Construction of industrial strains for the efficient consolidated bioprocessing of raw starch.

BACKGROUND

Consolidated bioprocessing (CBP) combines enzyme production, saccharification and fermentation into a one-step process. This strategy represents a promising alternative for economic ethanol production from starchy biomass with the use of amylolytic industrial yeast strains.

RESULTS

Recombinant Y294 laboratory strains simultaneously expressing an α-amylase and glucoamylase gene were screened to identify the best enzyme combination for raw starch hydrolysis. The codon optimised glucoamylase encoding gene () and the native α-amylase encoding gene () were selected for expression in two industrial yeast strains, namely Ethanol Red™ (hereafter referred to as the ER) and M2n. Two δ-integration gene cassettes were constructed to allow for the simultaneous multiple integrations of the and genes into the yeasts' genomes. During the fermentation of 200 g l raw corn starch, the amylolytic industrial strains were able to ferment raw corn starch to ethanol in a single step with high ethanol yields. After 192 h at 30 °C, the ER T12 and M2n T1 strains (containing integrated and gene cassettes) produced 89.35 and 98.13 g l ethanol, respectively, corresponding to estimated carbon conversions of 87 and 94%, respectively. The addition of a commercial granular starch enzyme cocktail in combination with the amylolytic yeast allowed for a 90% reduction in exogenous enzyme dosage, compared to the conventional simultaneous saccharification and fermentation (SSF) control experiment with the parental industrial host strains.

CONCLUSIONS

A novel amylolytic enzyme combination has been produced by two industrial strains. These recombinant strains represent potential drop-in CBP yeast substitutes for the existing conventional and raw starch fermentation processes.