Green and sustainable manufacture of chemicals from biomass: state of the art

http://kfrserver.natur.cuni.cz/studium/prednasky/vyberclanku/pdf/p68_ucit/7_LIMAYEM.pdf

Lignocellulosic biomass for bioethanol production: current perspectives, potential issues and future prospects.

Global climate change linked to the accumulation of greenhouse gases has caused concerns regarding the use of fossil fuels as the major energy source. To mitigate climate change while keeping energy supply sustainable, one solution is to rely on the ability of microorganisms to use renewable resources for biofuel synthesis. In this Review, we discuss how microorganisms can be explored for the production of next-generation biofuels, based on the ability of bacteria and fungi to use lignocellulose; through direct CO2 conversion by microalgae; using lithoautotrophs driven by solar electricity; or through the capacity of microorganisms to use methane generated from landfill. Furthermore, we discuss how to direct these substrates to the biosynthetic pathways of various fuel compounds and how to optimize biofuel production by engineering fuel pathways and central metabolism.

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Fuelling the future: microbial engineering for the production of sustainable biofuels

More than 70 years ago, the filamentous ascomycete Trichoderma reesei was isolated on the Solomon Islands due to its ability to degrade and thrive on cellulose containing fabrics. This trait that relies on its secreted cellulases is nowadays exploited by several industries. Most prominently in biorefineries which use T. reesei enzymes to saccharify lignocellulose from renewable plant biomass in order to produce biobased fuels and chemicals. In this review we summarize important milestones of the development of T. reesei as the leading production host for biorefinery enzymes, and discuss emerging trends in strain engineering. Trichoderma reesei has very recently also been proposed as a consolidated bioprocessing organism capable of direct conversion of biopolymeric substrates to desired products. We therefore cover this topic by reviewing novel approaches in metabolic engineering of T. reesei.

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Cellulases and beyond: the first 70 years of the enzyme producer Trichoderma reesei.

Depolymerization of cellulose to glucose requires the synergistic action of three key cellulases, endoglucanase (E.C. 3.2.1.4), exoglucanase (E.C. 3.2.1.176) (E.C. 3.2.1.91) and β-glucosidase (E.C. 3.2.1.21). They belong to the glycoside hydrolase (GH) family and catalyze the hydrolysis of glyosidic linkages depolymerizing cellulose to fermentable sugars. Cellulases are naturally produced by a wide spectrum of bacteria and fungi. These enzymes usually exist as cellulosomes attached to the cell wall of bacteria but are secreted into environments in fungi. They exist either as monomers or multimers with each monomer having a simple architecture containing a cellulose binding domain (CBD) and a catalytic domain (CD) interlinked by a linker peptide. Thermophilic, mesophilic and psychrophilic cellulases are quite different in their structures and amino acid compositions. Post-translational modifications such as glycosylation contribute to enzyme function, multiplicity and stability. Recent advances in recombinant DNA technology allow fast identification of novel cellulase genes, large scale production of cellulases and their genetic modifications to make tailor-made enzymes for various applications. Cellulases have been traditionally used in food processing and textile industries. The rapid depletion of fossil fuels and production of cheap fermentable sugars from abundant renewable resources have increased the demand for cellulases in lignocellulose-based biorefinery. However, the lack of a better understanding of the mechanisms of individual cellulases, their synergistic actions and their high prices are the major bottlenecks yet to be overcome for large scale commercial applications of cellulases in lignocellulose-based biorefinery.

Microbial cellulases: Engineering, production and applications

The filamentous fungus T. reesei effectively degrades cellulose and is known to produce various cellulolytic enzymes such as β-glucosidase, endoglucanase, and cellobiohydrolase. The expression levels of each cellulase are controlled simultaneously, and their ratios and synergetic effects are important for effective cellulose degradation. However, in recombinant Saccharomyces cerevisiae, it is difficult to simultaneously control many different enzymes. To construct engineered yeast with efficient cellulose degradation, we developed a simple method to optimize cellulase expression levels, named cocktail δ-integration. In cocktail δ-integration, several kinds of cellulase expression cassettes are integrated into yeast chromosomes simultaneously in one step, and strains with high cellulolytic activity (i.e., expressing an optimum ratio of cellulases) are easily obtained. Although the total integrated gene copy numbers of cocktail δ-integrant strain was about half that of a conventional δ-integrant strain, the phosphoric acid swollen cellulose (PASC) degradation activity (64.9 mU/g-wet cell) was higher than that of a conventional strain (57.6 mU/g-wet cell). This suggests that optimization of the cellulase expression ratio improves PASC degradation activity more so than overexpression. To our knowledge, this is the first report on the expression of cellulase genes by δ-integration and optimization of various foreign genes by δ-integration in yeast. This method should be very effective and easily applied for other multi-enzymatic systems using recombinant yeast.

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Cocktail δ-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains

Background: Hydrolysis of cellulose requires the action of the cellulolytic enzymes endoglucanase, cellobiohydrolase and b-glucosidase. The expression ratios and synergetic effects of these enzymes significantly influence the extent and specific rate of cellulose degradation. In this study, using our previously developed method to optimize cellulase-expression levels in yeast, we constructed a diploid Saccharomyces cerevisiae strain optimized for expression of cellulolytic enzymes, and attempted to improve the cellulose-degradation activity and enable direct ethanol production from rice straw, one of the most abundant sources of lignocellulosic biomass. Results: The engineered diploid strain, which contained multiple copies of three cellulase genes integrated into its genome, was precultured in molasses medium (381.4 mU/g wet cell), and displayed approximately six-fold higher phosphoric acid swollen cellulose (PASC) degradation activity than the parent haploid strain (63.5 mU/g wet cell). When used to ferment PASC, the diploid strain produced 7.6 g/l ethanol in 72 hours, with an ethanol yield that achieved 75% of the theoretical value, and also produced 7.5 g/l ethanol from pretreated rice straw in 72 hours. Conclusions: We have developed diploid yeast strain optimized for expression of cellulolytic enzymes, which is capable of directly fermenting from cellulosic materials. Although this is a proof-of-concept study, it is to our knowledge, the first report of ethanol production from agricultural waste biomass using cellulolytic enzymeexpressing yeast without the addition of exogenous enzymes. Our results suggest that combining multigene expression optimization and diploidization in yeast is a promising approach for enhancing ethanol production from various types of lignocellulosic biomass.

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Naho Taniguchi

Papers

Cocktail δ-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains

Direct ethanol production from cellulosic materials using a diploid strain of Saccharomyces cerevisiae with optimized cellulase expression.

Direct bioethanol production from cellulose by the combination of cellulase-displaying yeast and ionic liquid pretreatment