WO2017096608A1

WO2017096608A1 - Construction method for co-expressing vhb hemogloblin and cellulase protein in pichia pastoris

Info

Publication number: WO2017096608A1
Application number: PCT/CN2015/097118
Authority: WO
Inventors: 孙付保; 白仁惠; 张震宇; 许银彪; 王春迪
Original assignee: 江南大学
Priority date: 2015-12-11
Filing date: 2015-12-11
Publication date: 2017-06-15

Abstract

A construction method for co-expressing VHb hemogloblin and a cellulase protein in Pichia pastoris comprises performing codon optimization on a gene of endoglucanase II (EG II) of Trichoderma reesei, and co-expressing the optimized gene of endoglucanase II (EG II) of Trichoderma reesei and Vitreoscilla hemogloblin (VHb) in Pichia pastoris.

Description

Instruction manual

Title: Construction method for co-expression of hemoglobin VHb and cellulase protein in Pichia pastoris

Technical field

[0001] The present invention relates to the construction of a recombinant Pichia pastoris system which co-expresses the Vitreoscilla hemoglobin gene (vgb) and cellulase proteins, and belongs to the field of bioengineering technology.

Background technique

[0002] Cellulase is a general term for a class of multi-component enzymes, which is capable of hydrolyzing cellulose which is difficult to be bio-utilized into glucose which is beneficial for bio-use, and is therefore considered to be one of the enzyme preparations having great potential. Cellulase is a glycoside hydrolase. Cellulase capable of degrading cellulose includes at least three components, namely exonuclease (CBH), endoglucanase (EG) and β-glucoside. Enzyme (BG). EG acts on the non-crystalline region of cellulose, hydrolyzes the β-1,4 glycosidic bond, and can cut off the linear cellulose polymer to form a large amount of small cellulose molecules; CBH hydrolyzes the 1,4-β-ϋ-glucosidic bond, acting on The ends of the linear cellulose polymer form a cellobiose molecule; BG hydrolyzes cellobiose to glucose. Through the synergy of the cellulase complex enzyme components, the most abundant biopolymer _cellulose on the earth can be converted into glucose that can be easily utilized by microorganisms, and biofuels can be produced by fermentation to alleviate the current energy shortage problem.

[0003] In recent years, heterologous expression of cellulase genes to obtain single enzyme components is a research hotspot. Pichia pastoris can achieve post-translational modification, such as glycosylation, disulfide bond formation, allowing proteins to fold correctly and obtain active target proteins. In addition, Pichia does not produce endogenous lignocellulase groups. The extracellular protein component is not complicated, and the recombinant Pichia pastoris fermentation supernatant can be directly used as a single enzyme preparation without purification. Therefore, Pichia pastoris is the preferred host bacteria to achieve cellulase composition. Source expression to obtain high concentration and high purity cellulase protein components has become a research hotspot.

[0004] The data indicate that Vitreoscilla hemoglobin (VHb) is produced from the strict aerobic bacteria Vitreoscilla, and VHb is an automatic first aid protein in the hypoxic state of Vitreoscilla. The hypothesis about its function suggests that VHb can capture oxygen in a hypoxic environment, supplying oxidases associated with respiration, thereby increasing respiratory efficiency and promoting ATP production. Heterologous expression of cellulase protein components by Pichia pastoris as a host strain, industrialization The premise is that high-density culture with insufficient oxygen supply must have high potential. By realizing the co-expression of VHb and cellulase protein in Pichia pastoris, the enzyme production level and industrial application potential of recombinant Pichia pastoris can be improved.

technical problem

[0005] Cellulases are widely available, and cellulase-producing organisms include insects, mollusks, protozoa, bacteria, and fungi. However, these self-produced enzymes inherently have a certain proportion of defects. For the most widely used cellulase-producing fungus, Trichoderma reesei, the cellulase system lacks the exonuclease CBH II and The β-glucosidase BG results in inefficient enzymatic hydrolysis of its cellulase system. In addition, whether it is derived from the organism's self-produced enzyme system or artificially produced commercial cellulase system, it contains more than 80 kinds of proteins, and even more, resulting in lower core enzyme components than enzyme activity, the same It is also the reason for the low efficiency of cellulase enzymatic hydrolysis. More importantly, the ratios of cellulose, hemicellulose, and lignin in different lignocellulosic materials are significantly different, resulting in the same type of commercial cellulase preparation or self-produced enzyme that does not exert optimal enzymatic hydrolysis. Therefore, optimizing the custom cellulase to achieve the best ratio of each component, in order to obtain the optimal hydrolysis effect with the minimum amount of enzyme. Therefore, in order to optimize the custom cellulase, it is critical to obtain a cellulase single enzyme component.

