Self-Redirection of Metabolic Flux toward Squalene and Ethanol Pathways by Engineered Yeast

Abstract

We have previously reported that squalene overproducing yeast self-downregulate the expression of the ethanol pathway (non-essential pathway) to divert the metabolic flux to the squalene pathway. In this study, the effect of co-production of squalene and ethanol on other non-essential pathways (fusel alcohol pathway, FA) of Saccharomyces cerevisiae was evaluated. However, before that, 13 constitutive promoters, like IRA1p, PET9p, RHO1p, CMD1p, ATP16p, USA3p, RER2p, COQ1p, RIM1p, GRS1p, MAK5p, and BRN1p, were engineered using transcription factor bindings sites from strong promoters HHF2p (−300 to −669 bp) and TEF1p (−300 to −579 bp), and employed to co-overexpress squalene and ethanol pathways in S. cerevisiae. The FSE strain overexpressing the key genes of the squalene pathway accumulated 56.20 mg/L squalene, a 16.43-fold higher than wild type strain (WS). The biogenesis of lipid droplets was stimulated by overexpressing DGA1 and produced 106 mg/L squalene in the FSE strain. AFT1p and CTR1p repressible promoters were also characterized and employed to downregulate the expression of ERG1, which also enhanced the production of squalene in FSE strain up to 42.85- (148.67 mg/L) and 73.49-fold (255.11 mg/L) respectively. The FSE strain was further engineered by overexpressing the key genes of the ethanol pathway and produced 40.2 mg/mL ethanol in the FSE1 strain, 3.23-fold higher than the WS strain. The FSE1 strain also self-downregulated the expression of the FA pathway up to 73.9%, perhaps by downregulating the expression of GCN4 by 2.24-fold. We demonstrate the successful tuning of the strength of yeast promoters and highest coproduction of squalene and ethanol in yeast, and present GCN4 as a novel metabolic regulator that can be manipulated to divert the metabolic flux from the non-essential pathway to engineered pathways.

Recent advances in the microbial production of squalene

“…Later, two iron and copper repressible promoters P AFT1 and P CTR1 , respectively are characterized and used to repress the ERG1 gene in S. cerevisiae towards squalene bioproduction. ERG1 repression with metal ion repressible P CTR1 promoter has enhanced squalene up to twofold when compared to P AFT1 promoter (Manzoor et al 2020). In the same study, TFBS TEF1 and TFBS HHF2 were engineered into different constitutive promoters thereby enhancing the strength of the yeast constitutive promoters.…”

“…ERG1 gene codes for squalene monooxygenase in the MVA pathway downstream of squalene in S. cerevisiae. Being an essential gene, disruption of ERG1 leads to a lethal phenotype and therefore repression of ERG1 gene by promoter replacement has become a prominent strategy in the development of cell factories towards squalene biosynthesis (Hull et al 2014;Manzoor et al 2020;Liu et al 2020a, b;Zhu et al 2021). Also, the usage of repressible promoters provides better economic feasibility when compared to the enzyme inhibitors at an industrial level.…”

“…The engineered promoters such as P HHF2 , P IRA1 , P RHO1 , and P PET9 with TFBS TEF1 were utilized to drive the transcription of HMG1, IDI1, ERG20, and ERG9 genes respectively. This study resulted in a strain with squalene improvement to nearly 74-fold (Manzoor et al 2020). Further, P HXT1 is a glucose inducible promoter and causes repression when the glucose levels in the medium become low or zero.…”

Advanced Strategies for the Efficient Production of Squalene by Microbial Fermentation

“…The coaccumulation and co-occurrence of terpenoids in LDs provide an opportunity to achieve the high-yield production and storage of terpenoids in LDs. Through the overexpression of diacylglycerol acyltransferase ( DGA1 ) to stimulate the biogenesis of lipid droplets and inhibition of ERG1 expression by employing AFT1p and CTR1p repressible promoters, the squalene titer reached 255.11 mg/L, which was 73.49 times that of the wild-type strain. , Another study showed that the overexpression of DGA1 in the engineered strain of S. cerevisiae with the overexpression of tHMGR can enhance the lipid bodies biosynthesis, leading to 12-fold increase in the squalene production compared with the original strain . These results provided a feasible scheme for the further efficient synthesis of hydrophobic compounds in S. cerevisiae .…”