plasmid 3 by Bgl II and BamH I to obtain an expression cassette 2, carrying out single enzyme digestion on the recombinant plasmid which co-displays and expresses xylanase DSB and glucanase EG II by BamH I, and inserting the expression cassette 2 into a BamH I site of the recombinant plasmid which co-displays and expresses xylanase DSB and glucanase EG II to obtain a recombinant plasmid which co-displays and expresses xylanase DSB, glucanase EG II and pectinase PG 5;

7) and (3) transforming the recombinant plasmids which co-display and express xylanase DSB, pectinase PG5 and glucanase EG II into a yeast host cell to obtain the recombinant yeast which co-display three NSP enzymes.

Preferably, wherein the expression vector is any one of pPICZ α A, pPICZ α B, pPICZ α C, pGAPZ α A, pGAPZ α B, pGAPZ α C. Wherein, the first three vectors or the last three vectors only have one enzyme cutting site at the multiple cloning site which is different, and the expression effect is not influenced when the three vectors are used for expressing the foreign protein; the first three vectors are different from the last three vectors in the used promoters, wherein the first three are inducible promoters, and the last three are constitutive promoters, and the promoters are selected according to the needs when in specific use.

Preferably, the yeast host cell is any one of Pichia pastoris SMD1168, Pichia pastoris GS115, Pichia pastoris X33, Pichia pastoris KM 71. The pichia pastoris hosts are all common hosts, have different genotypes and phenotypes, have different screening markers, and are selected according to the needs during specific use.

In order to achieve the aim, the invention also provides the recombinant yeast which displays the three NSP enzymes together, wherein the recombinant yeast uses pichia pastoris cell wall protein as an anchoring protein, and the surface of the recombinant yeast is displayed and expressed with xylanase DSB, glucanase EG II and pectinase PG5 together.

Preferably, the recombinant yeast is constructed by the method described above.

In order to achieve the aim, the invention also provides a whole-cell catalyst for displaying three NSP enzymes together, the recombinant yeast for displaying three NSP enzymes together is cultured, and thalli are collected to obtain the whole-cell catalyst for displaying three NSP enzymes together with catalytic activity.

Preferably, the whole-cell catalyst has the xylanase activity of 13236U/g (highest), the glucanase activity of 2056U/g (highest) and the pectinase activity of 3227U/g (highest) during shake flask fermentation at the temperature of 28-30 ℃ and the speed of 200-250 rpm.

In order to achieve the purpose, the invention provides an application of the whole-cell catalyst which totally displays three NSP enzymes in the degradation of NSP of forage wheat.

Preferably, wherein the application comprises: three NSP enzyme co-display type whole-cell catalysts are used for degrading and treating NSP in wheat bran samples, when the amount of the wheat bran samples is 0.3g, 0.6mg of the three NSP enzyme co-display type whole-cell catalysts are added, and the reducing sugar production amount can reach 60.1 mg/g.

Compared with the prior art, the technical scheme of the invention has the beneficial effects that: the invention constructs three NSP enzyme cell surface co-display recombinant yeast engineering bacteria, co-displays and expresses three NSP enzymes of xylanase DSB, glucanase EG II and pectinase PG5 on the surface of a pichia pastoris cell, collects thalli after shake flask fermentation culture of the recombinant bacteria to obtain three NSP enzyme cell surface co-display whole cell catalysts, the whole cell catalysts have xylanase enzyme activity of 13236U/g, glucanase enzyme activity of 2056U/g and pectinase enzyme activity of 3227U/g, and the three enzymes have obvious synergistic promotion effect when wheat bran serving as a feed raw material is degraded. The three NSP enzyme co-display recombinant yeast constructed by the invention can simultaneously and efficiently display and express 3 NSP enzymes, realizes one-bacterium multi-enzyme, has the advantages of good stability, avoiding complicated separation and purification, simple preparation method, easy recovery and recycling, reducing production cost and the like compared with free enzyme, and lays a certain foundation for efficiently producing complex enzyme with excellent performance.

Drawings

FIG. 1: schematic representation of the three NSP enzymes co-display expression recombinant plasmid.

FIG. 2: the recombinant yeast which displays three NSP enzymes and 3 strains independently display the growth curve and the enzyme activity curve of the recombinant yeast which expresses the three NSP enzymes; wherein A is the growth curve of 4 recombinant yeasts, B is the xylanase enzyme activity producing curve of the co-display expression recombinant yeast and the display expression DSB recombinant yeast, C is the glucanase enzyme activity producing curve of the co-display expression recombinant yeast and the display expression EG II recombinant yeast, and D is the pectinase enzyme activity producing curve of the co-display expression recombinant yeast and the display expression PG5 recombinant yeast.

FIG. 3: and (3) comparing the reducing sugar content of the degraded wheat bran serving as the feed raw material with the reducing sugar content of three single-display type whole-cell catalysts which display three NSP enzymes and have the same enzyme activity unit.

