CN113549644B

CN113549644B - Recombinant yeast displaying three NSP enzymes together, construction method and application thereof

Info

Publication number: CN113549644B
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: 2024-02-02
Anticipated expiration: 2041-05-14
Also published as: CN113549644A

Abstract

The invention discloses a recombinant yeast for displaying three NSP enzymes together, and a preparation method and application thereof, wherein the recombinant yeast uses pichia pastoris cell wall protein as anchoring protein, three NSP enzymes of xylanase DSB, glucanase EG II and pectinase PG5 are displayed and expressed on the surface of pichia pastoris cell together, three NSP enzyme cell surface co-display recombinant yeast engineering bacteria are constructed, three NSP enzyme cell surface co-display type whole cell catalysts are prepared, the xylanase activity of the whole cell catalysts is up to 13236U/g, the glucanase activity is up to 2056U/g, and the pectinase activity is up to 3227U/g during shake flask fermentation. The three NSP enzyme co-display recombinant yeast engineering bacteria can display and express 3 NSP enzymes simultaneously and efficiently, so that 'one bacteria and multiple enzymes' are realized, compared with free enzymes, the method has the advantages of good stability, simple preparation method, easy recovery and regeneration, low production cost and the like, and when NSP in the wheat bran serving as a feed raw material is subjected to degradation treatment by the three NSP enzyme co-display type whole cell catalyst, the three enzymes have obvious synergistic promotion effects.

Description

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

Technical Field

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

Background

Non-starch polysaccharides (NSP) are a general term for most polysaccharide carbohydrates in plant tissues except starch, mainly comprise 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 specificity of NSP structure, the animal digestive tract does not have endogenous enzyme which can hydrolyze the NSP directly, and can only be partially fermented and utilized by microorganisms in the organism, so NSP can cause anti-nutrition effect, increase the food viscosity of animal intestinal tracts, reduce the digestibility of animals to nutrient substances, and limit the wide application of a plurality of agricultural and sideline products in livestock and poultry feeds.

In order to reduce or eliminate the anti-nutrition effect of NSP in the feed, besides adopting the processing methods of crushing, naOH treatment, steam tabletting and the like, NSP enzyme preparations such as cellulase, xylanase, mannanase, pectase and the like can be added in the feed, the NSP enzyme can damage the special structure of NSP, so that the utilization rate of the feed is improved, and when the complex enzyme is added, the effect of synergistically degrading NSP exists among single enzymes, so that the effects of reducing the anti-nutrition effect, improving the growth performance of livestock and improving the intestinal health of the livestock are obviously superior to those of adding the single enzyme, and therefore, the method for improving the anti-nutrition effect of NSP by adding the complex enzyme in the feed is a recognized efficient and safe method.

At present, most NSP complex enzymes are produced in a compound mode, a plurality of single enzymes are required to be produced firstly, the production is complex, the types of enzyme preparations are various, the adding modes and adding levels of various enzymes are different, and in addition, the temperature of the granulating process in the feed production is high, so that the activity and 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

Therefore, the main purpose of the invention is to provide a recombinant yeast displaying three NSP enzymes together, and a construction method and application thereof, wherein the three NSP enzymes are efficiently displayed and expressed in the same host bacteria, so as to realize 'one-bacteria multi-enzyme'.

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

1) complete gene synthesis of three NSP enzyme genes after codon optimization, including xylanase DSB gene (SEQ ID NO: 1), glucanase EG II gene (SEQ ID NO: 2) and pectinase PG5 gene (SEQ ID NO: 3);

2) The gene of the anchor protein is obtained by PCR amplification from 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 the nucleotide sequence of xylanase DSB into the multiple cloning site of the expression vector, and inserting the nucleotide sequence of the anchoring protein GCW61 into the multiple cloning site of the expression vector, so that the gene of xylanase DSB and the gene of anchoring protein GCW61 form a fusion gene DSB-GCW61, and obtaining xylanase DSB cell surface display expression recombinant plasmid 1 taking GCW61 as anchoring protein;

4) Inserting the nucleotide sequence of the glucanase EG II into a multiple cloning site of an expression vector, and inserting the nucleotide sequence of the dockerin GCW51 into the multiple cloning site of the expression vector, so that a fusion gene EG II-GCW 51 is formed by the gene of the glucanase EG II and the gene of the dockerin GCW51, and the glucanase EG II cell surface display expression recombinant plasmid 2 taking the GCW51 as dockerin is obtained;

