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Open AccessArticle

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

1
School of Life Science, Beijing Institute of Technology, Beijing 100081, China
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Department of Tuberculosis, Bolan University of Medical and Health Sciences, Quetta 87300, Pakistan
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Department of Biological Sciences, International Islamic University, Islamabad 45550, Pakistan
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Department of Microbiology, University of Balochistan, Quetta 87300, Pakistan
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Department of Biochemistry, University of Karachi, Karachi 75530, Pakistan
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Department of Industrial Biotechnology, Government College University, Lahore 53801, Pakistan
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Department of Botany, Government College University, Lahore 53801, Pakistan
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Department of Bioscience, COMSATS Institute of Information Technology (CIIT), Islamabad 45550, Pakistan
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Institute for Synthetic Biosystem, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 100081, China
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Institute of Biochemistry, University of Balochistan, Quetta 87300, Pakistan
*
Authors to whom correspondence should be addressed.
Metabolites 2020, 10(2), 56; https://doi.org/10.3390/metabo10020056
Received: 18 December 2019 / Revised: 28 January 2020 / Accepted: 30 January 2020 / Published: 1 February 2020
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.
Keywords: Saccharomyces cerevisiae; engineered promoters; repressible promoters; squalene; fusel alcohol; ethanol Saccharomyces cerevisiae; engineered promoters; repressible promoters; squalene; fusel alcohol; ethanol
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Manzoor, R.; Ahmed, M.; Riaz, N.; Kiani, B.H.; Kaleem, U.; Rashid, Y.; Nawaz, A.; Awan, M.U.F.; Khan, H.; Imtiaz, U.; Rasheed, Y.; Kaleem, I.; Rasool, A. Self-Redirection of Metabolic Flux Toward Squalene and Ethanol Pathways by Engineered Yeast. Metabolites 2020, 10, 56.

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