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ACS Synth Biol. 2016 Feb 19;5(2):172-86. doi: 10.1021/acssynbio.5b00199. Epub 2015 Dec 11.

A Toolbox of Diverse Promoters Related to Methanol Utilization: Functionally Verified Parts for Heterologous Pathway Expression in Pichia pastoris.

Author information

1
Institute of Molecular Biotechnology, NAWI Graz, Graz University of Technology , Petersgasse 14, Graz 8010, Austria.
2
Austrian Centre of Industrial Biotechnology (ACIB GmbH) , Petersgasse 14, Graz 8010, Austria.
3
Omics Center Graz , Stiftingtalstrasse 24, 8036 Graz, Austria.

Abstract

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.

KEYWORDS:

Pichia pastoris; heterologous pathway expression; promoters; toolbox of modular parts; transcriptional fine-tuning; transcriptional terminators

PMID:
26592304
DOI:
10.1021/acssynbio.5b00199
[Indexed for MEDLINE]
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