Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered Saccharomyces cerevisiae

作者：Bingyin Peng, Sarah J. Weintraub, Zeyu Lu, Samuel Evans, Qianyi Shen, Liam McDonnell, Manuel R. Plan, Thomas Collier, Li Chen Cheah, Lei Ji, Christopher B. Howard, Will Anderson, Matt Trau, Geoff Dumsday, Erin Bredeweg, Eric M. Young, Robert Speight, Claudia E. Vickers · 发表于：ACS Synthetic Biology · 年份：2023 · DOI：10.1021/acssynbio.3c00363 · 被引用次数：9 · 研究领域：Microbial Metabolic Engineering and Bioproduction、Fungal and yeast genetics research、Biofuel production and bioconversion

The variability in phenotypic outcomes among biological replicates in engineered microbial factories presents a captivating mystery. Establishing the association between phenotypic variability and genetic drivers is important to solve this intricate puzzle. We applied a previously developed auxin-inducible depletion of hexokinase 2 as a metabolic engineering strategy for improved nerolidol production in Saccharomyces cerevisiae, and biological replicates exhibit a dichotomy in nerolidol production of either 3.5 or 2.5 g L –1 nerolidol. Harnessing Oxford Nanopore’s long-read genomic sequencing, we reveal a potential genetic cause─the chromosome integration of a 2μ sequence-based yeast episomal plasmid, encoding the expression cassettes for nerolidol synthetic enzymes. This finding was reinforced through chromosome integration revalidation, engineering nerolidol and valencene production strains, and generating a diverse pool of yeast clones, each uniquely fingerprinted by gene copy numbers, plasmid integrations, other genomic rearrangements, protein expression levels, growth rate, and target product productivities. Τhe best clone in two strains produced 3.5 g L –1 nerolidol and ∼0.96 g L –1 valencene. Comparable genotypic and phenotypic variations were also generated through the integration of a yeast integrative plasmid lacking 2μ sequences. Our work shows that multiple factors, including plasmid integration status, subchromosomal location, gene copy number, sesquiterpene synt...