Detailed Description

To further illustrate the technical means and effects of the present invention adopted to achieve the predetermined objects, the following description is given with reference to the preferred embodiments. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The experimental methods in the present invention are conventional methods unless otherwise specified, and the gene cloning procedures can be specifically described in molecular cloning instruction manual compiled by J. Samsburg et al and Pichia pastoris expression instruction manual by Invitrogen corporation. The following materials or reagents, unless otherwise specified, are all commercially available.

Interpretation of terms

LBL plate: 0.5 wt% of NaCl, 1 wt% of peptone, 0.5 wt% of yeast extract powder and 2 wt% of agar powder.

LBL liquid medium: 0.5 wt% NaCl, 1 wt% peptone and 0.5 wt% yeast extract powder.

YPDS plates: 2 wt% of glucose, 2 wt% of peptone, 1 wt% of yeast extract powder, 18.2 wt% of sorbitol and 2 wt% of agar powder.

YPD liquid medium: 2 wt% of glucose, 2 wt% of peptone and 1 wt% of yeast extract powder.

BMGY medium: 1 wt% yeast extract powder, 2 wt% tryptone, 1.34 wt% YNB, 4X 10^-5Biotin at wt%, glycerol at 1 wt%, 100mM, pH6.0 phosphate buffer.

BMMY medium: 1 wt% yeast extract powder, 2 wt% tryptone, 1.34 wt% YNB, 4X 10^-5Biotin at wt%, methanol at 1 wt%, 100mM, pH6.0 phosphate buffer.

Example 1: construction of xylanase DSB cell surface display recombinant yeast engineering bacteria

(1) Cloning of the dockerin GCW61 Gene

200. mu.L of Pichia pastoris X33 (available from Invitrogen) suspension (OD)₆₀₀Value about 20) was inoculated into YPD liquid medium, cultured at 30 ℃ for 24 hours on a shaker at 200rpm, and the cells were collected and extracted with the genomic DNA Kit Yeast DNA Kit from Omega Bio-tek to obtain the genomic DNA of Pichia pastoris X33.

A synthetic primer is designed according to the gene sequence SEQ ID NO:4 of the anchoring protein GCW 61:

P1：5’-GCGGTACCAACAACCTATCAAACGAGAG-3’(SEQ ID NO:7)

P2：5’-GCCTGAGCGGCCGCTTAAATCAATAGAGCAACA-3’(SEQ ID NO:8)

wherein the underlined part of primer P1 is the KpnI cleavage site and the underlined part of primer P2 is the Not I cleavage site. And (3) taking the extracted genome DNA of the pichia pastoris X33 as a template and P1/P2 as primers, and carrying out PCR amplification to obtain a gene fragment of the dockerin GCW 61.

(2) Construction of xylanase DSB surface display recombinant plasmid

Trusting Kinry Biotech company to perform codon optimization and total gene synthesis (SEQ ID NO:1) on xylanase DSB gene to obtain recombinant plasmid pPICZ alpha A/DSB, performing double digestion on the recombinant plasmid pPICZ alpha A/DSB and the gene fragment of the anchoring protein GCW61 obtained by PCR amplification by using Kpn I and Not I respectively, then connecting the double digestion product of GCW61 and the double digestion product of pPICZ alpha A/DSB, and using a chemical method (CaCl)₂Method) to E.coli Top10 competent cells, 200. mu.L of bacterial suspension is coated on an LBL plate containing 25. mu.g/mL Zeocin antibiotics for screening, a single transformant is selected to be cultured in an LBL liquid medium containing 25. mu.g/mL Zeocin antibiotics in a shaker at 37 ℃ and 200rpm for 15h, after plasmid extraction, Kpn I and Not I are used for double enzyme digestion identification and sequencing verification, and the recombinant plasmid pPICZ alpha A/DSB-GCW61 (recombinant plasmid 1) is successfully constructed.

(3) Construction of xylanase DSB surface display recombinant bacteria

Linearization is carried out on recombinant plasmid pPICZ alpha A/DSB-GCW61 by using restriction endonuclease SacI, then electric transformation method (electric shock voltage is 1.5KV, electric shock time is 5ms) is used for transforming into pichia pastoris X33 competent cells, 200 mu L of bacterial suspension is taken and coated on YPDS plates containing 100 mu g/mL Zeocin antibiotics for screening, single transformant is picked and cultured in YPD liquid culture medium at 30 ℃ and 250rpm shaking table for 24h, bacteria liquid PCR identification is carried out by using identification primer P3/P4, and the result shows that DSB-GCW61 fusion gene is integrated into pichia pastoris X33 genome, and the recombinant bacterium X33/DSB-GCW61 for displaying and expressing DSB is successfully constructed.

P3：5’-GGCTGGCACGATGGTTATTA-3’(SEQ ID NO:9)

P4：5’-CACGGAAGAAGTATGGTTGGAG-3’(SEQ ID NO:10)

Example 2: construction of recombinant yeast engineering bacteria displayed on cell surface of glucanase EG II

(1) Cloning of the dockerin GCW51 Gene

Pichia pastoris X33 was inoculated into YPD liquid medium, cultured at 30 ℃ for 24 hours on a shaker at 200rpm, and the cells were collected, and extracted with the genome extraction Kit Yeast DNA Kit from Omega Bio-tek to obtain the genomic DNA of Pichia pastoris X33.

