Signal Peptide Optimization Increases Glucose Oxidase Expression in Pichia pastoris

doi:10.13304/j.nykjdb.2020.0219

Abstract

Abstract:

Glucose oxidase （GOD） is widely used in food processing， feed additives， biosensors and industrial production. Compared to Aspergillus， Pichia pastoris is a microorganism that has the potential to produce GOD more efficiently， with fewer by-products and simpler separation purification. However， due to factors such as exogenous genes， gene dosage， and secretion signals， the GOD yield of Pichia pastoris is still low， which limits the large-scale application of GOD in food processing and other fields. In this study， based on Pichia pastoris codon preference and mRNA secondary structure energy， the alpha factor signal peptide in Pichia pastoris recombinant expression cassette was modified to obtain 4 Pichia recombinant strains containing different signal peptides. The results showed that the GOD activity of the recombinant strain containing the highest codon frequency signal peptide α¹was 2.01 times that of the starting strain， while the GOD enzyme activity of the recombinant strain containing low codon frequency signal peptide α⁴ was only 41%. The above results also indicated that the arrangement of the signal peptide nucleic acid sequence in Pichia pastoris expression vector could affect the expression level of recombinant GOD， which provided a new research idea for increasing the expression level of Pichia pastoris recombinant protein.

Key words: glucose oxidase, signal peptide, Pichia pastoris, protein expression

摘要：

葡萄糖氧化酶（glucose oxidase，GOD）广泛用于食品加工、饲料添加剂、生物传感器及工业生产等领域。较曲霉而言，毕赤酵母是更具高效生产GOD潜力的微生物，其副产物少，分离纯化简单。但由于外源基因、基因剂量以及分泌信号等因素的影响，毕赤酵母GOD产量仍然较低，限制了GOD在食品加工等领域的大规模应用。基于毕赤酵母密码子偏好性和mRNA二级结构能量，对毕赤酵母重组表达盒中的α因子信号肽进行改造，得到4种含有不同信号肽的毕赤酵母重组菌株。结果表明，使用含最高密码子频率信号肽α¹重组菌株的GOD酶活力是出发菌株的2.01倍；而使用含低密码子频率信号肽α⁴重组菌株的GOD酶活力仅为出发菌株的41%。综上所述，毕赤酵母表达载体中的信号肽核酸序列排布能够影响重组GOD的表达量，为提高毕赤酵母重组蛋白表达量提供了新的研究思路。

关键词: 葡萄糖氧化酶, 信号肽, 毕赤酵母, 蛋白表达

CLC Number:

Na ZHANG, Yaru YAN, Yun WU, Yuhong ZHANG, Wei ZHANG. Signal Peptide Optimization Increases Glucose Oxidase Expression in Pichia pastoris[J]. Journal of Agricultural Science and Technology, 2023, 25(2): 211-219.

张娜, 闫亚茹, 武运, 张宇宏, 张伟. 信号肽优化提高葡萄糖氧化酶在毕赤酵母中的表达量[J]. 中国农业科技导报, 2023, 25(2): 211-219.

Figures/Tables 8

Table 1 Primers for gene amplification

引物名称Primer name	引物序列Primer sequence（5’-3’）
α-signal-F	ACAACTAATTATTCGAAACGAGATCAAAAAACAACTAATTATTCG
α-signal-R	TCGATTCCATTGGAGAATTCAGCTTCAGCCTCTCTTTTCTCGAGA
ZT-pPICZαA-F	AGATCAAAAAACAACTAATTATTCGAAACG
ZT-pPICZαA-R	CAATGAAGCTTCGATTCCATTGGAGAATTC

Fig. 1 DNA sequence alignment of five different signal peptide

Fig. 2 Secondary structure of five α-signal peptide mRNAs

Fig. 3 mRNA secondary structure energy of five α-signal peptides

Fig. 4 Codon adaptation indices of five α-signal peptides

Fig. 5 GOD enzyme activity of recombinant strains with different signal peptides and recombinant strains containing wild-type signal peptides

Fig. 6 Enzyme activity average

Fig. 7 SDS-PAGE analysis of recombinant strains GODNote： M-Protein molecular weight marker.

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