Evaluating and engineering Saccharomyces cerevisiae promoters for increased amylase expression and bioethanol production from raw starch.

Abstract

Bioethanol production from starchy biomass via consolidated bioprocessing (CBP) will benefit from amylolytic Saccharomyces cerevisiae strains that produce high levels of recombinant amylases. This could be achieved by using strong promoters and modification thereof to improve gene expression under industrial conditions. This study evaluated eight endogenous S. cerevisiae promoters for the expression of a starch-hydrolysing α-amylase gene. A total of six of the native promoters were modified to contain a promoter-proximal intron directly downstream of the full-length promoter. Varying results were obtained; four native promoters outperformed the ENO1P benchmark under aerobic conditions and two promoters showed better expression under simulated CBP conditions. The addition of the RPS25A intron significantly improved the expression from most promoters, displaying increased transcript levels, protein concentrations and amylase activities. Raw starch-utilising strains were constructed through co-expression of selected α-amylase cassettes and a glucoamylase gene. The amylolytic strains displayed improved fermentation vigour on raw corn starch and broken rice, reaching 97% of the theoretical ethanol yield and converting 100% of the available carbon to products within 120h in small-scale CBP fermentations on broken rice. This study showed that enhanced amylolytic strains for the conversion of raw starch to ethanol can be achieved through turnkey promoter selection and/or engineering.