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.

FIG 1

The impact of ΔYPL062W on terpenoid production. Recombinant strains expressing terpenoid production pathways were created and assayed to investigate the effect of ΔYPL062W on their corresponding production titers. Geraniol, amorphadiene, geranylgeraniol, zymosterol, and lycopene are representative of monoterpenoid, sesquiterpenoid, diterpenoid, triterpenoid, and tetraterpenoid, respectively. The improvement in production titer is indicated as Δ/C, which is the ratio of terpenoid production titer of the ΔYPL062W strain to that of the control/parent strain.

FIG 2

Transcriptional changes of genes involved in fatty acid catabolism, MVA formation, and energy metabolism by ΔYPL062W, whose reactions mainly occur in the peroxisome (A), cytoplasm (B), and mitochondrion (C), respectively. The key metabolites in each pathway are marked in bold. Transcriptional data are boxed next to each gene. Strain names are abbreviated as follows: C, SyBE_Sc14C10, and Δ, ΔYPL062W strain SyBE_Sc14C02. Strains were cultured under both 2% (mass/vol) (2% G) and 4% (mass/vol) (4% G) glucose conditions. The time reading after the “@” symbol in the legend indicates the sampling time within the glucose consumption phase (i.e., 4 h) or the ethanol consumption phase (i.e., 12 h). The relative transcription level for each gene is indicated as Δ/C, which is the ratio of the transcription level in the ΔYPL062W to that in the control strain. Genes that are significantly upregulated and downregulated and genes without significant transcriptional differences are colored red, blue, and gray, respectively.

FIG 3

The effect of ΔYPL062W on heterologous gene expression. (A) The transcriptional changes of CrtE, CrtB, and CrtI in lycopene-producing strains revealed by RNA-seq analysis. Strains SyBE_Sc14C07 (control strain, Lycopene_C) and SyBE_Sc14C23 (ΔYPL062W strain, Lycopene_Δ) were cultured under both 2% (mass/vol) (2% G) and 4% (mass/vol) (4% G) glucose conditions. The time reading after the “@” symbol in the legend indicates the sampling time within the glucose consumption (the top line of the table) and ethanol consumption phases (the bottom line of the table). The relative transcription level for each gene is indicated as Lycopene_Δ/C, which is the ratio of the transcription level of the recombinant lycopene pathway in the ΔYPL062W strain to that in the control strain. Genes that are significantly upregulated and downregulated and genes without significant transcriptional differences are colored red, blue, and gray, respectively. (B and C) The effects of ΔYPL062W on P_GAL1 and P_GAL10 activities when cultured under 2% (mass/vol) glucose (B) or 4% (mass/vol) glucose (C). Promoter activities are represented as relative fluorescence intensities of RFP in the control strain (SyBE_Sc14C02, C) and ΔYPL062W strain (SyBE_Sc14C10, Δ) without lycopene synthesis. (D, E, and F) The transcription level of genes CrtE (D), CrtB (E), and CrtI (F) in lycopene-producing strains (Lycopene_C and Lycopene_Δ) as determined by real-time PCR. Cells were cultured under 2% (mass/vol) glucose, and samples were taken within the glucose consumption phase (i.e., 4 h) or the ethanol consumption phase (i.e., 12 h). The relative transcription level for each gene was determined as 2^−ΔΔCT using gene ALG9 for normalization. All data are from at three or more experimental replicates. Statistically significant differences are indicated as follows: *, P < 0.05, and **, P < 0.01 (two-tailed Student t test).