Problem solution

Technical solution

[0006] The object of the present invention is to construct a Pichia pastoris which co-expresses the Vitreoscilla hemoglobin gene and the cellulase protein gene.

The specific steps of the Pichia cell system for co-expressing VHb protein and endoglucanase II provided by the present invention are as follows [0008] 1. Optimization of EG II codon bias:

The present invention achieves EG II codon bias optimization, the original codon sequence of which is as follows:

SEQ ID NO. 1

1 ATGAACAAGT CCGTGGCTCC ATTGCTGCTT GCAGCGTCCA

TACTATATGG CGGCGCCGTC

[0012] 61 GCACAGCAGA CTGTCTGGGG CCAGTGTGGA GGTATTGGTT

GGAGCGGACC TACGAATTGT DVDIDVIOOD DIDVVDVOVD

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IOOIOIOOOV ODIOVDIDIV DODVIOVVVD IVDOOIIOVD 199 [ 00]

ODIOOIIIDD OVODVDIIVV DIDOIVVIDV IDDDOODOOO VDIOOIIVDI VOOOIOODVV 109 [I ZOO]

OOIVODIDOI VIIVVDVDDI VDVODIODIV DOIDVIVDOD OOOIDIDIOI OIOOOO V I S [OZOO]

IIOIIDOVDI VOIVIOVVDD IIIVDOVODV DDIIVOIIDI VVDOODOOOI IIVVDVVDVV 181/ [6100]

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DDOVOOODDI IVIOIVVDOD

OrVirVlID IVVDIDDDVO DIIOIIDOVD IDOOIDDIDO IZl [£100] ll .60/ST0ZN3/X3d 809960/ .Ϊ0Ζ OAV CGCTTGCCAC TTGGCTCCGA GCAACGTTCA GTCCTGCATA

CAGATGTCTA TCTTGGCTAT

1141 GTTGGTTGGG GTGCCGGATC ATTTGATAGC ACGTATGTCC

TGACGGAAAC ACCGACTGGC

1201 AGTGGTAACT CATGGACGGA CACATCCTTG GTCAGCTCGT

GTCTCGCAAG AAAGTAG

[0032] The EG II amino acid sequence before codon optimization is shown in SEQ ID NO.

SEQ ID NO. 2

1 MNKSVAPLLL AASILYGGAV AQQTVWGQCG GIGWSGPTNC

APGSACSTLN PYYAQCIPGA

[0035] 61 TTITTSTRPP SGPTTTTRAT STSSSTPPTS SGVRFAGVNI AGFDFGCTTD

GTCVTSKVYP

[0036] 121 PLKNFTGSNN YPDGIGQMQH FVNEDGMTIF RLPVGWQYLV

NNNLGGNLDS TSISKYDQLV

[0037] 181 QGCLSLGAYC IVDIHNYARW NGGIIGQGGP TNAQFTSLWS

QLASKYASQS RVWFGIMNEP

241 HDVNINTWAA TVQEVVTAIR NAGATSQFIS LPGNDWQSAG

AFISDGSAAA LSQVTNPDGS

[0039] 301 TTNLIFDVHK YLDSDNSGTH AECTTNNIDG AFSPLATWLR

QNNRQAILTE TGGGNVQSCI

[0040] 361 QDMCQQIQYL NQNSDVYLGY VGWGAGSFDS TYVLTETPTG

SGNSWTDTSL VSSCLARK

[0041] The present invention utilizes Gene Designer (DNA2.0, Menlo Park, CA, USA) to optimize the codon bias of the EG II nucleotide sequence shown in SEQ ID NO. 1, and the optimized nucleotide sequence is SEQ. ID NO. 3 .

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[0064] After codon optimization, the EG II amino acid sequence is identical to SEQ ID NO.

[0065] 2. Constructing the expression vector pPIC9K-eg2 _:

[0066] Using the plasmid pPIC9K as a vector, the optimized EG II gene was inserted into the promoter AOX1 downstream, the 5'-end cleavage site was EcoR I, and the 3'-end cleavage site was Not I, and the pPIC9K-eg2 expression vector was constructed. .

[0067] 3. Obtaining a recombinant strain:

[0068] Sac l was used as a _P PIC9K-eg2 expression vector linearization site, and the expression vector was linearized and transformed into Pichia pastoris by electroporation.

