PMID- 31148217
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20230201
IS  - 1097-0061 (Electronic)
IS  - 0749-503X (Print)
IS  - 0749-503X (Linking)
VI  - 36
IP  - 9
DP  - 2019 Sep
TI  - Pichia pastoris protease-deficient and auxotrophic strains generated by a novel, 
      user-friendly vector toolbox for gene deletion.
PG  - 557-570
LID - 10.1002/yea.3426 [doi]
AB  - Targeted gene knockouts play an important role in the study of gene function. For 
      the generation of knockouts in the industrially important yeast Pichia pastoris, 
      several protocols have been published to date. Nevertheless, creating a targeted 
      knockout in P. pastoris still is a time-consuming process, as the existing 
      protocols are labour intensive and/or prone to accumulate nucleotide mutations. 
      In this study, we introduce a novel, user-friendly vector-based system for the 
      generation of targeted knockouts in P. pastoris. Upon confirming the successful 
      knockout, respective selection markers can easily be recycled. Excision of the 
      marker is mediated by Flippase (Flp) recombinase and occurs at high frequency 
      (>/=95%). We validated our knockout system by deleting 20 (confirmed and putative) 
      protease genes and five genes involved in biosynthetic pathways. For the first 
      time, we describe gene deletions of PRO3 and PHA2 in P. pastoris, genes involved 
      in proline, and phenylalanine biosynthesis, respectively. Unexpectedly, knockout 
      strains of PHA2 did not display the anticipated auxotrophy for phenylalanine but 
      rather showed a bradytroph phenotype on minimal medium hinting at an alternative 
      but less efficient pathway for production of phenylalanine exists in P. pastoris. 
      Overall, all knockout vectors can easily be adapted to the gene of interest and 
      strain background by efficient exchange of target homology regions and selection 
      markers in single cloning steps. Average knockout efficiencies for all 25 genes 
      were shown to be 40%, which is comparably high.
CI  - (c) 2019 The Authors Yeast Published by John Wiley & Sons Ltd.
FAU - Ahmad, Mudassar
AU  - Ahmad M
AD  - Institute of Molecular Biotechnology, Graz University of Technology, Graz, 
      Austria.
FAU - Winkler, Christine M
AU  - Winkler CM
AD  - Institute of Molecular Biotechnology, Graz University of Technology, Graz, 
      Austria.
FAU - Kolmbauer, Markus
AU  - Kolmbauer M
AD  - Institute of Molecular Biotechnology, Graz University of Technology, Graz, 
      Austria.
FAU - Pichler, Harald
AU  - Pichler H
AD  - Institute of Molecular Biotechnology, Graz University of Technology, Graz, 
      Austria.
AD  - Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria.
FAU - Schwab, Helmut
AU  - Schwab H
AD  - Institute of Molecular Biotechnology, Graz University of Technology, Graz, 
      Austria.
AD  - Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria.
FAU - Emmerstorfer-Augustin, Anita
AU  - Emmerstorfer-Augustin A
AUID- ORCID: 0000-0002-3392-8839
AD  - Institute of Molecular Biotechnology, Graz University of Technology, Graz, 
      Austria.
LA  - eng
GR  - Pakistan Scholarship Programme HRDI-UESTP-Austria/
GR  - J3787 & W901/Austrian Science Fund/
PT  - Journal Article
DEP - 20190730
PL  - England
TA  - Yeast
JT  - Yeast (Chichester, England)
JID - 8607637
SB  - IM
PMC - PMC6771850
OTO - NOTNLM
OT  - P. pastoris
OT  - auxotrophic strains
OT  - gene disruption
OT  - knockout plasmids
OT  - proteases-deficient strains
COIS- The authors declare no conflict of interest.
EDAT- 2019/05/31 06:00
MHDA- 2019/05/31 06:01
PMCR- 2019/10/01
CRDT- 2019/06/01 06:00
PHST- 2019/03/28 00:00 [revised]
PHST- 2019/02/01 00:00 [received]
PHST- 2019/05/26 00:00 [accepted]
PHST- 2019/05/31 06:00 [pubmed]
PHST- 2019/05/31 06:01 [medline]
PHST- 2019/06/01 06:00 [entrez]
PHST- 2019/10/01 00:00 [pmc-release]
AID - YEA3426 [pii]
AID - 10.1002/yea.3426 [doi]
PST - ppublish
SO  - Yeast. 2019 Sep;36(9):557-570. doi: 10.1002/yea.3426. Epub 2019 Jul 30.