Engineering of cis-Element in Saccharomyces cerevisiae for Efficient Accumulation of Value-Added Compound Squalene via Downregulation of the Downstream Metabolic Flux

Abstract

Transcriptional downregulation is widely used for metabolic flux control. Here, marO, a cis-element of Escherichia coli mar operator, was explored to engineer promoters of Saccharomyces cerevisiae for downregulation. First, the ADH1 promoter (P ADH1 ) and its enhanced variant P UADH1 were engineered by insertion of marO into different sites, which resulted in decrease in both gfp5 transcription and GFP fluorescence intensity to various degrees. Then, marO was applied to engineer the native ERG1 and ERG11 promoters due to their importance for accumulation of value-added intermediates squalene and lanosterol. Elevated squalene content (4.9-fold) or lanosterol content (4.8-fold) and 91 or 28% decrease in ergosterol content resulted from the marO-engineered promoter P ERG1(M5) or P ERG11(M3) , respectively, indicating the validity of the marO-engineered promoters in metabolic flux control. Furthermore, squalene production of 3.53 g/L from cane molasses, a cheap and bulk substrate, suggested the cost-effective and promising potential for squalene production.

Enhancing fluxes through the mevalonate pathway in Saccharomyces cerevisiae by engineering the HMGR and β‐alanine metabolism

“…In our previous study, 100.31 AE 9.90 mg g À1 CDW of intracellular squalene was obtained after 24 h of cultivation in YPD medium, which was the highest specific synthesis rate reported to date at the shake flask stage (Zhou et al, 2021). Here, squalene content in strain SQ3-5 reached 152.01 AE 4.52 mg g À1 DCW, indicating the effectiveness of the used engineering strategies.…”

“…Haploid S. cerevisiae YS58 (Teunissen et al, 1993) was used to investigate the roles of target genes. Industrial S. cerevisiae strain SQ-3 (Zhou et al, 2021) was used Table 1. Yeast strains used in this study.…”

“…Plasmid pFA6a-kanMX4 was used as the template for amplification of G418-resistant cassette KanMX. Plasmid pGMZC was used to amplify selective marker P GAL1 -mazF-zeoR (GMZ) including zeocin-resistant cassette P TEF1 -zeoR-T CYC1 and mazF expression cassette P GAL1 -mazF-T AOX1 (Zhou et al, 2021). To compare promoter strength, ERG1 promoter was amplified from S. cerevisiae, H. polymorpha, P. pastoris and P. rhodozyma genomes, respectively, and then was ligated into plasmid pYC-GA (Zhou et al, 2021) to generate plasmids pYC-SEGA, pYC-HEGA, pYC-PEGA and pYC-XEGA with green fluorescence protein (GFP) as the reporter.…”

“…Plasmid pGMZC was used to amplify selective marker P GAL1 -mazF-zeoR (GMZ) including zeocin-resistant cassette P TEF1 -zeoR-T CYC1 and mazF expression cassette P GAL1 -mazF-T AOX1 (Zhou et al, 2021). To compare promoter strength, ERG1 promoter was amplified from S. cerevisiae, H. polymorpha, P. pastoris and P. rhodozyma genomes, respectively, and then was ligated into plasmid pYC-GA (Zhou et al, 2021) to generate plasmids pYC-SEGA, pYC-HEGA, pYC-PEGA and pYC-XEGA with green fluorescence protein (GFP) as the reporter. For expression of b-caryophyllene synthase (CPS), plasmid pYE-CPS was constructed with the codon-optimized coding sequence of b-caryophyllene synthase gene QHS1 regulated by promoter P IRA1 and terminator T SUP4 .…”

Cat8 Response to Nutritional Changes and Interaction With Ehrlich Pathway Related Factors

“…Based on YS58-HA and YS58-CAT8-HA strains, YS58-HA Δsnf1 and YS58-CAT8-HA Δsnf1 strains were obtained by introducing hygromycin resistance and deleting SNF1 . The following strains with gene deletion were obtained using the scarless knockout method created by our laboratory ( Zhou et al, 2021 ), including the strains YS58-Δcat8, YS58-Δaro80, and YS58-Δgln3gat1. CAT8 self-promoter was further replaced with TEF2 promoter by the seamless replacement method to obtain YS58-CAT8 Δaro80 and YS58-CAT8 Δgln3gat1 strains.…”