According to the gene sequence SEQ ID NO of the anchoring protein GCW51, a synthetic primer is designed:

P5：5’-CCGGTACCGATGACGATGACTCATTAC-3’(SEQ ID NO:11)

P6：5’-TATATAGCGGCCGCCTAGATCAATAGGGCAAT-3’(SEQ ID NO:12)

wherein the underlined part of primer P5 is the KpnI cleavage site and the underlined part of primer P6 is the Not I cleavage site. And (3) taking the extracted genome DNA of the pichia pastoris X33 as a template and P5/P6 as primers, and carrying out PCR amplification to obtain a gene fragment of the dockerin GCW 51.

(2) Construction of dextranase EG II surface display recombinant plasmid

Trusting Kinry Biotech company to perform codon optimization and total gene synthesis (SEQ ID NO:2) on the gene of dextranase EG II to obtain recombinant plasmid pPICZ alpha A/EG II, performing double digestion on the recombinant plasmid pPICZ alpha A/EG II and the gene fragment of the anchoring protein GCW51 obtained by PCR amplification by using Kpn I and Not I respectively, then connecting the double digestion product of GCW51 and the double digestion product of pPICZ alpha A/EG II, and using a chemical method (CaCl)₂Method) to E.coli Top10 competent cells, 200. mu.L of the bacterial suspension is coated on an LBL plate containing 25. mu.g/mL Zeocin antibiotics for screening, a single transformant is selected to be cultured in an LBL liquid medium containing 25. mu.g/mL Zeocin antibiotics in a shaker at 37 ℃ and 200rpm for 15h, after plasmid extraction, Kpn I and Not I are used for double enzyme digestion identification and sequencing verification, and the recombinant plasmid pPICZ alpha A/EG II-GCW 51 (recombinant plasmid 2) is successfully constructed.

(3) Construction of recombinant bacterium displayed on surface of glucanase EG II

Linearization is carried out on recombinant plasmid pPICZ alpha A/EG II-GCW 51 by using restriction endonuclease SacI, then electric transformation method (electric shock voltage is 1.5KV, electric shock time is 5ms) is used for transforming into Pichia pastoris X33 competent cells, 200 mu L of bacterial suspension is taken and coated on YPDS plates containing 100 mu g/mL Zeocin antibiotics for screening, single transformant is picked and cultured in YPD liquid culture medium at 30 ℃ and 250rpm shaking table for 24h, bacteria liquid PCR identification is carried out by using identification primer P7/P8, and the result shows that EG II-GCW 51 fusion gene is integrated into Pichia pastoris X33 genome, and recombinant bacterium X33/EG II-GCW 51 for displaying and expressing EG II is successfully constructed.

P7：5’-TTCCACTCCACCAACTTCAT-3’(SEQ ID NO:13)

P8：5’-TGCTTCCAGTAGTCGTCCCT-3’(SEQ ID NO:14)

Example 3: construction of pectinase PG5 cell surface display recombinant yeast engineering bacteria

(1) Cloning of the dockerin GCW21 Gene

200 mu L of a Pichia pastoris X33 frozen stock solution is inoculated into a YPD liquid medium, cultured in a shaker at 30 ℃ and 200rpm for 24h, and cells are collected and extracted by using a genome extraction Kit Yeast DNA Kit of Omega Bio-tek company to obtain the genome DNA of Pichia pastoris X33.

Through analyzing that 94-99 th base in the gene sequence of the anchoring protein GCW21 is KpnI site, designing and synthesizing overlap extension PCR primer according to the gene sequence SEQ ID NO. 6 of the anchoring protein GCW 21:

P9：5’-AAGGTACCACAACTGAGTTGGAGCCAATCT-3’(SEQ ID NO:15)

P10：5’-TGTGCCAGTAGTACCAGTACCAGTACCGGTTCCA-3’(SEQ ID NO:16)

P11：5’-TACTGGTACTACTGGCACAGGTACAGAAACTGGTA-3’(SEQ ID NO:17)

P12：5’-TAGGTAGCGGCCGCTCAAATTAACATAGCGACGAAG-3’(SEQ ID NO:18)

wherein the underlined part of primer P9 is the KpnI cleavage site and the underlined part of primer P12 is the Not I cleavage site. Taking the extracted genome DNA of the pichia pastoris X33 as a template and P9/P10 as primers, and obtaining an A gene fragment of the anchor protein GCW21 by PCR amplification; the B gene segment of the anchor protein GCW21 is obtained by PCR amplification by taking the extracted genome DNA of the pichia pastoris X33 as a template and P11/P12 as primers; and (2) mixing the A gene fragment and the B gene fragment according to a molar ratio of 1: 1 as a template for overlap extension PCR, and performing overlap extension PCR using P9/P12 as primers to obtain a gene fragment of dockerin GCW21 with the KpnI site removed.

