Match of functional module with chassis in 7-dehydrocholesterol synthesis

Abstract:

The key challenge to generate engineered cells by synthetic biology for producing 7-dehydrocholesterol (7-DHC) in a high titer is the match between functional module and chassis. Our study focused on solving this problem by combining different promoters and yeast chassis to increase 7-DHC production. To optimize the chassis in order to accumulate zymosterol, the substrate for 7-DHC synthesis, we overexpressed truncated HMG-CoA reductase (tHmg1p) and squalene epoxidase (Erg1p), both are key genes of yeast endogenous zymosterol biosynthetic pathway. In addition, we knocked out C-24 methyl transferase (Erg6p) and C-22 dehydrogenase (Erg5p) to inhibit the conversion of zymosterol to ergosterol. By introducing heterologous C-24 reductase under three promoters with different strengths, namely TDH3p, PGK1p and TDH1p, we constructed functional modules of diverse activities. Nine engineeredcells were generated based on the combination of these three modules and three chassis. The result shows that the engineered cell composed of functional module regulated by TDH3p and chassis SyBE_000956 had the highest 7-DHC production, indicating a better match than others. This study provides evidences for importance of match and empirical support for rational design of subsequent researches.