5) Inserting the nucleotide sequence of pectase PG5 into the multiple cloning site of the expression vector, and inserting the nucleotide sequence of anchored protein GCW21 into the multiple cloning site of the expression vector, so that the gene of pectase PG5 and the gene of anchored protein GCW21 form fusion gene PG5-GCW21, and obtaining the pectase PG5 cell surface display expression recombinant plasmid 3 taking GCW21 as anchored protein;

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

7) And transforming the recombinant plasmid co-displaying and expressing xylanase DSB, pectinase PG5 and glucanase EG II into a yeast host cell to obtain the recombinant yeast co-displaying the three NSP enzymes.

Preferably, the expression vector is any one of pPICZ alpha A, pPICZ alpha B, pPICZ alpha C, pGAPZ alpha A, pGAPZ alpha B, pGAPZ alpha 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 affected when the first three vectors or the last three vectors are used for expressing exogenous proteins; the first three vectors and the second three vectors are different in that the promoters used are different, the first three are inducible promoters, the second three are constitutive promoters, and the specific use is selected according to the needs.

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

In order to achieve the above purpose, the invention also provides a recombinant yeast displaying three NSP enzymes together, wherein the recombinant yeast uses pichia pastoris cell wall protein as anchoring protein, and xylanase DSB, glucanase EG II and pectinase PG5 are displayed on the surface of the recombinant yeast.

Preferably, wherein 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 altogether, the recombinant yeast for displaying the three NSP enzymes altogether is cultured, and thalli are collected to obtain the whole cell catalyst for displaying the three NSP enzymes altogether with catalytic activity.

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

In order to achieve the aim, the invention provides application of the whole cell catalyst displaying three NSP enzymes in degrading NSP of wheat for feeding.

Preferably, wherein the application comprises: when the wheat bran sample amount is 0.3g, 0.6mg of the three NSP enzyme co-display type whole cell catalyst is added, and the reducing sugar generation amount can reach 60.1mg/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, three NSP enzymes of xylanase DSB, glucanase EG II and pectinase PG5 are co-displayed and expressed on the surface of pichia pastoris cells, and after shaking flask fermentation culture of the recombinant bacteria, bacterial bodies are collected to obtain three NSP enzyme cell surface co-display whole cell catalysts, wherein the xylanase activity of the whole cell catalysts reaches 13236U/g, the glucanase activity reaches 2056U/g, the pectinase activity reaches 3227U/g, and when wheat bran serving as a feed raw material is degraded, the three enzymes have obvious synergistic effect. The constructed three NSP enzyme co-display recombinant yeasts can simultaneously and efficiently display and express 3 NSP enzymes, realize 'one bacterium and multiple enzymes', have the advantages of good stability, avoiding complicated separation and purification, simple preparation method, easy recovery and regeneration, low production cost and the like compared with free enzymes, and lay a certain foundation for efficient production of complex enzymes with excellent performance.

Drawings

Fig. 1: schematic representation of three NSP enzymes co-displaying expression recombinant plasmids.

Fig. 2: the recombinant yeast displaying three NSP enzymes and 3 recombinant yeast displaying the three NSP enzymes separately have growth curves and enzyme activity curves; wherein A is the growth curve of 4 recombinant yeasts, B is the xylanase production enzyme activity curve of the co-display expression recombinant yeasts and the display expression DSB recombinant yeasts, C is the glucanase production enzyme activity curve of the co-display expression recombinant yeasts and the display expression EG II recombinant yeasts, and D is the pectinase production enzyme activity curve of the co-display expression recombinant yeasts and the display expression PG5 recombinant yeasts.

Fig. 3: comparison of reducing sugar content after wheat bran degradation treatment of diet raw material by total cell catalysts displaying three NSP enzymes and three independent display type total cell catalysts displaying the same enzyme activity units.

Detailed Description

In order to further describe the technical means and effects adopted by the present invention to achieve the intended purpose, the following description is made with reference to the preferred embodiments. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications. The experimental methods in the present invention are conventional methods unless otherwise specified, and specific reference may be made to the "molecular cloning Experimental Manual" by J.Sam Broker et al and the "Pichia pastoris expression operation Manual" by Invitrogen corporation for the genetic cloning operations. The following materials or reagents, unless otherwise specified, are all commercially available.