(2) Construction of recombinant plasmid for surface display of pectinase PG5

Trusting Kinry Biotech Co., Ltd to perform codon optimization and whole gene synthesis (SEQ ID NO:3) on the gene of pectinase PG5 to obtain recombinant plasmid pPICZ alpha A/PG5, performing double digestion on the recombinant plasmid pPICZ alpha A/PG5 and the gene fragment of dockerin GCW21 obtained by overlap extension PCR amplification by using Kpn I and Not I respectively, then connecting the double digestion product of GCW21 and the double digestion product of pPICZ alpha A/PG5, and using a chemical method (CaCl)₂Method) to E.coli Top10 competent cells, 200. mu.L of bacterial suspension is coated on LBL plates containing 25. mu.g/mL Zeocin antibiotics for screening, single transformant is selected to be cultured in LBL liquid culture medium containing 25. mu.g/mL Zeocin antibiotics in a shaking table at 37 ℃ and 200rpm for 15h, after plasmid extraction, Kpn I and Not I are used for double enzyme digestion identification and sequencing verification, and the recombinant plasmid pPICZ alpha A/PG5-GCW21 (recombinant plasmid 3) is successfully constructed.

(3) Construction of recombinant bacterium displayed on surface of pectinase PG5

The recombinant plasmid pPICZ alpha A/PG5-GCW21 is linearized by using restriction endonuclease Sac I, then is transformed into pichia pastoris X33 competent cells by using an electrical transformation method (the electric shock voltage is 1.5KV, the electric shock time is 5ms), is coated on a YPDS plate containing 100 mu g/mL Zeocin antibiotics for screening, is picked up and cultured in a YPD liquid culture medium for 24h in a shaker at 30 ℃ and 250rpm, and is subjected to bacterial liquid PCR identification by using an identification primer P13/P14, and the result shows that a PG5-GCW21 fusion gene is integrated into a pichia pastoris X33 genome, so that the recombinant bacterium X33/PG5-GCW21 for expressing PG5 is successfully constructed.

P13：5’-CTGGACCTTAGCGATCTGGC-3’(SEQ ID NO:19)

P14：5’-GTGGGATGGTATGGTTGGAGTG-3’(SEQ ID NO:20)

Example 4: construction of three NSP enzyme cell surface co-display recombinant yeast engineering bacteria

(1) Construction of three NSP enzyme co-display expression recombinant plasmid

The expression vector pPICZ alpha A contains a Bgl II restriction endonuclease site at the initial position of a promoter, a BamHI restriction endonuclease site at the end of a terminator, and Bgl II and BamHI are isocaudarner enzymes, the recombinant plasmid pPICZ alpha A/EG II-GCW 51 (recombinant plasmid 2) is subjected to double digestion by Bgl II and BamHI to obtain an expression cassette 1, the recombinant plasmid pPICZ alpha A/DSB-GCW61 (recombinant plasmid 1) is subjected to single digestion by BamHI, and the expression cassette 1 is inserted into the BamHI site of the recombinant plasmid pPICZ alpha A/DSB-GCW61 (recombinant plasmid 1) to obtain a recombinant plasmid pPICZ alpha A/DSB (GCW61) -EG II (GCW51) which co-displays and expresses xylanase B and glucanase II. The recombinant plasmid pPICZ alpha A/PG5-GCW21 (recombinant plasmid 3) was subjected to double digestion with Bgl II and BamH I to obtain expression cassette 2, and pPICZ alpha A/DSB (GCW61) -EG II (GCW51) was subjected to single digestion with BamH I to insert expression cassette 2 into BamH I site of pPICZ alpha A/DSB (GCW61) -EG II (GCW51) to obtain recombinant plasmid pPICZ alpha A/DSB (GCW61) -EG II (GCW51) -PG5(GCW21) which exhibited three enzymes in common.

(2) Construction of recombinant Yeast Co-displaying expression of three NSP enzymes

Extracting recombinant plasmid pPICZ alpha A/DSB (GCW61) -EG II (GCW51) -PG5(GCW21), concentrating the concentration of the recombinant plasmid to 1 mu g/mu L by using a vacuum centrifugal concentrator, performing electric transformation (the electric shock voltage is 1.5KV, the electric shock time is 5ms) on 10 mu L of the recombinant plasmid to pichia pastoris X33 competent cells, 200 mu L of bacterial suspension is taken and coated on a YPDS plate containing 100 mu g/mL Zeocin antibiotics for screening, a single transformant is selected and cultured in a YPD liquid culture medium for 24h in a shaking table at 30 ℃ and 250rpm, bacterial liquid PCR identification is respectively carried out by using identification primers P3/P4, P7/P8 and P13/P14, the result shows that a DSB-GCW61 fusion gene, an EG II-GCW 51 fusion gene and a PG5-GCW21 fusion gene are all integrated into a Pichia pastoris X33 genome, the co-display expression recombinant bacterium X33/DSB (GCW61) -EG II (GCW51) -PG5(GCW21) is successfully constructed.

