Abstract
Background
The 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.
Results
Based 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.
Conclusion
The 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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Data Availability
The data supporting the conclusions of this article are included with the article and its additional file.
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This project is financed by the Natural Science Foundation of Fujian Province (2022J01132183) and Fujian Normal University, Fujian Provincial Science Fund for Distinguished Young Scholar (2020J01311402).
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YH conceived and designed the experiments. YZ and SW performed the majority of the laboratory work. LL and CZ completed the cloning of promoters. FC constructed plasmids. SW and YH wrote the manuscript. YL revised the manuscript. All authors have read and approved the final manuscript.
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Zhang, Y., Wang, S., Lu, L. et al. Isolation and evaluation of strong endogenous promoters for the heterologous expression of proteins in Pichia pastoris. World J Microbiol Biotechnol 38, 226 (2022). https://doi.org/10.1007/s11274-022-03412-3
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DOI: https://doi.org/10.1007/s11274-022-03412-3