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J Biotechnol. 2016 Oct 10;235:84-91. doi: 10.1016/j.jbiotec.2016.06.004. Epub 2016 Jun 7.

Pichia pastoris mutants as host strains for efficient secretion of recombinant branched chain aminotransferase (BCAT).

Author information

1
Austrian Center of Industrial Biotechnology (ACIB), Petersgasse 14, 8010 Graz, Austria.
2
TU Graz, Institute of Molecular Biotechnology, Petersgasse 14, 8010 Graz, Austria.
3
Medical University, Institute of Pathology, Research Unit Functional Proteomics and Metabolic Pathways, and BioTechMed-Graz, Omics Center Graz, Stiftingtalstrasse 24, 8010 Graz, Austria.
4
Ingenza Ltd., Roslin Biocentre, Midlothian, UK.
5
TU Graz, Institute of Molecular Biotechnology, Petersgasse 14, 8010 Graz, Austria. Electronic address: andrea_camattari@bti.a-star.edu.sg.

Abstract

Branched chain aminotransferase (BCAT) is one of the enzymatic tools of choice for the production of chiral amines or amino acids; especially, non-natural amino acids are of interest as building blocks for the pharmaceutical industry. The expression and subsequent secretion of BCAT counteracts limited cell permeability of target substrates and facilitates downstream processing. Since Pichia pastoris secretes a negligible amount of native proteins and was previously shown to efficiently secrete recombinant proteins, it was chosen as the expression host. We examined different promoters and glycosylation states and also engineered the host strain by disrupting genes encoding proteins related to cell wall assembly (Scw10, Cwp1) and glycosylation (Och1). Finally, we were able not only to increase the extracellular BCAT production, but also to achieve a more homogenous product in terms of glycosylation and identified a deletion strain, which counteracts typical cell clustering in the Δoch1 strain.

KEYWORDS:

Aminotransferases; Chiral amino acid; Expression; Pichia pastoris; Protein secretion

PMID:
27287536
DOI:
10.1016/j.jbiotec.2016.06.004
[Indexed for MEDLINE]

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