Example 5: shake flask culture and enzyme production analysis of recombinant yeasts

(1) Shake flask culture of recombinant bacteria

The 4 constructed recombinant yeasts X33/DSB-GCW61, X33/EG II-GCW 51, X33/PG5-GCW21 and X33/DSB (GCW61) -EG II (GCW51) -PG5(GCW21) are respectively cultured at the shake flask level. Inoculating each recombinant bacterium into a container 25mL BMGY medium in 250mL triangular flask, 30 ℃, 250rpm under the conditions of overnight culture to OD₆₀₀The value reaches 2-6, and about 4-12mL of thallus is collected by centrifugation at 6000rpm at room temperature and is resuspended in a 250mL triangular flask filled with 25mL of BMMY liquid culture medium, so that the initial OD of the bacterial suspension is ensured₆₀₀The value is 1, the culture is carried out at 30 ℃ and 250rpm for 168 hours, 0.25mL is sampled every 24 hours, and 1% (v/v) of methanol is supplemented for induction expression. After the shake flask culture is finished, centrifuging at room temperature and 6000rpm to remove supernatant, and collecting thalli which are the whole-cell catalyst with catalytic activity.

(2) Monitoring of growth of each recombinant bacterium

The concentration of the thallus is represented by the absorbance value of the bacterial suspension at the wavelength of 600nm, and the absorbance value OD of each bacterial suspension at the wavelength of 600nm is determined after sampling every 24h_600nmThe OD of each recombinant bacterium was plotted on the abscissa as time_600nmValues are plotted as the ordinate against the growth curve.

(3) Enzyme activity determination of xylanase produced by X33/DSB-GCW61 and X33/DSB (GCW61) -EG II (GCW51) -PG5(GCW21)

And (3) centrifuging 200 mu L of bacterial liquid at room temperature at 10000rpm for 1min, removing the supernatant, adding 200 mu L of 50mmol/L disodium hydrogen phosphate-citric acid buffer solution with the pH value of 6.5 to wash the bacteria, repeating the steps for three times, and then resuspending the bacteria by using 200 mu L of buffer solution to obtain the sample for determining the enzyme activity. Measuring enzyme activity of xylanase in each sample by absorbance method, using 0.5% (m/v) xylan as substrate, mixing 100 μ L diluted sample with 900 μ L substrate, reacting at 65 deg.C for 10min, immediately adding 1.5mL DNS to terminate reaction and boiling for 5min for color development, placing test tube in cold water bath for cooling completely, and measuring OD₅₄₀And (4) calculating the enzyme activity. 1 enzyme activity unit is defined as the amount of xylanase required to hydrolyse 0.5 wt% of a xylan substrate to yield 1. mu. mol xylose within 1min at 65 ℃ and pH 6.5.

(4) Enzyme activity determination of X33/EG II-GCW 51 and X33/DSB (GCW61) -EG II (GCW51) -PG5(GCW21) glucanase production

Centrifuging 200 μ L of bacterial solution at room temperature at 10000rpm for 1min, discarding supernatant, adding 200 μ L of 50mmol/L disodium hydrogen phosphate-citric acid buffer solution with pH4.8 to wash thallus, repeating for three times, and resuspending thallus with 200 μ L of buffer solutionAnd (3) measuring the enzyme activity of the sample. Measuring enzyme activity of dextranase of each sample by absorbance method, using 1% (m/v) carboxymethyl cellulose as substrate, mixing diluted sample 100 μ L and substrate 900 μ L, reacting at 75 deg.C for 10min, immediately adding 1.5mL DNS to terminate reaction and boiling for 5min for color development, placing test tube in tap water, cooling completely, and measuring OD₅₄₀And (4) calculating the enzyme activity. 1 enzyme activity unit is defined as the amount of glucanase required to hydrolyze 1% of a carboxymethyl cellulose substrate to produce 1. mu. mol of glucose within 1min at 75 ℃ and pH 4.8.

(5) Enzyme activity assay of X33/PG5-GCW21 and X33/DSB (GCW61) -EG II (GCW51) -PG5(GCW21) pectinase production

And (3) centrifuging 200 mu L of bacterial liquid at room temperature at 10000rpm for 1min, removing the supernatant, adding 200 mu L of 100mmol/L disodium hydrogen phosphate-citric acid buffer solution with pH of 3.5 to wash the bacteria, repeating the steps for three times, and then resuspending the bacteria by using 200 mu L of buffer solution, namely the sample for determining the enzyme activity. Measuring enzyme activity of pectinase in each sample by absorbance method, using 0.3% (m/v) pectin as substrate, mixing 100 μ L diluted sample with 900 μ L substrate, reacting at 70 deg.C for 10min, immediately adding 1.5mL DNS to terminate reaction and boiling for 5min for color development, placing the test tube in cold water bath, cooling completely, and measuring OD₅₄₀And (4) calculating the enzyme activity. 1 enzyme activity unit is defined as the amount of pectinase required to hydrolyse 0.3% of the pectin substrate to produce 1. mu. mol of polygalacturonic acid within 1min at 70 ℃ and pH 3.5.