FIG 4

The effect of ΔYPL062W on ergosterol biosynthesis. (A) Transcriptional profiles of ergosterol biosynthesis genes in host strains (SyBE_Sc14C02, C) and (SyBE_Sc14C10, Δ) and lycopene-producing strains (SyBE_Sc14C07, Lycopene_C) and (SyBE_Sc14C23, Lycopene_Δ) on 2% (mass/vol) glucose. Transcriptional data are boxed next to each gene. The time reading after the “@” symbol in the legend indicates the sampling time within the glucose consumption phase (i.e., 4 h) or the ethanol consumption phase (i.e., 12 h). The relative transcription level for each gene is denoted as Lycopene_Δ/C or Δ/C, which are the ratios of the transcription levels in the ΔYPL062W strain to that in the no-deletion control strain for either lycopene-producing recombinant strains or nonproducing parental strains. Genes that are significantly upregulated and downregulated and genes without significant transcriptional differences are colored red, blue, and gray, respectively. (B) Visual microscopic analysis of ΔYPL062W strains with/without lycopene synthesis (Lycopene_Δ and Δ). Cells were cultured under 2% (mass/vol) glucose for 46 h. The contents of squalene, zymosterol, and ergosterol in (C) host strains (C and Δ) and (D) lycopene-producing strains (Lycopene_C and Lycopene_Δ) cultured on 2% (mass/vol) glucose are quantified from experimental triplicates.

FIG 5

The effect of ΔYPL062W on fatty acid biosynthesis. (A) Transcriptional profiles of fatty acid biosynthesis genes in host strains control (C, SyBE_Sc14C02) and ΔYPL062W (Δ, SyBE_Sc14C10) and corresponding lycopene-producing strains (Lycopene_C, SyBE_Sc14C07 and Lycopene_Δ, SyBE_Sc14C23) cultured on 2% (mass/vol) glucose. Transcriptional data are boxed next to each gene. The time reading after the “@” symbol in the legend indicates the sampling time within the glucose consumption phase (i.e., 4 h) or the ethanol consumption phase (i.e., 12 h). The relative transcription level for each gene is denoted as Lycopene_Δ/C or Δ/C, which are the ratios of the transcription levels in the ΔYPL062W strain to that in the no-deletion control strain for either lycopene-producing recombinant strains or nonproducing parental strains. Genes that are significantly upregulated and downregulated and genes without significant transcriptional differences are colored red, blue, and gray, respectively. (B and C) Fatty acid contents in host strains (C and Δ [B]) and lycopene-producing strains (Lycopene_C and Lycopene_Δ [C]) cultured on 2% (mass/vol) glucose. are quantified from experimental triplicates. (D and E) Unsaturation index of host strains (C and Δ [D]) and lycopene-producing strains (C and Δ [E]) cultured on 2% (mass/vol) glucose. Unsaturation index was calculated as the ratio of unsaturated fatty acid content to that of total fatty acids.

FIG 6

Redefinition and reconstitution of YPL062W. (A) YPL062W is not expressed, and its deletion negatively impacts ALD6 expression. Transcriptional profiles of Chr XVI (bp 429929 to 434090). Schematic diagram of genome structure spanning YPL062W locus (Chr XVI [bp 429929 to 434090]). Transcriptional profile of gene contexts (from TIM50 through YPL062W to ALD6) in host strains (SyBE_Sc14C02, C and SyBE_Sc14C10, Δ) on 2% (mass/vol) glucose. Cells were sampled at 4 h and 12 h. (B) Characterization of YPL062W as the core promoter driving transcription of ALD6. Various constructs (P_A to P_L) containing all or part of the sequence spanning the YPL062W locus were fused to RFP, and their activities were measured by relative fluorescence intensities at 48 h of cultivation on 2% (mass/vol) glucose. P_A is a full-length promoter. P_B to P_D, P_F, and P_H toP_K each contain increasingly larger truncations of the conserved region. P_E and P_G contain transversion mutations in conserved domain VI (marked in orange). The null-expression control P_L has YPL062W entirely replaced by a KanMX cassette. The correlation between the transcription levels of ALD6 and terpenoid production titers. Geraniol (monoterpenoid) (C), amorphadiene (sesquiterpenoid) (D), geranylgeraniol (diterpenoid) (E), zymosterol (triterpenoid) (F), and lycopene (tetraterpenoid) (G) were selected to test the correlation between ALD6 transcription levels and terpenoid production. The relative production for each terpenoid was determined as the ratio of product titer in each strain harboring a truncated conserved region to that of the control strain containing and intact P_A region. The relative transcription level of ALD6 was determined as the ratio of the transcription level of ALD6 controlled by corresponding truncated promoter to that of ALD6 regulated by the intact P_A region.