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

Nucleic Acids Research, gkab 535, 01 July 2021

 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 2: Enhancing homologous recombination (HR) for precise genome editing. (A) Screening HR related genes for improving seamless deletion. The heterologous HR related genes from S. cerevisiae or endogenous P. pastoris genes were overexpressed and be followed by deleting GUT1 in P. pastoris. The strains carried the plasmid expressing Cas9 and gRNA for targeting GUT1. The deletion cassette was constructed by fusing the upstream and downstream homologous arms of 1 kb. Wild-type strain GS115 without overexpressing RAD genes was used as a control. Eight colonies were picked for colony PCR analysis with primer pair Check-GUT1-F/Check-GUT1-R. (B) PpRAD52 overexpression improved the seamless deletion of FAA1 compared with KU70 deletion. (C) Relative expression level of KU70 and RAD52 in S. cerevisiae or P. pastoris. The housekeeping actin gene was chosen as baseline for quantification of expression level. Relative expression level of these genes was calculated by normalized the expression level of RAD52 in the respective strains. (D) PpRAD52 overexpression improved the seamless deletion of GUT1 in P. pastoris. The P_GAP and P_ADH2 were used to overexpress RAD52 and the relative expression levels of RAD52 were analyzed by qPCR. (E) PpRAD52 overexpression improved the seamless deletion of GUT1, FAA1, and FAA2. (F) HR rates for FAA1 deletion with various HA lengths in strains of PpRAD52 overexpression. (G) HR rates for eGFP overexpression cassette integration into PNSI-1 site with various HA lengths in strains of PpRAD52 overexpression. (H) The positive rates of integration of three genes at three genome loci. Twenty colonies were picked for colony PCR analysis. 1 kb homologous arms was used in both integration and knockout experiments unless otherwise noted. Mean values and standard deviations of biological triplicates were shown in 2B to 2H.