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Sexual crossing of thermophilic fungus Myceliophthora heterothallica improved enzymatic degradation of sugar beet pulp.

ABSTRACT: BACKGROUND:Enzymatic degradation of plant biomass requires a complex mixture of many different enzymes. Like most fungi, thermophilic Myceliophthora species therefore have a large set of enzymes targeting different linkages in plant polysaccharides. The majority of these enzymes have not been functionally characterized, and their role in plant biomass degradation is unknown. The biotechnological challenge is to select the right set of enzymes to efficiently degrade a particular biomass. This study describes a strategy using sexual crossing and screening with the thermophilic fungus Myceliophthora heterothallica to identify specific enzymes associated with improved sugar beet pulp saccharification. RESULTS:Two genetically diverse M. heterothallica strains CBS 203.75 and CBS 663.74 were used to generate progenies with improved growth on sugar beet pulp. One progeny, named SBP.F1.2.11, had a different genetic pattern from the parental strains and had improved saccharification activity after the growth on 3 % sugar beet pulp. The improved SBP saccharification was not explained by altered activities of the major (hemi-)cellulases. Exo-proteome analysis of progeny and parental strains after 7-day growth on sugar beet pulp showed that only 17 of the 133 secreted CAZy enzymes were more abundant in progeny SBP.F1.2.11. Particularly one enzyme belonging to the carbohydrate esterase family 5 (CE5) was more abundant in SBP.F1.2.11. This CE5-CBM1 enzyme, named as Axe1, was phylogenetically related to acetyl xylan esterases. Biochemical characterization of Axe1 confirmed de-acetylation activity with optimal activities at 75-85 °C and pH 5.5-6.0. Supplementing Axe1 to CBS 203.75 enzyme set improved release of xylose and glucose from sugar beet pulp. CONCLUSIONS:This study identified beneficial enzymes for sugar beet pulp saccharification by selecting progeny with improved growth on this particular substrate. Saccharification of sugar beet pulp was improved by supplementing enzyme mixtures with a previously uncharacterized CE5-CBM1 acetyl xylan esterase. This shows that sexual crossing and selection of M. heterothallica are the successful strategy to improve the composition of enzyme mixtures for efficient plant biomass degradation.

SUBMITTER: Aguilar-Pontes MV

PROVIDER: S-EPMC4761134 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Sexual crossing of thermophilic fungus Myceliophthora heterothallica improved enzymatic degradation of sugar beet pulp.

Aguilar-Pontes Maria Victoria MV Zhou Miaomiao M van der Horst Sjors S Theelen Bart B de Vries Ronald P RP van den Brink Joost J

Biotechnology for biofuels 20160220

<h4>Background</h4>Enzymatic degradation of plant biomass requires a complex mixture of many different enzymes. Like most fungi, thermophilic Myceliophthora species therefore have a large set of enzymes targeting different linkages in plant polysaccharides. The majority of these enzymes have not been functionally characterized, and their role in plant biomass degradation is unknown. The biotechnological challenge is to select the right set of enzymes to efficiently degrade a particular biomass. ...[more]

PMID: 26900400

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Project description:Background:Fumaric acid is widely used in food and pharmaceutical industries and is recognized as a versatile industrial chemical feedstock. Increasing concerns about energy and environmental problems have resulted in a focus on fumaric acid production by microbial fermentation via bioconversion of renewable feedstocks. Filamentous fungi are the predominant microorganisms used to produce organic acids, including fumaric acid, and most studies to date have focused on Rhizopus species. Thermophilic filamentous fungi have many advantages for the production of compounds by industrial fermentation. However, no previous studies have focused on fumaric acid production by thermophilic fungi. Results:We explored the feasibility of producing fumarate by metabolically engineering Myceliophthora thermophila using the CRISPR/Cas9 system. Screening of fumarases suggested that the fumarase from Candida krusei was the most suitable for efficient production of fumaric acid in M. thermophila. Introducing the C. krusei fumarase into M. thermophila increased the titer of fumaric acid by threefold. To further increase fumarate production, the intracellular fumarate digestion pathway was disrupted. After deletion of the two fumarate reductase and the mitochondrial fumarase genes of M. thermophila, the resulting strain exhibited a 2.33-fold increase in fumarate titer. Increasing the pool size of malate, the precursor of fumaric acid, significantly increased the final fumaric acid titer. Finally, disruption of the malate-aspartate shuttle increased the intracellular malate content by 2.16-fold and extracellular fumaric acid titer by 42%, compared with that of the parental strain. The strategic metabolic engineering of multiple genes resulted in a final strain that could produce up to 17 g/L fumaric acid from glucose in a fed-batch fermentation process. Conclusions:This is the first metabolic engineering study on the production of fumaric acid by the thermophilic filamentous fungus M. thermophila. This cellulolytic fungal platform provides a promising method for the sustainable and efficient-cost production of fumaric acid from lignocellulose-derived carbon sources in the future.

Project description:Background:Cellulosic biomass is a promising resource for bioethanol production. However, various sugars in plant biomass hydrolysates including cellodextrins, cellobiose, glucose, xylose, and arabinose, are poorly fermented by microbes. The commonly used ethanol-producing microbe Saccharomyces cerevisiae can usually only utilize glucose, although metabolically engineered strains that utilize xylose have been developed. Direct fermentation of cellobiose could avoid glucose repression during biomass fermentation, but applications of an engineered cellobiose-utilizing S. cerevisiae are still limited because of its long lag phase. Bioethanol production from biomass-derived sugars by a cellulolytic filamentous fungus would have many advantages for the biorefinery industry. Results:We selected Myceliophthora thermophila, a cellulolytic thermophilic filamentous fungus for metabolic engineering to produce ethanol from glucose and cellobiose. Ethanol production was increased by 57% from glucose but not cellobiose after introduction of ScADH1 into the wild-type (WT) strain. Further overexpression of a glucose transporter GLT-1 or the cellodextrin transport system (CDT-1/CDT-2) from N. crassa increased ethanol production by 131% from glucose or by 200% from cellobiose, respectively. Transcriptomic analysis of the engineered cellobiose-utilizing strain and WT when grown on cellobiose showed that genes involved in oxidation-reduction reactions and the stress response were downregulated, whereas those involved in protein biosynthesis were upregulated in this effective ethanol production strain. Turning down the expression of pyc gene results the final engineered strain with the ethanol production was further increased by 23%, reaching up to 11.3 g/L on cellobiose. Conclusions:This is the first attempt to engineer the cellulolytic fungus M. thermophila to produce bioethanol from biomass-derived sugars such as glucose and cellobiose. The ethanol production can be improved about 4 times up to 11 grams per liter on cellobiose after a couple of genetic engineering. These results show that M. thermophila is a promising platform for bioethanol production from cellulosic materials in the future.

Dataset Information

Sexual crossing of thermophilic fungus Myceliophthora heterothallica improved enzymatic degradation of sugar beet pulp.

Publications

Sexual crossing of thermophilic fungus Myceliophthora heterothallica improved enzymatic degradation of sugar beet pulp.

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