High level secretion of cellobiohydrolases by Saccharomyces cerevisiae

Abstract

BackgroundThe main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP). Development of Saccharomyces cerevisiae for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases.ResultsWe expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by S. cerevisiae. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of cbh1 and cbh2 genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel™) to ethanol by CBH-producing S. cerevisiae strains with the addition of beta-glucosidase.ConclusionsGene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome.

Cellulose degradation and assimilation by the unicellular phototrophic eukaryote Chlamydomonas reinhardtii

“…It also provides an explanation why C. reinhardtii is able to occupy so many different habitats in nature. Most importantly, the ability of this microalga for substrate-mediated active secretion of endogenous cellulases fuels into recent biotechnologically driven efforts to engineer bioprocessing microbes such as S. cerevisiae and E. coli exactly with this phenotype to support cellulose digestion as a precondition for efficient production of biofuels 46,47 . Our new findings presented here will open up the opportunity of using a phototrophic microorganism for providing cellulases via secretion in suitable applications such as the sunlight-driven conversion of cellulosic paper waste material to biomass.…”

Advances in consolidated bioprocessing systems for bioethanol and butanol production from biomass: a comprehensive review

“…Thus, heterologous expression of these enzymes in the ethanologenic hosts would enhance efficient conversion of cellulose to ethanol (den Haan et al, 2013). Recently, relatively high levels of CBH1 (0.3 g/l) and CBH2 (1 g/l) production in S. cerevisiae have been reported (McBride et al, 2010;Ilmen et al, 2011). Ilmen et al (2011) developed a recombinant strain with a high ability to convert most of the glucan available in paper sludge to ethanol and displace about 60% of the enzymes usually required.…”

“…Recently, relatively high levels of CBH1 (0.3 g/l) and CBH2 (1 g/l) production in S. cerevisiae have been reported (McBride et al, 2010;Ilmen et al, 2011). Ilmen et al (2011) developed a recombinant strain with a high ability to convert most of the glucan available in paper sludge to ethanol and displace about 60% of the enzymes usually required. Furthermore, McBride et al (2010) developed a recombinant strain expressing three cellulases with high abilities to convert pre-treated corn stover to 2.6% (v/v) ethanol within 96 h (63% of the theoretical value) in one step without the addition of exogenous enzymes.…”

Fruit waste streams in South Africa and their potential role in developing a bio-economy

“…Mascoma is focusing on hardwoods and pulps as feedstocks. 107 In South Africa, there have been several breakthroughs in expressing cellulases in the yeast S. cerevisiae [108][109][110][111][112][113] and in the development of a consolidated bioprocessing yeast strain capable of converting pre-treated hardwood to ethanol with significantly reduced enzyme addition 114 . Pretreatment of different agricultural residues, with the aid of a 15-L reactor steam gun, has been evaluated with South African sponsored research funding in anticipation of an emerging cellulosic ethanol industry.…”