Construction of industrial Saccharomyces cerevisiae strains for the efficient consolidated bioprocessing of raw starch

Abstract

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 Saccharomyces cerevisiae 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 Talaromyces emersonii glucoamylase encoding gene (temG_Opt) and the native T. emersonii α-amylase encoding gene (temA) were selected for expression in two industrial S. cerevisiae 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 temG_Opt and temA genes into the yeasts' genomes. During the fermentation of 200 g l^-1 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 S. cerevisiae ER T12 and M2n T1 strains (containing integrated temA and temG_Opt gene cassettes) produced 89.35 and 98.13 g l^-1 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 S. cerevisiae strains. These recombinant strains represent potential drop-in CBP yeast substitutes for the existing conventional and raw starch fermentation processes.

Keywords: Amylases; Biofuels; Consolidated bioprocessing; Industrial yeast; Raw corn starch; Talaromyces emersonii.

Fig. 1

The amylolytic S. cerevisiae Y294 strains were evaluated at 30 °C under oxygen-limited conditions in 100 ml fermentation bottles containing 2 × SC^–URA broth supplemented with 5 g l⁻¹ glucose and 200 g l⁻¹ raw corn starch as carbohydrate sources. The a ethanol and b glucose production was monitored overtime. The S. cerevisiae Y294[TemG_Opt-TemA] strain was cultivated in a 2-l bioreactor, c ethanol and residual glucose concentrations at 26 and 30 °C, and d the percentage estimated carbon conversion at 26 and 30 °C. Values represent the mean of three repeats and error bars represent the standard deviation

Fig. 2

Construction of amylolytic S. cerevisiae M2n and ER industrial strains. The ENO1 temA and temG_Opt gene cassettes (a) were amplified using PCR and contained flanking regions homologous to the δ-integration sites. The TEF1_P–amdS-TEF1_T cassette was cloned onto yBBH1 (b) to generate the yBBH1–amdSYM yeast expression vector. Soluble starch plate assays to visualise hydrolysis zones surrounding the recombinant strains (c), following incubation on soluble starch at 30 °C. The S. cerevisiae M2n and ER parental strains displayed no extracellular amylase activity. Ethanol concentrations produced by S. cerevisiae recombinant strains during fermentation in YPD with 5 g l⁻¹ glucose and with 200 g l⁻¹ corn starch at 30 °C (d) were compared to the parental strains. Data are the mean of three repeats showing standard deviation

Fig. 3

S. cerevisiae ER and M2n strains during fermentation in 100 ml fermentation bottles with YPD containing 5 g l⁻¹ glucose and 200 g l⁻¹ corn starch. Ethanol concentrations produced by ER (a) and M2n strains (b) at 30 °C, as well as ER (c) and M2n strains (d) at 37 °C. Glucose concentrations in fermentation broth with ER (e) and M2n strains (f) at an incubation temperature of 37 °C. Selected GSHE dosages (µl) were used to supplement the ER T12 and M2n T1 CBP fermentations, as well as provide SSF conditions for the ER and M2n parental strains. Data are the mean of three repeats showing standard deviation