Pichia pastoris Alcohol Oxidase 1 (AOX1) Core Promoter
Engineering by High Resolution Systematic Mutagenesis
Rui M. C. Portela, Thomas Vogl,* Katharina Ebner, Rui Oliveira, and Anton Glieder
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
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
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 (appli-
cable in multi host vectors).
Eukaryotic promoters can generally be separated in two parts: an
upstream regulatory sequence (URS, also termed cis-regulatory
modules, CRMs, or enhancers
) and a
core promoter. CRMs contain transcription
factor binding sites (TFBSs), that are
bound by speciﬁc transcription factors
(TFs), conferring, for example, cell-cycle
or carbon source dependent regulation.
In contrast, the core promoter typically
controls transcription initiation, as RNA
polymerase II (RNAPII) and general TFs
bind to this region.
Gaining insights on
core promoter sequence-function relation-
ship is essential for understanding tran-
scription initiation and for generating core
promoters variants to increase or ﬁne-tune
expression for synthetic biology and meta-
bolic engineering applications
the design of general expression vectors.
In higher eukaryotes, synthetic core
promoters have been designed based on
commonly occurring motifs such as TATA
box, Inr (initator), DPE (downstream core
promoter element), and MTE (motif ten
In lower eukaryotes, namely
yeasts, the only clearly conserved motif in
core promoters appears to be the TATA
Several studies have demonstrated
the feasibility of synthetic core promoter
design in yeasts.
Different methodologies were applied
for promoter designs in Saccharomyces cerevisiae and Komaga-
taella phafﬁi(syn. Pichia pastoris), namely random mutagene-
indels (insertions and deletions) in the 5
and rationally designed short core pro-
or modiﬁcations based on nucleosome occupancy
Lubliner et al.
used a genome scale
bioinformatic analysis to examine sequence features that
correlate with core promoter strength in S. cerevisiae. These
data were also used by Portela et al. to generate synthetic core
promoters following a bottom up approach.
In another study
by Lubliner et al.,
the core promoter sequence was modiﬁed
to determine factors inﬂuencing promoter strength and Dvir
tested effects of the 5
UTR on expression. It was shown
that the sequence in the vicinity (10 bp upstream) of the start
codon is key to deﬁne the protein expression rate.
tested sequence, Dvir et al.
showed that the three nucleotides
upstream of the start codon, the mRNA secondary structure and
the presence of additional start codons were the key features
modulating protein levels. The authors also found a high
correlation between mRNA and protein levels (consistent with
previous results demonstrating that a decrease in translation
efﬁciency also has an impact on mRNA stability).
Dr. R. M. C. Portela
, Prof. R. Oliveira
REQUIMTE/LAQV, Departamento de Química,
Faculdade de Ci
encias e Tecnologia
Universidade Nova de Lisboa,
2829-516 Caparica, Portugal
Dr. T. Vogl
, K. Ebner, Prof. A. Glieder
Institute for Molecular Biotechnology,
NAWI Graz University of Technology,
Petersgasse 14/1, 8010 Graz, Austria
iBET, Instituto de Biologia Experimental e Tecnol
ogica, Apartado 12,
2780-901 Oeiras, Portugal.
Department of Computer Science and Applied Mathematics,
Weizmann Institute of Science, Rehovot, Israel.
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/biot.201700340.
Core Promoter Engineering www.biotechnology-journal.com
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