Raw starch conversion by Saccharomyces cerevisiae expressing Aspergillus tubingensis amylases

Abstract

BackgroundStarch is one of the most abundant organic polysaccharides available for the production of bio-ethanol as an alternative transport fuel. Cost-effective utilisation of starch requires consolidated bioprocessing (CBP) where a single microorganism can produce the enzymes required for hydrolysis of starch, and also convert the glucose monomers to ethanol.ResultsThe Aspergillus tubingensis T8.4 α-amylase (amyA) and glucoamylase (glaA) genes were cloned and expressed in the laboratory strain Saccharomyces cerevisiae Y294 and the semi-industrial strain, S. cerevisiae Mnuα1. The recombinant AmyA and GlaA displayed protein sizes of 110–150 kDa and 90 kDa, respectively, suggesting significant glycosylation in S. cerevisiae. The Mnuα1[AmyA-GlaA] and Y294[AmyA-GlaA] strains were able to utilise 20 g l-1 raw corn starch as sole carbohydrate source, with ethanol titers of 9.03 and 6.67 g l-1 (0.038 and 0.028 g l-1 h-1), respectively, after 10 days. With a substrate load of 200 g l-1 raw corn starch, Mnuα1[AmyA-GlaA] yielded 70.07 g l-1 ethanol (0.58 g l-1 h-1) after 120 h of fermentation, whereas Y294[AmyA-GlaA] was less efficient at 43.33 g l-1 ethanol (0.36 g l-1 h-1).ConclusionsIn a semi-industrial amylolytic S. cerevisiae strain expressing the A. tubingensis α-amylase and glucoamylase genes, 200 g l-1 raw starch was completely hydrolysed (saccharified) in 120 hours with 74% converted to released sugars plus fermentation products and the remainder presumably to biomass. The single-step conversion of raw starch represents significant progress towards the realisation of CBP without the need for any heat pretreatment. Furthermore, the amylases were produced and secreted by the host strain, thus circumventing the need for exogenous amylases.

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

“…The development of a CBP yeast towards the starch-to-ethanol route requires robust strains to be engineered for the production of raw starch hydrolysing enzymes in adequate quantities. The S. cerevisiae MEL2 and M2n strains that displayed promising industrial fitness (Favaro et al, 2013b, Viktor et al, 2013 were therefore chosen as hosts for the production of the recombinant enzymes. Since codon optimization can significantly improve gene expression levels and the subsequent functionality of the enzymes (Favaro et al, 2012b), the TLG1 (T. lanuginosus glucoamylase) and SFA1 (S. fibuligera a-amylase) genes were codon-optimized for expression in S. cerevisiae.…”

“…Efficient raw starch degrading enzymes (RSDE) can significantly reduce the energy requirements and simplify the production of starch-based biofuels (Robertson et al, 2006). However, a limited number of RSDE have been cloned and characterized, e.g., a-amylases from Lipomyces kononenkoae (Eksteen et al, 2004;Knox et al, 2004;Ramachandran et al, 2008), Streptomyces bovis (Yamada et al, 2010a), Cryptococcus and Bacillus (Gupta et al, 2003;Sun et al, 2010), and glucoamylases from Rhizopus oryzae (Yamada et al, 2010a), Corticium rolfsii, Saccharomycopsis fibuligera (Eksteen et al, 2004;Sun et al, 2010), Aspergillus awamori (Favaro et al, 2012b), and Aspergillus tubingensis (Viktor et al, 2013). Cost-effective conversion of raw starch to biofuels requires the production of starch-hydrolysing enzymes by a fermenting yeast to achieve liquefaction, hydrolysis, and fermentation (Consolidated Bioprocessing, CBP) in a single organism.…”

“…A polyploid S. cerevisiae strain, secreting both the Aspergillus awamori GA1 and Debaryomyces occidentalis AMY, converted 80% of 200 g/L raw starch with 80 g/L ethanol produced after 6 days, equating to 0.56 g/L/h (Kim et al, 2011). Similarly, Viktor et al (2013) reported that the semi-industrial S. cerevisiae Mnua1 strain, expressing the A. tubingensis a-amylase and glucoamylase genes, completely hydrolysed 200 g/L raw corn starch within 5 days, producing 70 g/L ethanol (0.58 g/L/h). Both recombinant strains were only evaluated on small-scale, whereas bioreactor experiments are essential to proof the concept of raw starch CBP.…”

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

“…fibuligera a All enzymes were secreted using their native secretion signal, with the exception of Pcbgl1B (using the T. reesei Xyn2 secretion signal). b RCC (recombinant cellulase cocktail) [31]. c EgA was expressed using the native DNA sequence, whereas all other genes were codon optimised for expression in S. cerevisiae.…”

Characterization of the starch degradation activity from newly isolated Rhizopus oryzae WCS-1 and mixed cultures with Saccharomyces cerevisiae for efficient ethanol production from starch

“…Normally, the glucoamylases from R. oryzae and other fungal strains exhibit an acidic optimum pH of 4.0-5.0 for starch degradation activity (23)(24)(25). Thermo and acido-stable properties of glucoamylase from R. oryzae and other fungal strains are suitable for biofuel production from starch (26)(27)(28)(29). In addition, partially purified enzymes of R. oryzae WCS-1 showed high thermo-stable starch degradation activities (Fig.…”