[0069] 4. Obtaining the vgb gene:

[0070] Obtaining the Vitreoscilla hemoglobin nucleotide sequence from NCBI (GenBank Accession No.)

M30794.1), the EcoR I restriction site was added at the 5' end, and the Not I restriction site was added at the 3' end to obtain the target gene by artificial synthesis.

[0071] 5. Constructing the expression vector pPICZaA-vgb: [0072] Using the plasmid pPICZotA as a vector, the vgb gene was inserted downstream of the promoter AOX1, and the 5' end cleavage site was

EcoR I, the 3' end cleavage site was Not I, and the pPICZaA-vgb expression vector was constructed.

[0073] 6. Obtaining a co-expressing recombinant strain:

[0074] Sac l was used as a pPICZotA-vgb expression vector linearization site to realize linearization of the expression vector, and electrotransformation was carried out into recombinant Pichia pastoris which already contained the eg2 gene.

[0075] 7, recombinant VHb protein activity detection:

[0076] Whether the recombinant Pichia pastoris secretes an active VHb protein is detected by a CO difference spectrum detection method.

[0077] 8, fermentation enzyme production and enzyme production levels:

[0078] The enzyme production levels of the EG II recombinant strain and the EG II and VHb co-expressing strains were compared by enzyme production by shake flask fermentation.

Advantageous effects of the invention

Beneficial effect

[0079] Advantageous Effects of the Invention:

[0080] 1. The invention realizes the optimization of the codon bias of the Trichoderma reesei endoglucanase II (EG II) gene, and the biased object is Pichia pastoris, and the GC content is reduced during the optimization process, from 55.9 %. The decrease was 46.2%, and the expression efficiency of recombinant Pichia pastoris was improved.

2. In the process of optimizing the codon bias of the Trichoderma reesei endoglucanase II (EG II) gene, the optimized nucleotide sequence of the RNA secondary structure folding software RNAstmcture is used in the present invention. The secondary structure is folded, and the base sequence is adjusted, so that the base of the initiation codon has an anthracene ring structure, which is favorable for ribosome binding and improves the expression efficiency of the recombinant Pichia pastoris.

[0082] 3. The invention realizes co-expression of cellulase protein and Vitreoscilla hemoglobin (VHb) in Pichia pastoris

The enzyme production efficiency of the recombinant Pichia pastoris in the low-oxygen high-density fermentation process is improved, and the industrial production potential of the recombinant Pichia pastoris is enhanced.

Brief description of the drawing

DRAWINGS

[0083] FIG. 1 : Experimental ideas and solutions;

Figure 2: Comparison of the eg2 nucleotide sequence before and after optimization;

Figure 3: Comparison of the eg2 amino acid sequence before and after optimization; Figure 4: _P PIC9K-eg2 expression vector map;

Figure 5: 1% nucleic acid gel electrophoresis:

[0088] 1: Sac l single digestion expression vector pPIC9K-eg2,

[0089] 2: EcoR I and Not l double digestion expression vector pPIC9K-eg2;

Figure 6: Screening of high-yield recombinant EG II protein strains;

Figure 7: Map of the pPICZaA-vgb expression vector;

Figure 8: 1% nucleic acid gel electrophoresis:

[0093] 1: Sac l single digestion expression vector pPICZaA-vgb,

2: EcoR I and Not l double digestion expression vector pPICZaA-vgb;

[0095] pPICZaA-vgb was successfully constructed, and the nucleic acid electrophoresis pattern after digestion

Figure 9: Screening of high-yield recombinant EG II protein co-expression strains

Figure 10: Colony PCR validation of co-expressing strains:

P: Pichia pastoris,

E: EG II recombinant Pichia,

VE1, VE25, VE31, etc.: EG II and VHb co-expressing strains;

Figure 11: Full-wavelength scanning of the fermentation supernatant of EG II and VHb co-expressing strains after CO gas treatment;

Figure 12: Comparison of growth and enzyme production abilities of EG II recombinant strains with EG II and VHb co-expressing strains:

A: Comparison of the ability of the strain to produce enzymes,

B: Comparison of strain growth ability.

Embodiments of the invention

The present invention constructs a Pichia pastoris which coexpresses the Vitreoscilla hemoglobin gene and the T. reesei endoglucanase II gene, thereby improving the oxygen utilization efficiency of the recombinant strain, thereby increasing the concentration of the bacteria and producing the enzyme. The ability to build ideas is shown in Figure 1.