The results of comparison of the growth curves of the recombinant bacteria and the enzyme activities of the three enzymes are shown in FIG. 2, and it can be seen from FIG. 2 that the growth trends of the 4 strains of recombinant bacteria in the culture process are not obviously different (FIG. 2A); the xylanase-producing enzyme activities of X33/DSB (GCW61) -EG II (GCW51) -PG5(GCW21) and X33/DSB-GCW61 are not obviously different (FIG. 2B); the slightly lower glucanase and pectinase activities of X33/DSB (GCW61) -EG II (GCW51) -PG5(GCW21) than X33/EG II-GCW 51 and X33/PG5-GCW21 were probably due to steric hindrance between enzyme molecules displayed on the cell surface (FIGS. 2C and 2D). The results show that the co-display expression of three NSP enzymes in the same host bacteria can not influence the growth, and X33/DSB (GCW61) -EG II (GCW51) -PG5(GCW21) can efficiently and simultaneously produce the three enzymes, wherein the highest enzyme activity of xylanase is 13236U/g, the highest enzyme activity of glucanase is 2056U/g, and the highest enzyme activity of pectinase is 3227U/g (when the three enzymes are subjected to shake flask fermentation at 30 ℃ and 250 rpm), so that the three NSP enzymes have better industrial application prospects.

Example 6: application of whole-cell catalyst for displaying three NSP enzymes

The wheat is an important feed raw material, however, because the wheat bran contains a large amount of NSP (non-nutritive protein) including xylan, glucan, pectin and the like, the wheat has certain anti-nutritional characteristics, so that the wide application of the wheat in feeds is limited, and the prepared whole-cell catalyst which displays three NSP enzymes together is applied to the degradation treatment of the NSP in the wheat bran.

(1) Preparation of a sample of de-starched wheat bran

Weighing 100g of wheat bran into a 1L beaker, adding 500mL of 50mmol/L disodium hydrogen phosphate-citric acid buffer solution with the pH value of 6.0, simultaneously adding 6g of amylase and 6g of protease, placing the mixture in a water bath at 50 ℃ for treatment for 3h, repeatedly washing the wheat bran, and then placing the mixture in an oven at 80 ℃ for drying until the weight is constant to obtain a starch-removed wheat bran sample.

(2) Application of whole-cell catalyst for degrading NSP (wheat bran NSP) for feed by totally displaying three NSP enzymes

Weighing 0.3g of wheat bran sample into a 100mL triangular flask, adding 30mL of 50mmol/L disodium hydrogen phosphate-citric acid buffer solution with the pH value of 6.0, adding 0.6mg of the whole-cell catalyst which is obtained in example 5 and shows three NSP enzymes in total, placing the mixture into a water bath shaker with the temperature of 60 ℃ and the rpm of 150 for treatment for 30min, treating the wheat bran sample by using three single-display type whole-cell catalysts with the same enzyme activity unit as a control, measuring the reducing sugar content of each sample after the treatment is finished, and calculating the relative reducing sugar content of other samples by using the highest reducing sugar content as 100%. The yield of reducing sugar after wheat bran is treated by the whole-cell catalyst which totally displays three NSP enzymes reaches 60.1mg/g, and the content of the reducing sugar after wheat bran samples are treated by the whole-cell catalyst which totally displays three NSP enzymes is obviously higher than that of the three independent display type whole-cell catalysts and is higher than the sum of the content of the reducing sugar after wheat bran samples are treated by the three independent display type whole-cell catalysts (figure 3), which shows that the three enzymes have a synergistic promotion effect in the degradation of the NSP of the wheat bran samples.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are still within the scope of the technical solution of the present invention.

Sequence listing

<110> university of the south China nationality

<120> recombinant yeast displaying three NSP enzymes together and construction method and application thereof

<130> 210080-I-CP-NZJ

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 585

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tgcgcaaccc ccaactcgga gggctggcac gatggttatt actattcctg gtggagtgac 60

