A Toolbox of Diverse Promoters Related to Methanol Utilization: Functionally Verified Parts for Heterologous Pathway Expression in Pichia pastoris
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- 作者：Thomas Vogl ; Lukas Sturmberger ; Thomas Kickenweiz ; Richard Wasmayer ; Christian Schmid ; Anna-Maria Hatzl ; Michaela A. Gerstmann ; Julia Pitzer ; Marlies Wagner ; Gerhard G. Thallinger ; Martina Geier ; Anton Glieder
- 刊名：ACS Synthetic Biology
- 出版年：2016
- 出版时间：February 19, 2016
- 年：2016
- 卷：5
- 期：2
- 页码：172-186
- 全文大小：896K
- ISSN：2161-5063
文摘
The heterologous expression of biosynthetic pathways for pharmaceutical or fine chemical production requires suitable expression hosts and vectors. In eukaryotes, the pathway flux is typically balanced by stoichiometric fine-tuning of reaction steps by varying the transcript levels of the genes involved. Regulated (inducible) promoters are desirable to allow a separation of pathway expression from cell growth. Ideally, the promoter sequences used should not be identical to avoid loss by recombination. The methylotrophic yeast Pichia pastoris is a commonly used protein production host, and single genes have been expressed at high levels using the methanol-inducible, strong, and tightly regulated promoter of the alcohol oxidase 1 gene (PAOX1). Here, we have studied the regulation of the P. pastoris methanol utilization (MUT) pathway to identify a useful set of promoters that (i) allow high coexpression and (ii) differ in DNA sequence to increase genetic stability. We noticed a pronounced involvement of the pentose phosphate pathway (PPP) and genes involved in the defense of reactive oxygen species (ROS), providing strong promoters that, in part, even outperform PAOX1 and offer novel regulatory profiles. We have applied these tightly regulated promoters together with novel terminators as useful tools for the expression of a heterologous biosynthetic pathway. With the synthetic biology toolbox presented here, P. pastoris is now equipped with one of the largest sets of strong and co-regulated promoters of any microbe, moving it from a protein production host to a general industrial biotechnology host.