CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris

Abstract

Background Pichia pastoris (syn. Komagataella phaffii ) is a widely used generally recognized as safe host for heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value pharmaceuticals and chemicals. Nevertheless, limited availability of selective markers and low efficiency of homologous recombination make this process difficult and time-consuming, particularly in the case of multistep biosynthetic pathways. Therefore, it is crucial to develop an efficient and marker-free multiloci gene knock-in method in P. pastoris . Results A non-homologous-end-joining defective strain (Δ ku70 ) was first constructed using the CRISPR–Cas9 based gene deficiency approach. It was then used as a parent strain for multiloci gene integration. Ten guide RNA (gRNA) targets were designed within 100 bp upstream of the promoters or downstream of terminator, and then tested using an eGFP reporter and confirmed as suitable single-locus integration sites. Three high-efficiency gRNA targets (P AOX1 UP-g2, P TEF1 UP-g1, and P FLD1 UP-g1) were selected for double- and triple-locus co-integration. The integration efficiency ranged from 57.7 to 70% and 12.5 to 32.1% for double-locus and triple-locus integration, respectively. In addition, biosynthetic pathways of 6-methylsalicylic acid and 3-methylcatechol were successfully assembled using the developed method by one-step integration of functional genes. The desired products were obtained, which further established the effectiveness and applicability of the developed CRISPR–Cas9-mediated gene co-integration method in P. pastoris . Conclusions A CRISPR–Cas9-mediated multiloci gene integration method was developed with efficient gRNA targets in P. pastoris . Using this method, multiple gene cassettes can be simultaneously integrated into the genome without employing selective markers. The multiloci integration strategy is beneficial for pathway assembly of complicated pharmaceuticals and chemicals expressed in P. pastoris.

Modular engineering for microbial production of carotenoids

“…Only a few bioprocesses focus on chromosomal genes and most still use plasmids for gene regulation, which are less stable, requires antibiotics and bear a higher metabolic burden (Furubayashi et al., 2015). Several strategies have been employed to solve these problems, such as novel marker-free genetic integration tools based on the CRISPR-Cas system (Klompe et al., 2019; Liu et al., 2019), stable gene expression systems based on an incoherent feedforward loop (iFFL) (Segall-Shapiro et al., 2018) and antibiotic-free plasmids based on Stable and TunAble PLasmid (STAPL) system that encode for translation initiation factors required by a corresponding auxotroph (Kang et al., 2018). Second, most enzymes of carotenoid biosynthesis are from plants and have a low catalytic efficiency in microorganisms.…”

Efficient expression of multiple guide RNAs for CRISPR/Cas genome editing

“…This BiP-driven multiplexed CRISPR/Cas9 system demonstrated successful multiloci integration where multiple gene cassettes simultaneously integrated into Pichia pastoris genome (Fig. 1C) (Liu et al 2019). Use of the constitutive rice BiP1 (OsBiP1) promoter was shown to achieve 86-93% editing efficiencies with tRNA or Csy4 gRNA processing system in rice (Fig.…”

“…In addition to TCTU and STU CRISPR/Cas expression systems, bidirectional promoters (BiPs) have been recently developed to drive Cas9 and gRNA expression in opposite direction in methylotrophic yeast and rice (Liu et al 2019;Ren et al 2019). P HTX1 , a bidirectional Pol II promoter, was designed to co-regulate the expression of Cas9 and gRNAs flanked by HH and HDV ribozymes with different terminators (Liu et al 2019). This BiP-driven multiplexed CRISPR/Cas9 system demonstrated successful multiloci integration where multiple gene cassettes simultaneously integrated into Pichia pastoris genome (Fig.…”

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

“…CRISPR-Cas9 was also used to develop a one-step multiloci gene integration method without the requirement of selective markers. This method can be used for pathway assembly of complicated pharmaceuticals expressed in P. pastoris (Liu Q. et al, 2019). The key factors that can enhance recombinant protein production in P. pastoris were well-described recently, and it was reported that up to 120 g DCW per liter of culture can be achieved using a chemically defined medium (García-Ortega et al, 2019).…”