ggtggagcgt gcgccacgta caccaacctg gaaggcggca cctacgagat cagctgggga 120

gatggcggta acctcgtcgg tggaaagggc tggaaccccg gcctgaacgc aagagccatc 180

cactttgagg gtgtttacca gccaaacggc aacagctacc ttgcggtcta cggttggacc 240

cgcaacccgc tggtcgagta ttacatcgtc gagaactttg gcacctatga tccttcctcc 300

ggtgctaccg atctaggaac tgtcgagtgc gacggtagca tctatcgact cggcaagacc 360

actcgcgtca acgcacctag catcgacggc acccaaacct tcgaccaata ctggtcggtc 420

cgccaggaca agcgcaccag cggtaccgtc cagacgggct gccacttcga cgcctgggct 480

cgcgctggtt tgaatgtcaa cggtgaccac tactaccaga tcgttgcaac ggagggctac 540

ttcagcagcg gctatgctcg catcaccgtt gctgacgtgg gctaa 585

<210> 2

<211> 1191

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

caacaaactg tttggggtca gtgtggtggt attggttggt ctggtccaac taactgtgct 60

ccaggttctg cttgttccac tttgaaccct tactacgctc aatgtatccc aggtgctact 120

actatcacta cttccacaag accaccatct ggtcctacta ctactacaag agctacttcc 180

acttcttctt ccactccacc aacttcatcc ggtgttagat tcgctggtgt taacattgct 240

ggtttcgact tcggttgtac tactgacggt acttgtgtta cttccaaggt ttacccacca 300

ttgaagaact tcactggttc caacaactac ccagacggta ttggtcaaat gcagcacttc 360

gttaacgagg acggtatgac tatcttcaga ttgccagttg gttggcagta cttggttaac 420

aacaacttgg gtggtaactt ggattccact tccatctcca agtacgacca attggttcag 480

ggttgtttgt ctttgggtgc ttactgtatc gttgacatcc acaactacgc aagatggaac 540

ggtggtatta ttggtcaagg tggaccaact aacgctcagt tcacttcctt gtggtcccaa 600

ttggcttcta agtacgcttc ccagtccaga gtttggttcg gtatcatgaa cgaaccacac 660

gacgttaaca tcaacacttg ggctgctact gttcaagagg ttgttactgc tatcagaaac 720

gctggtgcta cttcccaatt catctccttg ccaggtaacg attggcaatc tgctggtgct 780

ttcatttctg acggttccgc tgctgctttg tcccaagtta caaacccaga cggttccact 840

acaaacttga tcttcgacgt tcacaagtac ttggattccg acaactctgg tactcacgct 900

gagtgtacta caaacaacat cgacggtgct ttttctccat tggctacttg gttgagacag 960

aacaacagac aggctatctt gactgaaact ggtggtggta acgttcagtc ctgtatccaa 1020

gacatgtgtc agcagatcca gtacttgaac cagaactccg acgtttactt gggttacgtt 1080

ggttggggtg ctggttcttt cgattccact tacgttttga ctgagacacc aacatcttcc 1140

ggtaactctt ggactgacac ttccttggtt tcatcctgtt tggcaagaaa g 1191

<210> 3

<211> 1134

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgctgaaac taataggttc tctcgtgctc ctcgcctctg cggccgaggt gattgcctct 60

cccctcgctg agtcagttgc gccgtccata actctggaga agcgcgcatc ttgcacattc 120

tccgggtcca acggcgccgc tgctgcgatg gcttctcaga aggcttgctc cactatcgtc 180

ctgtcaaacg tggctgttcc ggctggcacg acgctggacc ttagcgatct ggcagatggc 240

accacagtca tcttcgaggg ggagacaacc tggggctaca aggagtggtc cggtcctcta 300

ctgcagattt ccggcaaaaa catcaaggtg gagggtgcgt cgggtgccac gctgaacccc 360

gacggcgccc gctggtggga cggccagggc ggcaacggtg gcaagacgaa gcccaagttc 420

ttcgccgcgc acggcctcac ctcgtcgtca tccatcacta atctgcacat cctgaacacc 480

cccgtccaag cagttagcat caacggatgc gatggcctga ccgtcaccga catgacgatc 540

gacgattccg ccggtgacac ccaaggcggc cacaacactg acgccttcga tattggatcc 600

agctccaaca ttatcatttc aggcgccaag gtctacaacc aggacgactg tgtcgctgtc 660

aactccggca caggtatcac ctttaccggt gggctctgct ccggtggcca tggcctgtcg 720

attggtagcg ttggtggccg gtctgataat accgtcgaga atgtgtcctt tacgaactcc 780

caggtgacga agtccgacaa tggtctccgc atcaaggcct ccaaagggaa gactggcaca 840

atcaagggaa tcacctactc aggcatcacc ctgagctcca tccgcaagta cggtatcctc 900

attgaacaaa actacgacgg tggcgatctc aagggtgacc cgacgtccgg catccccatc 960

accgacctga ccatggagaa catcagtggt aaaggcgccg tggcgtcaag cgggcacaat 1020

atcgctattg tctgcggcag tggggcctgc tcgaattgga cctggaagaa cgtcgaggtc 1080

accggcgggc agacctatgg gagctgcgag aacgttccta gcgttgccca atgc 1134

<210> 4

<211> 144

<212> DNA

<213> Pichia pastoris (Pichia pastoris GS115)

<400> 4

aacaacctat caaacgagag taatggtact aatcactcca accatacttc ttccgtgcca 60

actggagctg ccgttcgtgc ctctggtatg ggagctggct tgttgggagc tggtgttgta 120

gccggtgttg ctctattgat ttaa 144

<210> 5

<211> 585

<212> DNA

<213> Pichia pastoris (Pichia pastoris GS115)

<400> 5

gatgacgatg actcattacc ttttgttatt gttaactccg cgtcaggaga aacccatgat 60

ggaacttact ggggtgtgaa taacgccgga gcagtcgttc caagctccac tggagtgcga 120

tttgtggtca acgacgacgg tgagttggaa ggtaatgacg aggaagttga agtgacctca 180

aacgggtttt tgaccttaag agatgacaat gatgaaaatg aggggttttc tctcacagat 240

gatgatccac cggttttgct gttaaatcgt caaactccta ccgtgtggat atgtggatct 300

gatgatgacg cccgtattgc tctgggttca caatcgccac aagatgattg tgtagagtac 360

tccattgaag ttcagctgca aagtggttca agaagcgggt ccagcacaag aacaagcagt 420

agaacaactg gaaccagtgc aaccagtgca accagtgcaa ccagtggaac cagtgcaaca 480

gggacgacta ctggaagcac ctcgacagct actgatggag cccacaagct tgttggcggg 540

ctgtctggat tggctggtgg tgttgccatt gccctattga tctag 585

<210> 6

<211> 648

<212> DNA

<213> Pichia pastoris (Pichia pastoris GS115)

