Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichia pastoris

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.

The Bibliometric Landscape of Gene Editing Innovation and Regulation in the Worldwide

“…Tong, Y. et al developed a CRISPR system for actinomycete genomes that can efficiently and reversibly control target genes actIORF1 and actVB [ 80 ]. Cai, P. et al, edited Pichia pastoris genome and found that fatty alcohol production can be increased to 380 mg/L [ 81 ].…”

Advances and Opportunities of CRISPR/Cas Technology in Bioengineering Non-conventional Yeasts

“…The combined expression of ScRad51 , ScRad52 , and ScSae2 significantly improved the HR rate of O. polymorpha ( Gao et al, 2021 ). On the contrary, ScRad51/Rad52 expressed in P. pastoris ( Cai et al, 2021 ) and S. stipitis ( Cao et al, 2018 ) had no distinct improvement in HR efficiency, but overexpression of endogenous Rad51 and Rad52 resulted in higher HR activity of P. pastoris ( Cai et al, 2021 ). Presumably, the mechanism of DSB repair is mysterious and complex, the choice of proper genes and the expression strength of these genes are important to increase HR efficiency, which is essential for efficient operation of the multiple genes simultaneously in non-conventional yeasts.…”

Impact of overexpressing NADH kinase on glucoamylase production in Aspergillus niger

“…A combination of NADH kinase and aspartate aminotransferase may be more promising. Of course, the overexpression of these functional genes needs to find a suitable site, that is, an integration site that has little effect on the metabolism of A. niger after the site is knocked out, such as neutral sites (Cai et al, 2021 ). To quickly realize the multiple genetic manipulations of related genes in aconidial A. niger , a method is also mentioned in this paper, using AfpyrG-tagged cas9 plasmid for homologous recombination (Fig.…”