Primary and Secondary Metabolic Effects of a Key Gene Deletion (Δ YPL062W) in Metabolically Engineered Terpenoid-Producing Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae is an established cell factory for production of terpenoid pharmaceuticals and chemicals. Numerous studies have demonstrated that deletion or overexpression of off-pathway genes in yeast can improve terpenoid production. The deletion of YPL062W in S. cerevisiae, in particular, has benefitted carotenoid production by channeling carbon toward carotenoid precursors acetyl coenzyme A (acetyl-CoA) and mevalonate. The genetic function of YPL062W and the molecular mechanisms for these benefits are unknown. In this study, we systematically examined this gene deletion to uncover the gene function and its molecular mechanism. RNA sequencing (RNA-seq) analysis uncovered that YPL062W deletion upregulated the pyruvate dehydrogenase bypass, the mevalonate pathway, heterologous expression of galactose (GAL) promoter-regulated genes, energy metabolism, and membrane composition synthesis. Bioinformatics analysis and serial promoter deletion assay revealed that YPL062W functions as a core promoter for ALD6 and that the expression level of ALD6 is negatively correlated to terpenoid productivity. We demonstrate that ΔYPL062W increases the production of all major terpenoid classes (C₁₀, C₁₅, C₂₀, C₃₀, and C₄₀). Our study not only elucidated the biological function of YPL062W but also provided a detailed methodology for understanding the mechanistic aspects of strain improvement.IMPORTANCE Although computational and reverse metabolic engineering approaches often lead to improved gene deletion mutants for cell factory engineering, the systems level effects of such gene deletions on the production phenotypes have not been extensively studied. Understanding the genetic and molecular function of such gene alterations on production strains will minimize the risk inherent in the development of large-scale fermentation processes, which is a daunting challenge in the field of industrial biotechnology. Therefore, we established a detailed experimental and systems biology approach to uncover the molecular mechanisms of YPL062W deletion in S. cerevisiae, which is shown to improve the production of all terpenoid classes. This study redefines the genetic function of YPL062W, demonstrates a strong correlation between YPL062W and terpenoid production, and provides a useful modification for the creation of terpenoid production platform strains. Further, this study underscores the benefits of detailed and systematic characterization of the metabolic effects of genetic alterations on engineered biosynthetic factories.

Keywords: ALD6; Saccharomyces cerevisiae; YPL062W; terpenoids.