Isolation and evaluation of strong endogenous promoters for the heterologous expression of proteins in Pichia pastoris

发起人 异源的 毕赤酵母 报告基因 基因 基因表达 生物 抄写(语言学) 异源表达 分子生物学 遗传学 重组DNA 语言学 哲学
作者
Yafei Zhang,Shengyan Wang,Lingfang Lu,Chenshan Zhang,Fan Cai,Yao Lin,Yide Huang
出处
期刊:World Journal of Microbiology & Biotechnology [Springer Nature]
卷期号:38 (12) 被引量:5
标识
DOI:10.1007/s11274-022-03412-3
摘要

BackgroundThe heterologous expression of biosynthetic pathway genes for pharmaceutical or fine chemical production usually requires to express more than one gene in the host cells. In eukaryotes, the pathway flux is typically balanced by controlling the transcript levels of the genes involved. It is difficult to balance the stoichiometric fine-tuning of the reaction steps of the pathway by acting on one or two promoters. Furthermore, the promoter used should not be identical to avoid loss of inserted genes by recombination or dilute its transcription factors.ResultsBased on RNA-seq data, 18 candidate genes with the highest transcription levels at three carbon sources (glucose, glycerol and methanol) were selected and their promoter regions were isolated from GS115 genome. The performance of these promoters on the level of protein production was evaluated using LacZ and EGFP genes as the reporters, respectively. These isolated promoters all exhibited activity to express LacZ gene. Using LacZ as a reporter, of the 18 promoter candidates, 9 promoters showed higher expression levels for the reporter compare to pGAP, a strong promoter widely used for constitutive expression of heterologous proteins in Pichia pastoris. These promoters with high expression levels were further employed to evaluate secreted expression using EGFP as a reporter. 6 promoters exhibited stronger protein expression compare to pGAP. Interestingly, the protein expression driven by pFDH1 was slightly higher than that of commonly used pAOX1 at methanol, and methanol-induced expression of pFDH1 was not repressed by glycerol.ConclusionThe various promoters identified in this study could be used for heterologous expression of biosynthetic pathway genes for pharmaceutical or fine chemical production. the methanol-induced pFDH1 that is not repressed by glycerol is an attractive alternative to pAOX1 and may provide a novel way to produce heterologous proteins in Pichia pastoris.
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