阅读说明：本技术 一种β-甘露聚糖酶密码子优化序列及提高毕赤酵母工程菌表达水平的方法 (Beta-mannase codon optimization sequence and method for improving pichia pastoris engineering bacteria expression level ) 是由 郑宏臣 宋诙 徐健勇 甄杰 付晓平 于 2018-07-02 设计创作，主要内容包括：本发明涉及生物技术领域,具体公开了一种β-甘露聚糖酶密码子优化序列及提高毕赤酵母工程菌表达水平的方法。本发明为提高耐热酸性β-甘露聚糖酶Man5AS11R在毕赤酵母工程菌中的诱导表达量,首先应用Gene Designer(DNA2.0,Menlo Park,CA,USA)软件对man5AS11R基因的核苷酸序列进行密码子偏向性优化,通过pPIC9k-man5AS11R-opt重组载体,成功构建了毕赤酵母工程菌GS115/man5AS11R-opt。在此基础上,通过重组载体pPICZ-αA-vgb在菌株GS115/man5AS11R-opt中完成了血红蛋白VHb的共表达,显著促进了毕赤酵母工程菌发酵过程中的溶氧水平,提高了该工程菌发酵密度和产量,10L发酵罐发酵产酶达到20000U/mL以上,较原始菌株GS115/man5AS11R产量提高了一倍,显示了良好的产业化生产前景。(The invention relates to the technical field of biology, and particularly discloses a beta-mannase codon optimization sequence and a method for improving the expression level of pichia pastoris engineering bacteria. In order to improve the inducible expression quantity of the heat-resistant acidic beta-mannase Man5AS11R in pichia pastoris engineering bacteria, firstly, Gene Designer (DNA2.0, Menlo Park, CA, USA) software is applied to perform codon bias optimization on a nucleotide sequence of Man5AS11R Gene, and through a pPIC9k-Man5AS11R-opt recombinant vector, the pichia pastoris engineering bacteria GS115/Man5AS11R-opt is successfully constructed. On the basis, the co-expression of the hemoglobin VHb is completed in the strain GS115/man5AS11R-opt through the recombinant vector pPICZ-alpha A-vgb, the dissolved oxygen level in the fermentation process of the pichia pastoris engineering bacteria is obviously promoted, the fermentation density and the yield of the engineering bacteria are improved, the fermentation enzyme production of a 10L fermentation tank reaches over 20000U/mL, the yield is doubled compared with the yield of the original strain GS115/man5AS11R, and the good industrial production prospect is displayed.)

1. A codon optimized sequence of thermostable acidic beta-mannase Man5AS11R for Pichia pastoris host bacteria is characterized in that the sequence is shown AS SEQ ID NO. 1.

2. A construction method of a recombinant strain for improving the expression level of pichia pastoris engineering bacteria is characterized by comprising the following steps:

(1) carrying out codon optimization aiming at pichia pastoris on the nucleic acid sequence of the man5AS11R gene to obtain an optimized coding sequence man5AS 11R-opt;

(2) constructing a recombinant expression vector pPIC9k-man5AS11R-opt by applying the optimized sequence man5AS11R-opt, and transforming the recombinant expression vector pPIC9k-man5AS11R-opt into pichia pastoris GS115 to obtain a recombinant expression strain GS115/man5AS 11R-opt;

(3) the hemoglobin gene vgb of the Vitreoscilla is synthesized into a multiple cloning site of a vector pPICZ-alpha A, and the vector pPICZ-alpha A is transformed into GS115/man5AS11R-opt engineering bacteria to obtain high-yield engineering bacteria GS115/man5AS 11R-opt-VHb.

3. The haemoglobin VHb sequence of claim 2 obtained from NCBI (GenBank AY 278220).

4. The high-yield engineering bacterium GS115/man5AS11R-opt-VHb disclosed by claim 2 has the advantages that the yield is doubled compared with that of an original strain GS115/man5AS11R, and the enzyme activity of a 10L fermentation tank can reach more than 20000U/ml.

Technical Field

The invention belongs to the technical field of biology, and relates to a beta-mannase codon optimization sequence and a method for improving the expression level of pichia pastoris engineering bacteria.

Background

In recent years, with the development and utilization of natural hemicellulose resources, the elimination of mannan anti-nutritional factors in feeds and the discovery of pharmaceutical value of mannan-oligosaccharide, the demand of beta-mannase is increasing. At present, the commercial enzyme preparations of the beta-mannase in the market comprise Houseyi enzyme produced by American chengem company, Huafen enzyme produced by domestic doctor Olympic group, beta-mannase produced by challenge group and the like, but the application range of the enzyme is greatly reduced because the yield is small and the relative price of the product is very high.