Example 1 : Codon optimization of the T. reesei endoglucanase II (EG II) gene and construction of an expression vector.

[0107] 1. The present invention utilizes Gene Designer (DNA2.0, Menlo Park, CA, USA) to implement SEQ ID

The nucleotide sequence codon biasing of EG II (GenBank Accession No. DQ178347.1) shown in NO. 1 is optimized, and the optimized nucleotide sequence is shown in SEQ ID NO. Codon-optimized amino acid sequence and The original amino acid sequence is identical as shown in SEQ ID NO. Using 13⁄41^&&810]"18 08115 as the host, the nucleotide sequence is optimized by codon bias and the original nucleotide sequence is shown in Figure 2. The amino acid sequence is compared with the original amino acid sequence by codon bias optimization. 3.

[0108] 2. The 5' end cleavage site is EcoR I, and the 3' end cleavage site is Not

As a double restriction site, I inserted into the downstream of the pOX9K plasmid vector AOX1 promoter to construct the expression vector pPIC9 K-eg2. The expression vector map is shown in Fig. 4. The expression vector was verified by double enzyme digestion to confirm the successful construction of the expression vector _P PIC9K-eg2. The nucleic acid electrophoresis pattern after digestion was shown in Fig. 5.

Example 2: Linearization of expression vectors and electroporation and screening of high-yielding strains.

[0110] 1. Using Sac l as a linearization site of _P PIC9K-eg2 expression vector, linearize the expression vector, and verify the linearized plasmid by nucleic acid electrophoresis, as shown in FIG. 5 . By electrotransformation, Pichia pastori _S GS115 was transferred to obtain a recombinant strain, and multi-copy transformants were screened by using a geneticin G-418 concentration gradient, and high-yield strains were obtained by shake flask fermentation of colonies capable of normal growth on high-concentration resistant plates. The CMC enzyme activity of the fermentation supernatant was determined by the DNS method at 120 h, as shown in Fig. 6. One unit of enzyme activity unit was defined as the amount of enzyme capable of converting 1 μηιοΐ substrate in 1 minute under a water bath condition of 50 °C.

Example 3: Acquisition of the Vitreoscilla hemoglobin (VHb) gene and construction of an expression vector.

1. The Vitreoscilla hemoglobin gene (GenBank Accession No. M30794.1) was obtained from NCBI, the EcoR I restriction site was added at the 5' end, and the Not I restriction site was added at the 3' end, and artificial synthesis was performed. Obtain the gene of interest.

[0113] 2. The 5' end cleavage site is EcoR I, and the 3' end cleavage site is Not

I as a double restriction site, inserted into the pPICZotA plasmid vector downstream of the AOX1 promoter to construct the expression vector pPI

CZaA-vgb, the expression vector map is shown in Figure 7. The expression vector was verified by double enzyme digestion to confirm the successful construction of the expression vector pPICZotA-vgb, and the nucleic acid electrophoresis pattern after digestion was shown in Fig. 8.

Example 4: The expression vector pPICZotA-vgb was linearized and electrotransformed and screened for co-expressing high-yielding strains.

[0115] 1. The Sac l is used as a linearization site of the pPICZotA-vgb expression vector to realize linearization of the expression vector, and the linearized plasmid is verified by nucleic acid electrophoresis, as shown in FIG. 8 . The recombinant Pichia pastoris GS115 containing the eg2 gene was transformed by electroporation to obtain a co-expressing recombinant strain, and the multi-copy transformants were screened by the antibiotic Zeocin concentration gradient, and the colonies which can grow normally on the high-concentration resistant plate were fermented by shake flask to obtain high yield. The strain was sampled at 72 h and the CMC enzyme activity of the fermentation supernatant was determined by DNS method, as shown in Fig. 9. In addition, colony PCR was carried out on these strains to verify that the vgb gene and the eg2 gene coexist in the Pichia host chromosome, and the results are shown in FIG.

Example 5: Detection of recombinant VHb protein activity.

The CO and VHb proteins are capable of irreversible binding to form a stable porphyrin ring structure which has an absorption peak at a specific wavelength, and thus the recombinant VHb protein can be detected by this method.

[0118] The EG II recombinant strain and the EG II and VHb co-expressing strains were respectively introduced into the fermentation medium, and after fermentation for 120 h, the supernatant of the fermentation broth was taken, and the fermentation broth was treated with excess Na2S204 (safety powder) as a reducing agent for 10 min. After that, CO was introduced into the fermentation broth for 3 min. The fermentation supernatant of the EG II recombinant strain was used as a blank control, and the full-wavelength scanning was performed using a Hitachi U3900 spectrophotometer with a wavelength range of 300 to 500 nm. The result is shown in Figure 11.

