Abstract
Maintaining redox balance is critical for the production of heterologous secondary metabolites, whereas on various occasions the native cofactor balance does not match the needs in engineered microorganisms. In this study, 7-dehydrocholesterol (7-DHC, a crucial precursor of vitamin D3) biosynthesis pathway was constructed in Saccharomyces cerevisiae BY4742 with endogenous ergosterol synthesis pathway blocked by knocking out the erg5 gene (encoding C-22 desaturase). The deletion of erg5 led to redox imbalance with higher ratio of cytosolic free NADH/NAD+ and more glycerol and ethanol accumulation. To alleviate the redox imbalance, a water-forming NADH oxidase (NOX) and an alternative oxidase (AOX1) were employed in our system based on cofactor regeneration strategy. Consequently, the production of 7-dehydrocholesterol was increased by 74.4% in shake flask culture. In the meanwhile, the ratio of free NADH/NAD+ and the concentration of glycerol and ethanol were reduced by 78.0%, 50.7% and 7.9% respectively. In a 5-L bioreactor, the optimal production of 7-DHC reached 44.49(±9.63) mg/L. This study provides a reference to increase the production of some desired compounds that are restricted by redox imbalance.
Highlights
Maintaining redox balance plays an important role in the production of heterologous secondary metabolites [1] and cofactors act as redox carriers and energy transfer agents for the biochemical reactions [2]
Blocking ergosterol biosynthesis pathway was essential for 7-DHC production
The S.cerevisiae BY4742 erg5 null mutant strain ΔERG5 was chosen as chassis in this study to eliminate the competition of ergosterol biosynthesis pathway, and the result of GC-TOF-MS showed no detection of ergosterol (S2 Fig)
Summary
Maintaining redox balance plays an important role in the production of heterologous secondary metabolites [1] and cofactors act as redox carriers and energy transfer agents for the biochemical reactions [2]. The native cofactor balance does not match the needs in engineered microorganisms on various occasions [3]. The intracellular redox potential is primarily determined by the ratio of NADH/NAD+ rather than that of NADPH/NADP+ [4]. When glycolytic NADH is produced beyond the cellular capacity for its oxidation, reduced products like ethanol and glycerol will be generated [4]. Imbalanced oxidation-reduction potential leads to cell damage, carbon or energy waste, and even metabolic arrest [5]. Cofactor engineering through different biotechnological techniques could reshape the whole-cell
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