posted on 2024-02-21, 17:44authored byLouise
La Barbera Kastberg, Mykhaylo S. Petrov, Tomas Strucko, Michael K. Jensen, Christopher T. Workman
Promoters are crucial elements for engineering microbial
production
strains used in bioprocesses. For the increasingly popular chassis Komagataella phaffii (formerly Pichia pastoris), a limited number of well-characterized promoters constrain the
data-driven engineering of production strains. Here, we present an in silico approach for condition-independent de
novo identification of strong native promoters. The method
relies on tRNA-codon coadaptation of coding sequences in the K. phaffii genome and is based on two complementary scores:
the number of effective codons and the tRNA adaptation index. Genes
with high codon bias are expected to be translated efficiently and,
thus, also be under control of strong promoters. Using this approach,
we identified promising strong promoter candidates and experimentally
assessed their activity using fluorescent reporter assays characterizing
50 promoters spanning a 76-fold difference in expression levels in
a glucose medium. Overall, we report several promoters that should
be added to the molecular toolbox for engineering of K. phaffii and present an approach for identifying promoters in microbial genomes.