<400> 6

gaccagcgaa tctctgtcac cgttgttggt gatggtatca actcaggact gagatccgga 60

gggtctcatt ttgaggctgg accaaatgct gctggtaccc ccttagattt gatcctgtat 120

gagccatctg gtttcttggt agatgcagca gatgcttcca agtacgttgg ttgggatgtt 180

gcagctggca cttctttgac ttttttgcca caagaccaag gaggcaaaga ttggggaatc 240

gttgccggta acctcagatt caacgttgga ggtactacat tctatgcttg tgagactagg 300

accggtgttt gggaagtaaa gagttacgaa gctagtggat gcaaagctgt ggtactttcc 360

gtagctagtc acccagttcc ttcttccagt tcttccagtt cttcccatgc cccaacttcc 420

tctgttccat ctacttcgtc tcatgtgagc ccaactacca ctcaacctcc tcacacaacc 480

agttctcaca ccaaccacac atcaactacc ttgaccacat caggtaggaa tgactcgaac 540

cactccaacc ataccatccc accagttcca accggtgccg ctatgggagt ctctagcaac 600

tatggtttgt tggttgcagc tggaattgcc gccgctgctt tgttataa 648

<210> 7

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gcggtaccaa caacctatca aacgagag 28

<210> 8

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gcctgagcgg ccgcttaaat caatagagca aca 33

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ggctggcacg atggttatta 20

<210> 10

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cacggaagaa gtatggttgg ag 22

<210> 11

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ccggtaccga tgacgatgac tcattac 27

<210> 12

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tatatagcgg ccgcctagat caatagggca at 32

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ttccactcca ccaacttcat 20

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tgcttccagt agtcgtccct 20

<210> 15

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

aaggtaccac aactgagttg gagccaatct 30

<210> 16

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tgtgccagta gtaccagtac cagtaccggt tcca 34

<210> 17

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tactggtact actggcacag gtacagaaac tggta 35

<210> 18

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

taggtagcgg ccgctcaaat taacatagcg acgaag 36

<210> 19

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ctggacctta gcgatctggc 20

<210> 20

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gtgggatggt atggttggag tg 22

Claims

1. A construction method of recombinant yeast which displays three NSP enzymes together is characterized by comprising the following steps:

1) after codon optimization, the whole gene synthesizes three NSP enzyme genes, including xylanase DSB gene, glucanase EG II gene and pectase PG5 gene;

2) obtaining genes of the dockerin from a pichia pastoris X33 genome through PCR amplification, wherein the genes comprise GCW61, GCW51 and GCW 21;

7) and (3) electrically transforming the recombinant plasmids which co-display and express xylanase DSB, pectinase PG5 and glucanase EG II into a yeast host cell to obtain the recombinant yeast which co-display three NSP enzymes.

2. The construction method of claim 1, wherein in step 1), the nucleotide sequence of the gene of xylanase DSB is shown as SEQ ID NO. 1; the nucleotide sequence of the glucanase EG II gene is shown as SEQ ID NO. 2; the nucleotide sequence of the gene of the pectinase PG5 is shown as SEQ ID NO. 3.

3. The method according to claim 1, wherein in step 2), the nucleotide sequence of GCW61 is shown as SEQ ID NO. 4; the nucleotide sequence of GCW51 is shown as SEQ ID NO. 5; the nucleotide sequence of GCW21 is shown in SEQ ID NO. 6.

4. The method of constructing according to claim 1, wherein in step 3), the expression vector is any one of pPICZ α A, pPICZ α B, pPICZ α C, pGAPZ α A, pGAPZ α B, pGAPZ α C.

5. The method of claim 1, wherein in step 7), the yeast host cell is any one of Pichia pastoris SMD1168, Pichia pastoris GS115, Pichia pastoris X33, Pichia pastoris KM 71.

6. A recombinant yeast that displays three NSP enzymes in total, wherein the recombinant yeast is constructed by the method according to any one of claims 1 to 5.

7. The recombinant yeast according to claim 6, wherein the recombinant yeast is a pichia pastoris cell wall protein as an anchor protein, and the xylanase DSB, glucanase EG ii and pectinase PG5 are displayed and expressed on the surface of the recombinant yeast.

8. A whole-cell catalyst which exhibits three NSP enzymes in combination, characterized in that the whole-cell catalyst which exhibits three NSP enzymes in combination is obtained by culturing the recombinant yeast which exhibits three NSP enzymes in combination according to claim 6 or 7 and collecting the bacterial cells.

9. The whole-cell catalyst of claim 8, wherein the whole-cell catalyst has a xylanase activity of 13236U/g, a glucanase activity of 2056U/g and a pectinase activity of 3227U/g during shake flask fermentation at 28-30 ℃ and 200-250 rpm.

10. Use of a whole cell catalyst according to claim 8 or 9 which exhibits a total of three NSP enzymes in a feed enzyme preparation.