Interpretation of the terms

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

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

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

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

BMGY medium: 1wt% yeast extract, 2wt% tryptone, 1.34wt% YNB,4×10 ^-5 Biotin, 1% glycerol, 100mM phosphate buffer pH 6.0.

BMMY medium: 1wt% yeast extract, 2wt% tryptone, 1.34wt% YNB,4×10 ^-5 Biotin, 1% methanol, 100mM phosphate buffer pH 6.0.

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 X33 (purchased from Invitrogen) bacterial suspension (OD ₆₀₀ About 20) was inoculated into YPD liquid medium, and after culturing in a shaker at 30℃and 200rpm for 24 hours, cells were collected and extracted using the genome extraction Kit Yeast DNA Kit of Omega Bio-tek company to obtain the genomic DNA of Pichia pastoris X33.

Synthetic primers were designed based on 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 primer P1 underlined part is KpnI cleavage site, and the primer P2 underlined part is NotI cleavage site. The genomic DNA of the extracted Pichia pastoris X33 is used as a template, and P1/P2 is used as a primer, and the gene fragment of the anchoring protein GCW61 is obtained through PCR amplification.

(2) Construction of xylanase DSB surface display recombinant plasmid

The gene of xylanase DSB is subjected to codon optimization and total gene synthesis (SEQ ID NO: 1) by the entrusted Gossypium biotechnology company to obtain recombinant plasmid pPICZ alpha A/DSB, the recombinant plasmid pPICZ alpha A/DSB and the gene fragment of ankyrin GCW61 obtained by PCR amplification are simultaneously digested with KpnI and NotI respectively, then the digested product of GCW61 and digested product of pPICZ alpha A/DSB are ligated, and the recombinant plasmid pPICZ alpha A/DSB is subjected to chemical method (CaCl) ₂ Method) was transformed into E.coli Top10 competent cells, 200. Mu.L of the bacterial suspension was plated on LBL plates containing 25. Mu.g/mL of Zeocin antibiotic for selection, single transformants were picked up into LBL liquid medium containing 25. Mu.g/mL of Zeocin antibiotic, cultured in a shaker at 37℃and 200rpm for 15h, and after plasmid extraction, double restriction identification and sequencing verification were performed using KpnI and NotI, and recombinant plasmid pPICZαA/DSB-GCW61 (recombinant plasmid 1) was successfully constructed.

(3) Construction of xylanase DSB surface display recombinant bacterium

Linearizing recombinant plasmid pPICZalpha A/DSB-GCW61 by using restriction enzyme SacI, then transforming into pichia pastoris X33 competent cells by using an electrotransformation method (electric shock voltage is 1.5KV and electric shock time is 5 ms), coating 200 mu L of bacterial suspension on a YPDS plate containing 100 mu g/mL Zeocin antibiotics for screening, picking single transformant, culturing for 24h in YPD liquid medium at 30 ℃ and a 250rpm shaker, and carrying out bacterial liquid PCR identification by using identification primer P3/P4, wherein the result shows that the DSB-GCW61 fusion gene is integrated into pichia pastoris X33 genome, and successfully constructing recombinant bacterium X33/DSB-GCW61 for expressing DSB.

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

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

Example 2: construction of recombinant yeast engineering bacteria for glucanase EG II cell surface display

(1) Cloning of the dockerin GCW51 Gene

Pichia X33 was inoculated into YPD liquid medium, cultured in a shaker at 30℃and 200rpm for 24 hours, and then cells were collected, and genomic DNA of Pichia X33 was extracted using the genomic extraction Kit Yeast DNA Kit of Omega Bio-tek company.

Synthetic primers were designed based on the gene sequence SEQ ID NO. 5 of the anchoring protein GCW 51:

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

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

wherein the primer P5 underlined part is KpnI cleavage site, and the primer P6 underlined part is NotI cleavage site. The genomic DNA of the extracted pichia pastoris X33 is used as a template, and P5/P6 is used as a primer, and the gene fragment of the anchoring protein GCW51 is obtained through PCR amplification.

