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PFK2/FBPase-2 is a potential target for metabolic engineering in the filamentous fungus Myceliophthora thermophila

ABSTRACT: The PFK-2/ FBPase-2 are important regulatory enzymes in the glycolytic and are present in all major eukaryotic taxa. However, unlike the well-studied animal and plant PFK-2/ FBPase-2, their functions in filamentous fungi remain little investigated. As a typical filamentous fungus and industrial microorganisms, M. thermophila was choose to investigate the functions of this gene. At the same time, the application of regulating this gene in improving metabolites production was also studied.

ORGANISM(S): Thermothelomyces thermophilus ATCC 42464

PROVIDER: GSE214142 | GEO | 2022/09/28

REPOSITORIES: GEO

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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.

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PFK2/FBPase-2 is a potential target for metabolic engineering in the filamentous fungus Myceliophthora thermophila

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