组合策略促进碱性果胶酶在毕赤酵母中高效表达

High-Level Expression of Alkaline Polygalacturonate Lyase inPichia pastorisby Using a Combined Strategy

DOI：10.3969/j.issn.1673-1689.2019.02.006

中文关键词: 碱性果胶酶 毕赤酵母 密码子优化 分子伴侣 指数流加

英文关键词: alkaline polygalacturonate lyase,Pichia pastoris,codon-optimization,molecular chaper- one,exponential fed-batch

基金项目:

摘要点击次数: 243

全文下载次数: 312

中文摘要:

本研究组合了基因密码子优化及分子伴侣共表达策略提高碱性果胶酶（PGL）在毕赤酵母中的胞外产量。基于Pichia pastoris密码子偏好性，优化了来源于Bacillus sp.WSHB04-02 PGL高热稳定性突变体K314M基因，并克隆至pPIC9K的EcoR I-Not I得到PGL表达载体pPIC9K-PGL。pPIC9K-PGL转化Pichia pastoris GS115（his-）得到重组菌GS115/PGL 14#，胞外PGL酶活达到301.32 U/mL，较优化前提高25.1%。在此基础上，分别及同时共表达分子伴侣内质网蛋白折叠氧化还原辅助因子（ERO1）和泛素共轭酶（UBC1）。结果显示，同时表达ERO1和UBC1（GS115/PGLl-ERO1-UBC1 2#）使重组菌胞外PGL较共表达前提高49.3%，达到450.12 U/mL。应用指数流加策略对重组菌株GS115/PGLl-ERO1-UBC1 2#进行3 L罐发酵，诱导发酵96 h胞外PGL可达1 362.31 U/mL。研究结果对促进PGL产量的提升及其应用具有重要现实意义。

英文摘要:

In this study，codon usage optimization and co-expression of molecular chaperone are used to improve the extracellular production of alkaline polygalacturonate lyase（PGL） inPichia pastoris. According to the codon bias of P. pastoris，the gene of the stabilized mutant（K314M） of Bacillus sp. WSHB04-02 PGL is optimized and cloned into the EcoR I-Not I sites of pPIC9K-PGL，yielding the expression plasmid pPIC9K-PGL. pPIC9K-PGL is transformed into the P. pastoris GS115（his-），yielding the strain GS115/PGL 14#. GS115/PGL 14# produced 301.32 U/mL of extracellular PGL which is 25.1% higher than that of the strain with native PGL gene. On this basis，two molecular chaperones the protein folding factors endoplasmic reticulum oxidoreduction 1（ERO1） and ubiquitin-conjugating enzyme（UBC1） are co-expressed with the optimized K314M gene individually and simultaneously. It is showed that co-expression of ERO1 and UBC1 at the same time（GS115/PGLl-ERO1-UBC1 2#） increased the extracellular PGL by 49.3%，which is up to 450.12 U/mL. By using an exponential fed-batch strategy in 3L-fermenter，GS115/PGLl-ERO1- UBC1 2# produces 1 362.31 U/mL after the 96 h induction by methanol. The results here may benefit the industrial production and application of the PGL.

查看全文 查看/发表评论 下载PDF阅读器