(2) Construction of recombinant plasmid for glucanase EG II surface display

The recombinant plasmid pPICZ alpha A/EG II is obtained by carrying out codon optimization and total gene synthesis (SEQ ID NO: 2) on the gene of the glucanase EG II by the entrusted Gossypium biotechnology company, the gene fragment of the anchoring protein GCW51 obtained by amplifying the recombinant plasmid pPICZ alpha A/EG II and the PCR is simultaneously digested with KpnI and NotI respectively, then the digested product of GCW51 and the digested product of pPICZ alpha A/EG II are connected, and the recombinant plasmid pPICZ alpha A/EG II is obtained by a chemical method (CaCl) ₂ Method) was transformed into E.coli Top10 competent cells, 200. Mu.L of the bacterial suspension was plated on LBL plates containing 25. Mu.g/mL of Zeocin antibiotic for selection, single transformants were picked up into LBL liquid medium containing 25. Mu.g/mL of Zeocin antibiotic, cultured in a shaker at 37℃and 200rpm for 15 hours, and after plasmid extraction, double restriction identification and sequencing verification were performed using KpnI and NotI, and recombinant plasmid pPICZαA/EG II-GCW 51 (recombinant plasmid 2) was successfully constructed.

(3) Construction of recombinant bacteria for surface display of glucanase EG II

Linearizing recombinant plasmid pPICZalpha A/EG II-GCW 51 by using restriction enzyme SacI, then transforming into pichia pastoris X33 competent cells by using an electrotransformation method (electric shock voltage is 1.5KV and electric shock time is 5 ms), coating 200 mu L of bacterial suspension on a YPDS plate containing 100 mu g/mL Zeocin antibiotics for screening, picking single transformant, culturing for 24h in YPD liquid medium at 30 ℃ and a 250rpm shaker, and carrying out bacterial liquid PCR identification by using identification primer P7/P8, wherein the result shows that EG II-GCW 51 fusion gene is integrated into pichia pastoris X33 genome, and successfully constructing recombinant bacterium X33/EG II-GCW 51 for expressing EG II.

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

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

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

(1) Cloning of the dockerin GCW21 Gene

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

The 94 th to 99 th bases in the gene sequence of the anchoring protein GCW21 are KpnI sites, and overlapping extension PCR primers are designed and synthesized 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 primer P9 underlined part is KpnI cleavage site, and the primer P12 underlined part is NotI cleavage site. Taking the extracted genomic DNA of Pichia pastoris X33 as a template, and taking P9/P10 as a primer, and obtaining an A gene fragment of the anchoring protein GCW21 through PCR amplification; the extracted genomic DNA of Pichia pastoris X33 is used as a template, and P11/P12 is used as a primer, and a B gene fragment of the anchoring protein GCW21 is obtained through PCR amplification; the A gene fragment and the B gene fragment are mixed according to a molar ratio of 1:1, and performing overlap extension PCR by using P9/P12 as a primer to obtain a gene fragment of the anchor protein GCW21 with KpnI site removed.

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

The gene of pectase PG5 is codon optimized and total gene synthesized (SEQ ID NO: 3) to obtain recombinant plasmid pPICZ alpha A/PG5, the recombinant plasmid pPICZ alpha A/PG5 and the gene fragment of anchoring protein GCW21 obtained by overlap extension PCR amplification are simultaneously digested with KpnI and NotI respectively, then the digested product of GCW21 and digested product of pPICZ alpha A/PG5 are joined together, and the obtained mixture is chemically treated (CaCl) ₂ Method) was transformed into E.coli Top10 competent cells, 200. Mu.L of the bacterial suspension was plated on LBL plates containing 25. Mu.g/mL of Zeocin antibiotic for selection, single transformants were picked up into LBL liquid medium containing 25. Mu.g/mL of Zeocin antibiotic, cultured in a shaker at 37℃and 200rpm for 15h, and after plasmid extraction, double restriction identification and sequencing verification were performed using KpnI and NotI, successfully constructed as recombinant plasmid pPICZαA/PG5-GCW21 (recombinant plasmid 3).

(3) Construction of recombinant bacteria for surface display of pectase PG5

Linearizing recombinant plasmid pPICZαA/PG5-GCW21 by using restriction enzyme SacI, then transforming into pichia pastoris X33 competent cells by using an electrotransformation method (electric shock voltage is 1.5KV and electric shock time is 5 ms), coating the cells on YPDS plates containing 100 mug/mL Zeocin antibiotics for screening, picking up the transformants, culturing the transformants in YPD liquid medium at 30 ℃ for 24 hours in a shaking table at 250rpm, and carrying out bacterial liquid PCR identification by using identification primer P13/P14, wherein the result shows that PG5-GCW21 fusion genes are integrated into pichia pastoris X33 genome, and successfully constructing recombinant bacteria X33/PG5-GCW21 for expressing PG5.