Example 6: Comparison of enzyme production levels of EG II recombinant strains with EG II and VHb co-expressing strains.

[0120] 1. BMMY was used as the enzyme-producing medium, methanol was used as the inducer, and the enzyme was produced by 1.5% methanol addition. The enzyme activity and the concentration determination were performed for 72 h, 96 h and 120 h samples.

II recombinant strain and EG

The II and VHb co-expressing strains were compared in terms of biomass and enzyme activity, and the results are shown in Fig. 12.

[0121]

[0122]

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Claims

Claim

[Claim 1] A method for constructing co-expressed hemoglobin VHb and cellulase protein in Pichia pastoris, which is characterized by the following steps:

1) Codon bias optimization: The present invention implements the EG shown in SEQ ID NO. 1 using the software Gene Designer.

I〗

The nucleotide sequence codon bias is optimized, and the optimized nucleotide sequence is shown in SEQ ID N0.3.

2) Construction of pPIC9K-eg2 expression vector: The plasmid pPIC9K was used as a vector, and the optimized EG gene was inserted into the promoter downstream of AOX1, and the 5' end restriction site was EcoR.

I

, 3' end cleavage site is NW

I

, Construction of pPIC9K-eg2 expression vector.

3) Obtain the recombinant strain: "3⁄4c

I

As a linearization site of the _P PIC9K-eg2 expression vector, the expression vector was linearized and transferred to Pichia pastoris by electroporation.

4) Obtaining the vgb gene: Obtaining the Vitreoscilla hemoglobin gene from NCBI and adding EcoR at the 5' end

I

Enzyme cleavage site, add Noi 3 at the 3' end

The target gene is obtained by artificial synthesis.

5) Construction of pPICZaA-vgb expression vector: using plasmid pPICZaA as a vector, vgb-based Due to the insertion of the promoter downstream of AOX1, the 5' end cleavage site is EcoR Ϊ

, 3' end cleavage site is NW

I

, Construction of pPICZotA-vgb expression vector.

6) High-copy co-expressed strains were screened by high-concentration geneticin G-418 and bleomycin double resistance screening.

7) Comparison of the enzyme production levels of the EG recombinant strain and the EG and VHb co-expressing strains.

[Claim 2] A method for constructing co-expressed hemoglobin VHb and cellulase protein in Pichia pastoris according to claim 1, wherein step 1) codon bias optimization, codon mainly refers to encoding endo-cellulosic Enzymes, exocellulases, beta-grains and glycosidases, as well as xylanases and xylosidase, are also involved in hemicellulose side chain hydrolysis-related enzymes.

[Claim 3] A method for constructing co-expressed hemoglobin VHb and cellulase protein in Pichia pastoris according to claim 1, wherein the co-expressing recombinant strain refers to recombinant Pichia pastoris

[Claim 4] A method for constructing co-expressed hemoglobin VHb and cellulase protein in Pichia pastoris according to claim 1, wherein the recombinant strain stably and efficiently expresses cellulase protein, and co-expresses Pichia pastoris strain The homolog of the cellulase protein can produce active VHb protein

[Claim 5] The method for constructing co-expressed hemoglobin VHb and cellulase protein in Pichia pastoris according to claim 1, wherein the EG of step 1) is SEQ ID NO. 1 or SEQ ID N0.3 The T. reesei exonuclease gene encoded by the indicated nucleotide sequence.

[Claim 6] A method for constructing co-expressed hemoglobin VHb and cellulase protein in Pichia pastoris according to claim 1, wherein the EG II gene codon bias is optimized in step 1), and the optimized EG core The nucleotide sequence is shown in SEQ ID N0.3.

[Claim 7] A method for constructing a co-expressed hemoglobin VHb and a cellulase protein in Pichia pastoris according to claim 1, wherein the EG described in the step 1) The gene has an amino acid sequence as shown in SEQ ID N0.2.

[Claim 8] A method for constructing co-expressed hemoglobin VHb and cellulase protein in Pichia pastoris according to claim 1, wherein the expression vector described in the step 2) is a _P PIC9K-eg2 expression vector.

[Claim 9] A method for constructing co-expressed hemoglobin VHb and cellulase protein in Pichia pastoris according to claim 1, wherein the expression vector described in the step 5) is a pPIC9K-vgb expression vector.