| Summary: | 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 <i>Saccharomyces cerevisiae</i> was evaluated. However, before that, 13 constitutive promoters, like <i>IRA1p, PET9p, RHO1p, CMD1p, ATP16p, USA3p,</i> <i>RER2p, COQ1p, RIM1p, GRS1p, MAK5p,</i> and <i>BRN1p,</i> were engineered using transcription factor bindings sites from strong promoters <i>HHF2p</i> (−300 to −669 bp) and <i>TEF1p</i> (−300 to −579 bp), and employed to co-overexpress squalene and ethanol pathways in <i>S. cerevisiae.</i> 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 <i>DGA1</i> and produced 106 mg/L squalene in the FSE strain. <i>AFT1p</i> and <i>CTR1p</i> repressible promoters were also characterized and employed to downregulate the expression of <i>ERG1,</i> 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 <i>GCN4</i> 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 <i>GCN4</i> as a novel metabolic regulator that can be manipulated to divert the metabolic flux from the non-essential pathway to engineered pathways.
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