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 plasmids

The expression vector pPICZ alpha A contains a BglII restriction enzyme site at the start position of the promoter, a BamHI restriction enzyme site at the end of the terminator, and BglII and BamHI are homotail enzymes, the recombinant plasmid pPICZ alpha A/EG II-GCW 51 (recombinant plasmid 2) is double digested with BglII and BamHI to obtain expression cassette 1, the recombinant plasmid pPICZ alpha A/DSB-GCW61 (recombinant plasmid 1) is single digested with 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 recombinant plasmid pPICZ alpha A/DSB (GCW 61) -EG II (GCW 51) which co-displays the xylanase DSB and the glucanase EG II. The recombinant plasmid pPICZαA/PG5-GCW21 (recombinant plasmid 3) was digested with BglII and BamHI to obtain expression cassette 2, pPICZαA/DSB (GCW 61) -EG II (GCW 51) was digested with BamHI to obtain single enzyme, and the expression cassette 2 was inserted into the BamHI site of pPICZαA/DSB (GCW 61) -EG II (GCW 51) to obtain recombinant plasmid pPICZαA/DSB (GCW 61) -EG II (GCW 51) -PG5 (GCW 21) exhibiting three enzymes in total, see FIG. 1.

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

Recombinant plasmid pPICZ alpha A/DSB (GCW 61) -EG II (GCW 51) -PG5 (GCW 21) was extracted, the concentration of the recombinant plasmid was concentrated to 1. Mu.g/. Mu.L using a vacuum centrifugal concentrator, 10. Mu.L of recombinant plasmid was electrotransformed (shock voltage 1.5KV, shock time 5 ms) into Pichia pastoris X33 competent cells, 200. Mu.L of bacterial suspension was plated on YPDS plates containing 100. Mu.g/mL Zeocin antibiotics for screening, single transformants were picked up and cultured in YPD liquid medium at 30℃in a shaker for 24h, bacterial liquid PCR identification was performed with identification primers P3/P4, P7/P8, P13/P14, respectively, and the results showed that DSB-GCW61 fusion gene, EG II-GCW 51 fusion gene, PG5-GCW21 fusion gene were all integrated into Pichia pastoris X33 genome, and recombinant bacteria X33/DSB (GCW 61) -EG II (GCW 51) were successfully co-displayed.

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

(1) Shake flask culture of recombinant bacteria

4 recombinant yeasts X33/DSB-GCW61, X33 +.EG II-GCW 51, X33/PG5-GCW21 and X33/DSB (GCW 61) -EG II (GCW 51) -PG5 (GCW 21) were cultured at the shake flask level, respectively. Inoculating each recombinant strain into a 250mL triangular flask containing 25mL BMGY culture medium, and culturing at 30deg.C and 250rpm overnight to OD ₆₀₀ The bacterial suspension was resuspended in about 4-12mL of 250mL Erlenmeyer flask containing 25mL of BMMY liquid medium at room temperature at 6000rpm to give an initial OD ₆₀₀ The cells were incubated at 30℃and 250rpm for 168 hours, 0.25mL was sampled every 24 hours and 1% (v/v) methanol was added thereto for induction of expression. After the shake flask culture is finished, centrifuging at room temperature and 6000rpm to remove the supernatant, and collecting the thalli to obtain the whole cell catalyst with catalytic activity.

(2) Monitoring of growth of recombinant bacteria

The concentration of the bacterial suspension is characterized 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 measured after sampling every 24 hours _600nm Time is taken as an abscissa, and OD of each recombinant bacterium is taken as an abscissa _600nm The values are plotted as ordinate versus growth curve.

(3) Enzyme activity assay of xylanase produced by X33/DSB-GCW61 and X33/DSB (GCW 61) -EG II (GCW 51) -PG5 (GCW 21)

Taking 200 mu L of bacterial liquid, centrifuging for 1min at 10000rpm at room temperature, discarding the supernatant, adding 200 mu L of 50mmol/L disodium hydrogen phosphate-citric acid buffer solution with pH of 6.5 to wash the bacterial cells, and re-suspending the bacterial cells with 200 mu L of buffer solution after repeating for three times to obtain a sample for measuring the enzyme activity. Measuring enzyme activity of xylanase of each sample by absorbance method, using 0.5% (m/v) xylan as substrate, mixing diluted sample 100 μL with 900 μL substrate, reacting at 65deg.C for 10min, immediately adding 1.5mL DNS to stop reaction, boiling for 5min to develop color, placing test tube in cold water bath, cooling completely, and measuring OD ₅₄₀ And (3) calculating the enzyme activity. 1 enzyme activity unit is defined as the amount of xylanase required to hydrolyze 0.5wt% of the xylan substrate to 1. Mu. Mol xylose in 1min at 65℃and pH 6.5.

