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.

Enhancing Geranylgeraniol Production by Metabolic Engineering and Utilization of Isoprenol as a Substrate in Saccharomyces cerevisiae

“…In a previous study, the metabolic flux of GGPP and GGOH production was enhanced by deletion of ROX1 (transcriptional repressor of the sterol pathway), YPL 062 W (deletion of YPL 062 W increased levels of Acetyl-CoA), and YJL 064 W (potential distant genetic gene has interaction with the target pathway). ,− In the present study, to further enhance GGOH production, we attempted to increase the supply of GGPP by deleting these potential target genes. After ROX1 , YPL 062 W and YJL 064 W in G6 were inactivated individually, resulting in strains G8, G8P, and G8J (Tables and S3).…”

Identifying and engineering the ideal microbial terpenoid production host

“…Chen et al (2016) produced 55.56 mg g −1 DCW of lycopene in a S. cerevisiae strain harboring several knockouts, including the YPL062W locus whose function was unclear at that time. Only very recently, it was determined by the same group that this locus functions as a promoter with major influence on terpenoid production and that a knockout positively influences production levels of all terpenoid classes (Chen et al 2019). Respective lycopene production values for C. glutamicum or P. pastoris are markedly lower in the 1-digit mg g −1 DCW range (Bhataya et al 2009; Heider et al 2012).…”

High production of valencene in Saccharomyces cerevisiae through metabolic engineering

“…Moreover, some distantly located genetic loci might have potential interactions with some terpenoid pathway, such as ypl062w and yjl064w [7, 14]. Recent research pointed out that the ypl062w functioned as an important promoter for ald6 whose expression level was negatively correlated with terpenoid productivity [15]. Apart from pathway engineering, the selection of appropriate expression cassettes including promoter and terminator is also important for the optimization of valencene production [16].…”