Enhancement of the Gene Targeting Efficiency of Non-Conventional Yeasts by Increasing Genetic Redundancy

Abstract

In contrast to model yeasts, gene targeting efficiencies of non-conventional yeasts are usually low, which greatly limits the research and applications of these organisms. In this study, we aimed to enhance the gene targeting efficiency of non-conventional yeasts by improving the fitness of mutant strains, particularly by increasing the genetic redundancy of host cells. To demonstrate this process, OCH1 gene deletion in Pichia pastoris was performed. Extra copies of the OCH1 gene on a helper plasmid were provided for the P. pastoris GS115 strain before the native OCH1 gene in the genomic DNA was knocked out. The redundancy in OCH1 gene significantly eliminated the growth defects of the och1 mutant and increased the deletion efficiency of the OCH1 gene by two orders of magnitude with the same length of homologous flanks. The same strategy was used to delete the KU70 and SGS1 genes. The targeting efficiencies of KU70 and SGS1 were increased by 1- and 23-fold, respectively. Therefore, this study provided an efficient strategy for the deletion of “stubborn” genes in non-conventional yeasts. This study further showed that cellular fitness is potentially an important factor that can limit the efficiency of gene targeting.

Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing

“…1). The change in colony morphology indicated growth deficiencies, similar to the ones reported for OCH1 knock-out strains in P. pastoris and other non-conventional yeast203940. OCH1 codes for a mannosyltransferase and its loss leads to a lower cell wall integrity, negatively affecting growth and temperature sensitivity as well as an increased flocculation13.…”

“…1000 bp), a high variance in targeting efficiency is observed, indicating the prevalence of the non-homologous end joining (NHEJ) pathway in P. pastoris 181920. As a result, a high clonal variability is found after transformation, necessitating a time and labour-intensive screening process for the clone with the desired characteristics2122.…”

“…Many other yeasts, filamentous fungi and higher eukaryotes that are used in biotechnological applications display similar or more pronounced clonal variabilities due to a predominant NHEJ pathway252627, while in the model yeast Saccharomyces cerevisiae homologous recombination is dominant over the NHEJ pathway28. Different techniques to reduce the clonal variability in P. pastoris have been proposed14182029. However, disadvantages like high complexity, lower strain fitness or not yet fully realized methods for donor cassette integration have kept these techniques from replacing the established ones for the efficient construction of producer strains.…”

Effect of gene copy number and chaperone coexpression on recombinant hydrophobin HFBI biosurfactant production in Pichia pastoris

“…Specifically, in linearizing the 1‐copy vector in the AOX1 promoter, genomic integration by homologous recombination is targeted to the intrinsic AOX1 promoter in the yeast genome. In contrast, the inability to linearize the 2‐ or 3‐copy vectors within the AOX1 promoter means recombination could occur with higher likelihood at an off target locus by non‐homologous end joining (Chen et al, ; Näätsaari et al, ; Nett, Hodel, Rausch, & Wildt, ), potentially affecting the production of recombinant HFBI caused by differential integration.…”

Pichia pastoris protease‐deficient and auxotrophic strains generated by a novel, user‐friendly vector toolbox for gene deletion

“…Nonconventional yeasts such as P. pastoris often display a high ratio of NHEJ‐to‐HR activity, which is the main reason why targeted gene insertions are difficult to achieve (Näätsaari et al, ; Schwarzhans et al, ; Tsakraklides, Brevnova, Stephanopoulos, & Shaw, ). Several strategies were shown to improve HR activity in P. pastoris such as hydroxyurea‐mediated cell cycle arrest (Tsakraklides et al, ), increasing the genetic redundancy of host cells by providing extra copies of the gene to be deleted on a helper plasmid (Chen et al, ) or deletion of KU70 , a key player in NHEJ improved targeting (Näätsaari et al, ). Deletion of KU70 was also shown to drastically improved gene modifications using CRISPR/Cas9 tools (Weninger et al, ).…”