(4) Enzyme activity assay of glucanase produced by X33/EG II-GCW 51 and X33/DSB (GCW 61) -EG II (GCW 51) -PG5 (GCW 21)

Taking 200 mu L of bacterial liquid, centrifuging at 10000rpm for 1min at room temperature, and discardingAnd adding 200 mu L of 50mmol/L disodium hydrogen phosphate-citric acid buffer solution with pH of 4.8 into the supernatant to wash the thalli, and re-suspending the thalli with 200 mu L of buffer solution after repeating for three times to obtain a sample for measuring the enzyme activity. Measuring enzyme activity of dextranase of each sample by absorbance method, using carboxymethyl cellulose with substrate of 1% (m/v), mixing diluted sample 100 μL with substrate 900 μL, reacting at 75deg.C for 10min, immediately adding 1.5mL DNS to stop reaction, boiling for 5min to develop color, placing test tube in tap water, cooling completely, and measuring OD ₅₄₀ And (3) calculating the enzyme activity. 1 enzyme activity unit is defined as the amount of glucanase required to hydrolyze 1% of the carboxymethyl cellulose substrate to produce 1. Mu. Mol glucose in 1min at 75℃and pH 4.8.

(5) Enzyme activity assay of pectase produced by X33/PG5-GCW21 and X33/DSB (GCW 61) -EG II (GCW 51) -PG5 (GCW 21)

Taking 200 mu L of bacterial liquid, centrifuging for 1min at 10000rpm at room temperature, discarding the supernatant, adding 200 mu L of 100mmol/L disodium hydrogen phosphate-citric acid buffer solution with pH of 3.5 to wash the bacterial cells, and re-suspending the bacterial cells with 200 mu L of buffer solution after repeating for three times to obtain a sample for measuring the enzyme activity. Measuring enzyme activity of pectase of each sample by absorbance method, using pectin with substrate of 0.3% (m/v), mixing diluted sample 100 μL with substrate 900 μL, reacting at 70deg.C for 10min, immediately adding 1.5mL DNS to stop reaction, boiling for 5min to develop color, placing the test tube in cold water bath, cooling completely, and measuring OD ₅₄₀ And (3) calculating the enzyme activity. 1 enzyme activity unit is defined as the amount of pectinase required to hydrolyze 0.3% of the pectic substrate to produce 1. Mu. Mol polygalacturonic acid in 1min at 70℃and pH 3.5.

The comparison result of the growth curve of each recombinant bacterium and the enzyme activities of the three enzymes is shown in figure 2, and as can be seen from figure 2, the growth trend of 4 recombinant bacteria in the culture process is not obviously different (figure 2A); the xylanase-producing enzyme activities of X33/DSB (GCW 61) -EG II (GCW 51) -PG5 (GCW 21) and X33/DSB-GCW61 are not obviously different (FIG. 2B); the glucanase and pectinase production activities of X33/DSB (GCW 61) -EG II (GCW 51) -PG5 (GCW 21) were slightly lower than those of X33/EG II-GCW 51 and X33/PG5-GCW21, probably due to steric hindrance between the enzyme molecules displayed on the cell surface (FIGS. 2C and 2D). The results show that the co-display expression of the three NSP enzymes in the same host bacterium can not influence the growth, and X33/DSB (GCW 61) -EG II (GCW 51) -PG5 (GCW 21) can simultaneously produce three enzymes with high efficiency, wherein the xylanase activity is up to 13236U/g, the glucanase activity is up to 2056U/g, and the pectinase activity is up to 3227U/g (when shaking and fermenting at 30 ℃ and 250 rpm), and the method has good industrial application prospect.

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

Wheat is an important feed raw material, however, because the wheat bran contains a large amount of NSP (non-dairy products) 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.

