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Different construction strategies affected on the physiology of Pichia pastoris strains highly expressed lipase by transcriptional analysis of key genes.
Bioengineered ( IF 4.9 ) Pub Date : 2019-05-13 , DOI: 10.1080/21655979.2019.1614422
Jinjin Huang 1, 2 , Qing Wang 2, 3 , Wei Bu 1 , Lingxiao Chen 1 , Zhen Yang 2 , Weifa Zheng 1 , Ying Li 2 , Jilun Li 2
Affiliation  

We demonstrated previously that expression of Rhizomucor miehei lipase (RML) in Pichia pastoris could be significantly increased by addition of gene propeptide, optimized signal peptide codons and manipulation of gene dosage. In this study, effects of various strategies on the protein synthesis and secretion pathways were analyzed. Using nine strains previously constructed, we evaluated cell culture properties, enzymatic activities, and analyzed transcriptional levels of nine genes involved in protein synthesis and secretion pathways by qPCR. We observed that (i) Addition of propeptide decreased lipase folding stress by down-regulated four UPR-related genes. (ii) Signal peptide codons optimization had no effect on host with no change in the nine detected genes. (iii) Folding stress and limited transport capacity produced when rml gene dosage exceed 2. Different limiting factors on lipase expression in strains with different construction strategies were identified. This study provides a theoretical basis for further improving RML by transforming host.



中文翻译:

通过关键基因的转录分析,不同的构建策略对巴斯德毕赤酵母菌株的生理影响很大。

我们先前证明了毕赤酵母中的根瘤菌脂肪酶(RML)的表达可以通过添加基因前肽,优化的信号肽密码子和操纵基因剂量来显着增加。在这项研究中,分析了各种策略对蛋白质合成和分泌途径的影响。使用先前构建的九个菌株,我们评估了细胞培养特性,酶活性,并通过qPCR分析了参与蛋白质合成和分泌途径的九个基因的转录水平。我们观察到(i)前肽的添加通过下调四个UPR相关基因来降低脂肪酶折叠应力。(ii)信号肽密码子优化对宿主没有影响,检测到的9个基因没有变化。(iii)rml产生的折叠应力和有限的运输能力基因剂量超过2。鉴定了具有不同构建策略的菌株中限制脂肪酶表达的不同因素。该研究为通过转化宿主进一步提高RML提供了理论依据。

更新日期:2019-05-13
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