This work established a highly efficient CRISPR/Cas9 genome editing system in this diploid yeast, enabling marker‐free editing with efficiencies of up to 88% and routine generation of homozygous mutants and successfully expressed secretory mCherry and human IgG1 Fc fragment at different loci.
Abstract
Impact statement Nonconventional yeast Pichia kudriavzevii possesses attractive phenotypic traits for industrial biomanufacturing; however, it remains genetically intractable compared with established yeast chassis. Here, we present an integrated synthetic biology framework that renders P. kudriavzevii a programmable production host. We established a highly efficient CRISPR/Cas9 genome editing system in this diploid yeast, enabling marker‐free editing with efficiencies of up to 88% and routine generation of homozygous mutants. By integrating efficient marker‐free CRISPR/Cas9 genome editing and locus‐aware expression strategies, we successfully expressed secretory mCherry and human IgG1 Fc fragment at different loci. Our results demonstrate that P. kudriavzevii is a promising expression system for protein production.
Phtheirospermum japonicum
is a genetic model for parasitic Orobanchaceae, a family that includes agriculturally important weeds such as
Striga
,
Orobanche
, and
Phelipanche
. Functional genetic studies in these species are limited by the lack of stable transgenesis protocols, restricting heritable CRISPR/Cas...
H. Ruwe, Freya Inger Kleinelanghorst, Victoria Zimmer et al.· Plant, Cell and Environment· 0 citations
ABSTRACT Pichia kudriavzevii is gaining attention as a non‐conventional yeast with significant industrial potential, owing to its robust tolerance to low pH and diverse process‐related stresses. However, the lack of a reliable episomal expression system and the scarcity of well‐characterized promoters currently impede...
Fusarium venenatum is the most widely used filamentous fungus for biomass-based microbial protein production mainly used as meat analogs. However, insufficient iterative genome editing and lack of synthetic biology tools for fine-tuning gene expression limited engineering of F. venenatum for improved traits. Here, repl...
S. Gu, Na Wang, Ya-Dong Tu et al.· Metabolic Engineering· 0 citations
A one-step CRISPR-based transformation protocol developed in this study enables fast genome editing workflows with minimal hands-on time and demonstrates the versatility of the modified system for this industrially important genus.
Maximilian Hilkmann, Norma Welsch, M. F. Felle et al.· Applied Microbiology and Bio...· 0 citations
Nearly 100% knockout efficiency and over 86.67% integration efficiency were achieved in the described experimental cases with this novel pop-in/pop-out-based editing system, which provides a new gene-editing tool for synthetic biology in Pichia pastoris.
Guo-Wei Zhao, Liang-Cheng Jiao, Yun-Chong Li et al.· Microbiology Research· 0 citations
Cordycepin is a high-value bioactive compound with significant therapeutic potential. In this study, we report a transposon-based genome engineering strategy for establishing an allantoin-inducible cordycepin production platform in Saccharomyces cerevisiae. We first optimized the allantoin-inducible system by refining...
Chao-Jun Tang, Jun-Yi Wang, Xue-Yi Luo et al.· Engineering in Life Sciences· 0 citations
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