(1) Preparation of desstarch wheat bran samples

100g of wheat bran is weighed into a 1L beaker, 500mL of 50mmol/L disodium hydrogen phosphate-citric acid buffer solution with pH of 6.0 is added, 6g of amylase and 6g of protease are added simultaneously, the mixture is placed into a water bath with the temperature of 50 ℃ for 3 hours, the wheat bran is repeatedly washed, and then the mixture is placed into an oven with the temperature of 80 ℃ for drying until the weight is constant, and a starch-removed wheat bran sample is obtained.

(2) Application of whole-cell catalyst for degrading wheat bran NSP for feeding, which shows three NSP enzymes in total

Weighing 0.3g of wheat bran sample, putting the wheat bran sample into a 100mL triangular flask, adding 30mL of 50mmol/L of disodium hydrogen phosphate-citric acid buffer solution with pH of 6.0, adding 0.6mg of total cell catalyst which is obtained in example 5 and shows three NSP enzymes together, placing the whole cell catalyst in a water bath table at 60 ℃ and 150rpm for 30min, simultaneously treating the wheat bran sample by using three independent display type total cell catalysts with equal enzyme activity units as a reference, 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 maximum reducing sugar content as 100%. Total cell catalysts displaying three NSP enzymes total the amount of reducing sugar produced after wheat bran treatment reached 60.1mg/g, and total cell catalysts displaying three NSP enzymes total the amount of reducing sugar after wheat bran sample treatment was significantly higher than the total of the three individual display type total cell catalysts and greater than the sum of the amounts of reducing sugar after three individual display type total cell catalyst treatment (fig. 3), indicating that there was a synergistic promotion of the three enzymes in the degradation of NSP in wheat bran samples.

The above description is only of the preferred embodiments of the present invention, and is not intended to limit the present invention in any way, but any simple modification, equivalent variation and modification made to the above embodiments according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.

Sequence listing

<110> university of national south China

<120> recombinant yeast displaying three NSP enzymes altogether, 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 GS)

<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 GS)

<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 GS)

<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 method for constructing recombinant yeast displaying three NSp enzymes, comprising the following steps:

1) The genes of three NSP enzymes are synthesized through total genes after codon optimization, including the genes of xylanase DSB, glucanase EG II and pectinase PG 5; the nucleotide sequence of the xylanase DSB gene is shown as SEQ ID NO. 1; the nucleotide sequence of the gene of the glucanase EG II is shown as SEQ ID NO. 2; the nucleotide sequence of the gene of the pectase PG5 is shown as SEQ ID NO. 3;

2) PCR amplification of the Pichia pastoris X33 genome to obtain anchored protein genes including GCW61, GCW51 and GCW21; the nucleotide sequence of the GCW61 is shown as SEQ ID NO. 4; the nucleotide sequence of the GCW51 is shown as SEQ ID NO. 5; the nucleotide sequence of the GCW21 is shown as SEQ ID NO. 6;

7) And (3) electrically converting the recombinant plasmid co-displaying and expressing xylanase DSB, pectinase PG5 and glucanase EG II into pichia pastoris X33 to obtain recombinant yeast co-displaying the three NSP enzymes.

2. The method of claim 1, wherein in step 3), the expression vector is any one of ppiczα A, pPICZ α B, pPICZ α C, pGAPZ α A, pGAPZ α B, pGAPZ αc.

3. A recombinant yeast displaying three NSP enzymes altogether, wherein the recombinant yeast displaying three NSP enzymes altogether is constructed by the method of claim 1 or 2.

4. The recombinant yeast of claim 3, wherein the recombinant yeast has pichia pastoris cell wall protein as an anchor protein and has xylanase DSB, glucanase EG ii and pectinase PG5 expressed on its surface.

5. A whole cell catalyst displaying three NSP enzymes altogether, characterized in that the recombinant yeast displaying three NSP enzymes altogether according to claim 3 or 4 is cultivated, and thalli are collected to obtain the whole cell catalyst displaying three NSP enzymes altogether.

6. The whole cell catalyst according to claim 5, wherein the xylanase enzyme activity is 13236U/g, the glucanase enzyme activity is 2056U/g and the pectinase enzyme activity is 3227U/g when the whole cell catalyst is fermented in a shake flask at a temperature of 28-30 ℃ and a speed of 200-250 rpm.

7. Use of a whole cell catalyst according to claim 5 or 6 displaying a total of three NSP enzymes in a feeding enzyme preparation.