PMID- 29125227
OWN - NLM
STAT- MEDLINE
DCOM- 20180822
LR  - 20180822
IS  - 1860-7314 (Electronic)
IS  - 1860-6768 (Linking)
VI  - 13
IP  - 3
DP  - 2018 Mar
TI  - Pichia pastoris Alcohol Oxidase 1 (AOX1) Core Promoter Engineering by High 
      Resolution Systematic Mutagenesis.
PG  - e1700340
LID - 10.1002/biot.201700340 [doi]
AB  - Unravelling the core promoter sequence-function relationship is fundamental for 
      engineering transcription initiation and thereby a feasible "tuning knob" for 
      fine-tuning expression in synthetic biology and metabolic engineering 
      applications. Here a systematic replacement studies of the core promoter and 5' 
      untranslated region (5'UTR) of the exceptionally strong and tightly methanol 
      regulated Komagataella phaffii (syn. Pichia pastoris) alcohol oxidase 1 (AOX1) 
      promoter at unprecedented resolution is performed. Adjacent triplets of the 
      200 bp long core promoter are mutated at a time by changing the wild-type 
      sequence into cytosine or adenine triplets, resulting in 130 variants that are 
      cloned upstream of an eGFP reporter gene providing a library for expression 
      fine-tuning. Mutations in the TATA box motif, regions downstream of the 
      transcription start site or next to the start codon in the 5'UTR had a 
      significant effect on the eGFP fluorescence. Surprisingly, mutations in most 
      other regions are tolerated, indicating that yeast core promoters can show a high 
      tolerance toward small mutations, supporting regulatory models of degenerate 
      motifs, or redundant design. The authors exploited these neutral core promoter 
      positions, not affecting expression, to introduce extrinsic sequence elements 
      such as cloning sites (allowing targeted core promoter/5'UTR modifications) and 
      bacterial promoters (applicable in multi host vectors).
CI  - (c) 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
FAU - Portela, Rui M C
AU  - Portela RMC
AD  - REQUIMTE/LAQV, Departamento de Quimica, Faculdade de Ciencias e Tecnologia, 
      Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
FAU - Vogl, Thomas
AU  - Vogl T
AUID- ORCID: 0000-0002-3892-1740
AD  - Institute for Molecular Biotechnology, NAWI Graz University of Technology, 
      Petersgasse 14/1, 8010 Graz, Austria.
FAU - Ebner, Katharina
AU  - Ebner K
AD  - Institute for Molecular Biotechnology, NAWI Graz University of Technology, 
      Petersgasse 14/1, 8010 Graz, Austria.
FAU - Oliveira, Rui
AU  - Oliveira R
AD  - REQUIMTE/LAQV, Departamento de Quimica, Faculdade de Ciencias e Tecnologia, 
      Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
FAU - Glieder, Anton
AU  - Glieder A
AD  - Institute for Molecular Biotechnology, NAWI Graz University of Technology, 
      Petersgasse 14/1, 8010 Graz, Austria.
LA  - eng
PT  - Journal Article
DEP - 20171122
PL  - Germany
TA  - Biotechnol J
JT  - Biotechnology journal
JID - 101265833
RN  - EC 1.1.- (Alcohol Oxidoreductases)
RN  - EC 1.1.3.13 (alcohol oxidase)
SB  - IM
MH  - Alcohol Oxidoreductases/biosynthesis/*genetics
MH  - Gene Expression Regulation, Fungal
MH  - Genes, Reporter
MH  - *Metabolic Engineering
MH  - Mutagenesis, Site-Directed
MH  - Pichia/enzymology/genetics
MH  - Promoter Regions, Genetic
MH  - Protein Engineering/*methods
MH  - Saccharomyces cerevisiae/genetics
MH  - *Synthetic Biology
OTO - NOTNLM
OT  - AOX1
OT  - Pichia pastoris
OT  - core promoter library
OT  - gene expression
OT  - transcriptional fine-tuning
EDAT- 2017/11/11 06:00
MHDA- 2018/08/23 06:00
CRDT- 2017/11/11 06:00
PHST- 2017/05/08 00:00 [received]
PHST- 2017/10/09 00:00 [revised]
PHST- 2017/11/11 06:00 [pubmed]
PHST- 2018/08/23 06:00 [medline]
PHST- 2017/11/11 06:00 [entrez]
AID - 10.1002/biot.201700340 [doi]
PST - ppublish
SO  - Biotechnol J. 2018 Mar;13(3):e1700340. doi: 10.1002/biot.201700340. Epub 2017 Nov 
      22.