A programmable PCN platform for S. cerevisiae based on the endogenous 2μ plasmid achieves up to 20 copies per cell with enhanced homogeneity and stability and establishes a well quantified PCN regulation toolkit for yeast, addressing instability from multiple copies and enabling gene dosage control across DNA, RNA, and protein levels.
Abstract
Synthetic biology demands plasmid with tunable copy numbers. While dynamic plasmid copy number (PCN) regulation has been engineered in prokaryotes, a parallel capability remains unavailable in eukaryotic systems. Here, we develop a programmable PCN platform for S. cerevisiae based on the endogenous 2μ plasmid. The p2μ-Cir0 system achieves up to 20 copies per cell with enhanced homogeneity and stability, supporting 60-fold higher expression than chromosomal integration. Addition of a CEN element enables switchable PCN transitions from 1 to 38 copies, and Leu2d-based selection further raises copy number to ~70 and expression to ~110-fold. We demonstrate broad utility in tRNA phenotyping, GLP-1 precursor, 2-phenylethanol, β-carotenoid and ergothioneine production, and long-term strain preservation. These results establish a well quantified PCN regulation toolkit for yeast, addressing instability from multiple copies and enabling gene dosage control across DNA, RNA, and protein levels. Synthetic biology demands plasmid with tunable copy numbers, however this capability remains unavailable in eukaryotes. Here the authors develop a dynamic plasmid copy number system for S. cerevisiae and show bioproduction applications.
Plasmids are extra-chromosomal DNA molecules capable of autonomous replication, stable inheritance in a bacterial population, and horizontal transfer to other bacteria. Plasmids can harbour auxiliary genetic material that contributes to host bacterial fitness, the most prominent example being antimicrobial resistance (AMR) determinants, which remain the greatest threat to modern medicine. Since their discovery in the early 1950s, plasmids have been extensively studied due to their diversity, their capacity to spread between bacterial hosts, and their ability to carry and disseminate multiple AMR genes simultaneously. Recent advances in sequencing technology have transformed plasmid research, with transposon–insertion sequencing (TIS) enabling simultaneous analysis of millions of mutants and providing unprecedented scale, speed and resolution for studying plasmid biology. Here, we briefly outline a recommended methodology for generating plasmid transposon mutant libraries, which can be combined with TIS to investigate plasmid replication, maintenance and conjugation. We further summarise data from nine comprehensive plasmid TIS studies to date on five distinct plasmids, discuss alternative uses for plasmid libraries, challenges and future perspectives.
Steven J. Hancock, M. Phan, Jie-Lian Zheng· Microbiology Australia· 0 citations
The results support a model in which prolonged expression of a burdensome secreted recombinant protein can be associated with early non-genetic heterogeneity in intracellular mCherry fluorescence, growth advantages of low-fluorescence cells and evolution of plasmid variants that reduce expression.
Ming-Zhi Xu, Andrei Parker, K. Winzer et al.· Microbial Cell Factories· 0 citations
The eukaryotic cell cycle, with its inherent regulatory redundancy, provides an ideal target for exploring genome modularisation and minimisation through synthetic genomics. Building upon principles established by the Synthetic Yeast Genome (Sc2.0) project, we used CRISPR-mediated genome engineering to relocate nine key cell cycle genes into a synthetic gene cluster in the S. cerevisiae genome, to allow combinatorial study of these genes in yeast. We employed Cre/loxP recombination to rapidly generate hundreds of strains with different gene deletion combinations in the module, with the objective of identifying minimal gene sets that permit robust cell cycle function. Using FACS-sorting and POLAR (Pool of Long Amplified Reads) sequencing, we conducted high-throughput analysis of gene deletion combinations in large cell pools, detecting approximately 80% of theoretically possible gene combinations, including those predicted from prior mathematical modelling studies. Our findings demonstrate that the cell cycle gene set can be minimised while maintaining viability, though only select combinations of gene deletions ensure robust fitness across different conditions. This work establishes a framework for genome minimisation, opening the door to the design of simplified, modular synthetic genomes for diverse applications.
Anastasiya Malyshava, Klaudia Ciurkot, Lucas Cañizares Alonso et al.· bioRxiv· 0 citations
Genetic code expansion enables the site-specific installation of noncanonical amino acids (ncAAs) into proteins, but its limited efficiency in eukaryotes remains a major barrier to broader application. Here we establish a visual, plug-and-play screening platform to evolve 18S ribosomal DNA in Saccharomyces cerevisiae and identify ribosomal variants that improve ncAA incorporation. The best-performing strain, designated ribo-hyper, increased ncAA-dependent GFP production by 2.9-fold relative to the wild-type rDNA strain and enhanced incorporation across distinct orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Characterization of ribo-hyper showed that global translation activity and cellular growth were moderately reduced. Proteomic analysis further revealed changes in amino acid biosynthesis, translation-related proteins and stress-response pathways, indicating that the engineered ribosome reshapes cellular translation homeostasis. Perturbation of translation quality-control pathways, including the ribosome-rescue factors Dom34 and Hbs1 and the core mRNA exosome component Ski6, reduced ncAA-containing protein output, whereas disruption of ribosome quality-control factor Rqc2 had little effect. These findings support a role for ribosome rescue and associated mRNA turnover in efficient ncAA incorporation in the ribo-hyper strain. Together, our results establish eukaryotic ribosome engineering as a viable strategy for improving genetic code expansion in yeast.
Xiao-Xu Chen, Wentao Shen, Xian-Qing Chen et al.· Synthetic and Systems Biotec...· 0 citations
An RNA-guided bridge recombinase system is engineered through rational mutagenesis and AI-assisted directed evolution, enabling programmable chromosomal rearrangements in both plant and mammalian cells and achieving up to a 29.8-fold increase in activity.
Rui Gao, Jingjing Wei, Chao Sun et al.· Trends in Biotechnology· 0 citations
Advances in molecular biology tools are essential for streamlining and accelerating genetic engineering of cells across industrial and academic applications. While CRISPR-Cas improves genome editing efficiency, current systems have limitations and are often host specific, which restricts their versatility. This study describes a versatile CRISPR-Cas9 system for genome editing in industrially relevant Bacillus species. By adapting the well-established pJOE8999 vector-based CRISPR-Cas9 genome editing system, we constructed an inducer-independent, broad-host-range genome editing system. It maintains the benefits of low toxicity to the target cell and the cloning host as well as the ease to use of a single-plasmid CRISPR-Cas9 system. We utilized the constitutive Sigma70-type promoter from the conserved veg gene of Bacillus, to develop and test the suitability of promoter variants of different strengths for Cas9 expression. Successful gene deletions in three different Bacillus species demonstrated the versatility of the modified system for this industrially important genus. This was further confirmed by the integration of a reporter gene fusion and the introduction of a single point mutation in the genome of Bacillus licheniformis. This one-step CRISPR-based transformation protocol developed in this study enables fast genome editing workflows with minimal hands-on time. • Editing and screening of promoter variants for balanced Cas9 expression in Bacillus. • Development of a versatile inducer-independent, single-plasmid CRISPR-Cas-based system. • Verification of the modified CRISPR-based system for genome editing in different Bacilli.
Maximilian Hilkmann, Norma Welsch, M. F. Felle et al.· Applied Microbiology and Bio...· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