The pichia pastoris expression system has been developed into a mature foreign protein expression system, and is often used for large-scale expression of foreign proteins in industry. However, for some genes derived from bacteria, their expression levels are often low, even after codon optimization, and do not necessarily achieve the desired expression levels. In addition, high-density fermentation of pichia pastoris is also the key to realize high-level expression of foreign proteins, and pichia pastoris needs to consume a large amount of oxygen during high-density fermentation, and the level of dissolved oxygen often directly influences the expression level of the foreign proteins. Therefore, vitreoscilla hemoglobin (VHb) is used as an oxygen regulatory protein, and theoretically can bind free oxygen, thereby increasing the oxygen content in the fermentation broth, improving the cell growth conditions, and promoting the synthesis of total cell protein and the accumulation of foreign proteins.

Disclosure of Invention

1. The invention aims to provide a method for improving the expression level of heat-resistant acidic beta-mannase Man5AS11R in pichia pastoris engineering bacteria, which comprises the following specific steps:

(1) the nucleotide sequence of the man5AS11R gene is optimized by a codon aiming at pichia pastoris to obtain an optimized coding sequence man5AS11R-opt, which is shown AS SEQ ID NO. 1:

ACTGGTTTCTACGTTAACGGTGGTAAGTTGTACGACTCTACTGGTTGTCC 50

ATTCTACATCGTTGGTATCAACCACGGTCACTCTTGGTTCAAGAACGACA 100

CTGCTACTGCTATCCCAGCTATCGCTAAGACTGGTGCTAACACTGTTAGA 150

ATCGTTTTGTCTAACGGTACTCAATACACTAAGGACGACTTGAACTCTGT 200

TAAGAACATCATCAACTTGGCTGAAGAAAACAAGATCGACGCTGTTTTGG 250

AAGTTCACGACGCTACTGGTAAGGACGACTTCAACTCTTTGGACGCTGCT 300

GTTAACTACTGGATCTCTATCAAGGAAGCTTTGATCGGTAAGGAAGACAG 350

AGTTATCGTTAACATCGCTAACGAATGGTACGGTACTTGGAACGGTTCTG 400

CTTGGGCTGACGGTTACAAGAAGGCTATCCCAAAGTTGAGAGACGCTGGT 450

ATCAAGAACACTTTGATCGTTGACGCTGCTGGTTGGGGTCAATACCCACA 500

ATCTATCGTTGACTACGGTCAATCTGTTTTCGCTGCTGACTCTCAAAAGA 550

ACACTGCTTTCTCTATCCACATCTACGAATACGCTGGTAAGGACGCTGCT 600

ACTGTTAAGTCTAACATCGAAAACGTTTTGAACAAGGGTTTGGCTTTGAT 650

CGAAGGTGAATTCGGTGGTTACCACACTAACGGTGACGTTGACGAATACG 700

CTATCATCAAGTACGGTTTGGAAAAGGGTGTTGGTTGGTTGGCTTGGTCT 750

TGGTACGGTAACGGTATCAAGTGGAACTACTTGGACTTGGCTACTGGTCC 800

AAACGGTTCTTTGACTTCTTACGGTAACACTGTTGTTAACGACACTTACG 850

GTATCAAGAACACTTCTCAAAAGGCTGGTATCTTCTGTGGTGACGACGGT 900

GTTGGTGACGGTGGTCCAGGTGACTCTAACGGTACTAAGACTACTTTGTA 950

CAACTTCGAAACTGGTACTGAAGGTTGGTCTGGTAAGAACATCGAAACTG 1000

GTCCATGGTCTGTTAACGAATGGGCTGCTAAGGGTAACCACTCTTTGAAG 1050

GCTGACGTTAACTTGGGTGACAACTCTGAACACTACTTGAAGTTGACTCA 1100

AAACTTGAACTTCTCTGGTAAGTCTCAATTGACTGCTACTGTTAAGCACG 1150

CTGACTGGGGTAACTTCGGTGACGAAATCAACGCTAAGTTGTACGTTAAG 1200

ACTGAATCTGACTGGTAA 1218

(2) constructing a recombinant expression vector pPIC9k-man5AS11R-opt by applying the optimized sequence man5AS11R-opt, and transforming the recombinant expression vector pPIC9k-man5AS11R-opt into pichia pastoris GS115 to obtain a recombinant expression strain GS115/man5AS 11R-opt;

(3) the hemoglobin gene vgb of Vitreoscilla hybrida (GenBank AY 278220) is synthesized into a multiple cloning site of a vector pPICZ-alpha A, and is transformed into GS115/man5AS11R-opt engineering bacteria to obtain high-yield engineering bacteria GS115/man5AS 11R-opt-VHb.

2. The yield of the high-yield engineering bacteria GS115/man5AS11R-opt-VHb is doubled compared with the yield of the original strain GS115/man5AS11R, and the enzyme activity of a 10L fermentation tank can reach more than 20000U/ml.

Drawings

FIG. 1 construction of recombinant vector for Co-expression of Man5AS11R and VHb

FIG. 2 Man5AS11R codon-optimized 10L fermenter enzyme production

FIG. 3 enzyme production in 10L fermentor from Co-expression of Man5AS11R and VHb

FIG. 4 10L fermentor enzyme production profile co-expressed with VHb after codon optimization for Man5AS 11R.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.