Pichia pastoris mutants as host strains for efficient secretion of recombinant branched chain aminotransferase (BCAT).
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.
Austrian Center of Industrial Biotechnology (ACIB), Petersgasse 14, 8010 Graz, Austria.
Katrin
K
Weinhandl
Austrian Center of Industrial Biotechnology (ACIB), Petersgasse 14, 8010 Graz, Austria.
Melanie
M
Ballach
Austrian Center of Industrial Biotechnology (ACIB), Petersgasse 14, 8010 Graz, Austria.
Margit
M
Winkler
TU Graz, Institute of Molecular Biotechnology, Petersgasse 14, 8010 Graz, Austria.
Mudassar
M
Ahmad
TU Graz, Institute of Molecular Biotechnology, Petersgasse 14, 8010 Graz, Austria.
Anton
A
Glieder
Medical University, Institute of Pathology, Research Unit Functional Proteomics and Metabolic Pathways, and BioTechMed-Graz, Omics Center Graz, Stiftingtalstrasse 24, 8010 Graz, Austria.
Ruth
R
Birner-Gruenberger
Ingenza Ltd., Roslin Biocentre, Midlothian, UK.
Ian
I
Fotheringham
Ingenza Ltd., Roslin Biocentre, Midlothian, UK.
Franck
F
Escalettes
TU Graz, Institute of Molecular Biotechnology, Petersgasse 14, 8010 Graz, Austria. Electronic address: andrea_camattari@bti.a-star.edu.sg.
Andrea
A
Camattari
D055786:Gene Knockout Techniques; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*; D013237:Stereoisomerism; D000637:Transaminases / Q000235:genetics* / Q000378:metabolism*
D011994:Recombinant Proteins; D000637:Transaminases
Aminotransferases; Chiral amino acid; Expression; Pichia pastoris; Protein secretion