Consolidated bioprocessing of starchy substrates into ethanol by industrial Saccharomyces cerevisiae strains secreting fungal amylases

Abstract

The development of a yeast strain that converts raw starch to ethanol in one step (called Consolidated Bioprocessing, CBP) could significantly reduce the commercial costs of starch-based bioethanol. An efficient amylolytic Saccharomyces cerevisiae strain suitable for industrial bioethanol production was developed in this study. Codon-optimized variants of the Thermomyces lanuginosus glucoamylase (TLG1) and Saccharomycopsis fibuligera α-amylase (SFA1) genes were δ-integrated into two S. cerevisiae yeast with promising industrial traits, i.e., strains M2n and MEL2. The recombinant M2n[TLG1-SFA1] and MEL2[TLG1-SFA1] yeast displayed high enzyme activities on soluble and raw starch (up to 8118 and 4461 nkat/g dry cell weight, respectively) and produced about 64 g/L ethanol from 200 g/L raw corn starch in a bioreactor, corresponding to 55% of the theoretical maximum ethanol yield (g of ethanol/g of available glucose equivalent). Their starch-to-ethanol conversion efficiencies were even higher on natural sorghum and triticale substrates (62 and 73% of the theoretical yield, respectively). This is the first report of direct ethanol production from natural starchy substrates (without any pre-treatment or commercial enzyme addition) using industrial yeast strains co-secreting both a glucoamylase and α-amylase.

Utilisation of wheat bran as a substrate for bioethanol production using recombinant cellulases and amylolytic yeast

“…The resulting ethanol yield per gram of consumed starch was higher than 85% and 81% for MEL2 [TLG1-SFA1] and M2n[TLG1-SFA1], respectively, with productivity values comparable for the engineered strains (Table 3). Their starch-to-ethanol conversion efficiencies were similar to those recently described for the same engineered strains from raw corn starch, sorghum and triticale [12]. SEM of wheat bran samples during the SSF confirmed the ability of the recombinant yeast to break down the starch granules, which were abundantly present at the beginning of the fermentation (Fig.…”

“…The wild type S. cerevisiae MEL2 and M2n, with their respective recombinant strains MEL2[TLG1-SFA1] and M2n[TLG1-SFA1], were utilised for wheat bran fermentation. The engineered strains contained the TLG1 gene (glucoamylase from T. lanuginosus) expressed under the control of the ENO1 promoter and the SFA1 gene (a-amylase from S. fibuligera) expressed under the control of the PGK1 promoter sequences [12]. Both genes were codon optimised for expression in S. cerevisiae and integrated into the delta sequences on the genomes of the industrial S. cerevisiae MEL2 and M2n strains [12].…”

“…Fermentation medium is similar to the cultivation medium, but contained 0.5 g/L glucose and 10% (w/v) unmilled wheat bran. The Aspergillus niger D15[EgA] strain was maintained on spore plates and Glucoamylase (TLG1) T. lanuginosus [12] a-Amylase (SFA1)…”

“…Favaro and colleagues described the direct ethanol production from natural starchy substrates (corn, sorghum and triticale), using industrial yeast strains co-secreting glucoamylase and a-amylase enzymes [12]. Yamada et al [13] achieved the CBP of brown rice by the amylolytic laboratory strain MNIV/dGS producing almost 80 g/L of alcohol from 200 g/L of brown rice after 120 h. Although the above reports pave the way for the industrial CBP of raw starch to ethanol, their focus was on substrates composed only of starch, meanwhile many industrial starch-rich by-products are available in great quantities with different compositions in terms of cellulose and hemicellulose.…”

Whole-genome de novo sequencing, combined with RNA-Seq analysis, reveals unique genome and physiological features of the amylolytic yeast Saccharomycopsis fibuligera and its interspecies hybrid

“…Although the production of lignocellulosic ethanol is expected to increase rapidly as advanced biofuel in the renewable fuel industry, starch is still the most commonly used feedstock for the production of conventional biofuel (http://www.afdc.energy.gov/laws/RFS). A recombinant yeast strain for the consolidated bioprocessing of starch biomass to ethanol, which can carry out raw starch hydrolysis and fermentation without any pretreatment of commercial enzyme addition, was recently developed by introducing the S. fibuligera α-amylase ( SFA1 ) and Thermomyces lanuginosus glucoamylase ( TLG1 ) genes into the industrial S. cerevisiae strains [27]. In addition to the bioconversion of starchy biomass to useful bioproducts, such as biofuels and trehalose, the high amylolytic activity of S. fibuligera can be usefully exploited in the production of other starch derivatives as well, including corn syrup, detergents, paper, textiles, and adhesive [28].…”