Pichia pastoris mutants as host strains for efficient secretion of recombinant Branched Chain Aminotransferase (BCAT)

Katrin Weinhandl, Melanie Ballach, Margit Winkler, Ahmad Mudassar, Anton Glieder, Ruth Birner-Gruenberger, Ian Fotheringham, Franck Escalettes

Research output: Contribution to journalArticle

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.

LanguageEnglish
Pages84-91
Number of pages8
JournalJournal of biotechnology
Volume235
DOIs
StatusPublished - 2016

Fingerprint

Pichia
Glycosylation
Amino Acids
Drug Industry
Recombinant Proteins
Cell Wall
Amines
Cluster Analysis
Permeability
Proteins
branched-chain-amino-acid transaminase

Cite this

Pichia pastoris mutants as host strains for efficient secretion of recombinant Branched Chain Aminotransferase (BCAT). / Weinhandl, Katrin; Ballach, Melanie; Winkler, Margit; Mudassar, Ahmad; Glieder, Anton; Birner-Gruenberger, Ruth; Fotheringham, Ian; Escalettes, Franck.

In: Journal of biotechnology, Vol. 235, 2016, p. 84-91.

Research output: Contribution to journalArticle

Weinhandl, Katrin ; Ballach, Melanie ; Winkler, Margit ; Mudassar, Ahmad ; Glieder, Anton ; Birner-Gruenberger, Ruth ; Fotheringham, Ian ; Escalettes, Franck. / Pichia pastoris mutants as host strains for efficient secretion of recombinant Branched Chain Aminotransferase (BCAT). In: Journal of biotechnology. 2016 ; Vol. 235. pp. 84-91
@article{0c74e1b800ee47d8893e875b2f00d609,
title = "Pichia pastoris mutants as host strains for efficient secretion of recombinant Branched Chain Aminotransferase (BCAT)",
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.",
author = "Katrin Weinhandl and Melanie Ballach and Margit Winkler and Ahmad Mudassar and Anton Glieder and Ruth Birner-Gruenberger and Ian Fotheringham and Franck Escalettes",
note = "Copyright {\circledC} 2016 Elsevier B.V. All rights reserved.",
year = "2016",
doi = "10.1016/j.jbiotec.2016.06.004",
language = "English",
volume = "235",
pages = "84--91",
journal = "Journal of biotechnology",
issn = "0168-1656",
publisher = "Elsevier B.V.",

}

TY - JOUR

T1 - Pichia pastoris mutants as host strains for efficient secretion of recombinant Branched Chain Aminotransferase (BCAT)

AU - Weinhandl,Katrin

AU - Ballach,Melanie

AU - Winkler,Margit

AU - Mudassar,Ahmad

AU - Glieder,Anton

AU - Birner-Gruenberger,Ruth

AU - Fotheringham,Ian

AU - Escalettes,Franck

N1 - Copyright © 2016 Elsevier B.V. All rights reserved.

PY - 2016

Y1 - 2016

N2 - 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.

AB - 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.

U2 - 10.1016/j.jbiotec.2016.06.004

DO - 10.1016/j.jbiotec.2016.06.004

M3 - Article

VL - 235

SP - 84

EP - 91

JO - Journal of biotechnology

T2 - Journal of biotechnology

JF - Journal of biotechnology

SN - 0168-1656

ER -