Peng Cai, Xingpeng Duan, Xiaoyan Wu, Linhui Gao, Min Ye, Yongjin J Zhou

Nucleic Acids Research, Published 01 July 2021; gkab535, https://doi.org/10.1093/nar/gkab535

 Abstract

The industrial yeast Pichia pastoris has been harnessed extensively for production of proteins, and it is attracting attention as a chassis cell factory for production of chemicals. However, the lack of synthetic biology tools makes it challenging in rewiring P. pastoris metabolism. We here extensively engineered the recombination machinery by establishing a CRISPR-Cas9 based genome editing platform, which improved the homologous recombination (HR) efficiency by more than 54 times, in particular, enhanced the simultaneously assembly of multiple fragments by 13.5 times. We also found that the key HR-relating gene RAD52 of P. pastoris was largely repressed in compared to that of Saccharomyces cerevisiae. This gene editing system enabled efficient seamless gene disruption, genome integration and multiple gene assembly with positive rates of 68–90%. With this efficient genome editing platform, we characterized 46 potential genome integration sites and 18 promoters at different growth conditions. This library of neutral sites and promoters enabled two-factorial regulation of gene expression and metabolic pathways and resulted in a 30-fold range of fatty alcohol production (12.6–380 mg/l). The expanding genetic toolbox will facilitate extensive rewiring of P. pastoris for chemical production, and also shed light on engineering of other non-conventional yeasts.

See: https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkab535/6312753

Figure 1: Engineering recombination machinery and expanding genetic tool box for metabolic engineering of P. pastoris. (A) Enhancing recombination for precise genetic engineering by manipulation of the homologous recombination (HR) process and repressing the nonhomologous end joining (NHEJ). The genes marked as blue were deleted or overexpressed for enhancing HR efficiencies. (B) The neutral sites and promoters were profiled by using eGFP expression cassettes with the established genetic engineering platform. The red bar represents the location of neutral sites in the genome. (C) The variable transcription of promoters and neutral sites enables two-factorial regulation of gene expression and biosynthetic pathways.