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A novel thermostable D-amino acid oxidase of the thermophilic fungus Rasamsonia emersonii strain YA.

ABSTRACT: D-Amino acid oxidase (DAAO) is a valuable flavoenzyme capable of being used in various practical applications, such as in determining D-amino acids and producing a material for semisynthetic cephalosporins, requiring higher thermal stability, higher catalytic activity, and broad substrate specificity. In this study, we isolated the thermophilic fungus Rasamsonia emersonii strain YA, which can grow on several D-amino acids as the sole nitrogen source, from a compost and characterized DAAO (ReDAAO) of the fungus. ReDAAO expressed in Escherichia coli exhibited significant oxidase activity against various neutral and basic D-amino acids, in particular hydrophobic D-amino acids. In addition, the enzyme also significantly acted on cephalosporin C, a starting material for semisynthetic antibiotics, and D-Glu, a general substrate for D-aspartate oxidase but not for DAAO, showing its unique and practically useful substrate specificity. The apparent kcat and Km values of the enzyme toward good substrates were comparable to those of higher catalytic fungal DAAOs, and the thermal stability (T50 value of ~60?°C) was comparable to that of a thermophilic bacterial DAAO and significantly higher than that of other eukaryotic DAAOs. These results highlight the great potential of ReDAAO for use in practical applications.

SUBMITTER: Shimekake Y

PROVIDER: S-EPMC6697736 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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A novel thermostable D-amino acid oxidase of the thermophilic fungus Rasamsonia emersonii strain YA.

Shimekake Yuya Y Furuichi Takehiro T Abe Katsumasa K Kera Yoshio Y Takahashi Shouji S

Scientific reports 20190816 1

D-Amino acid oxidase (DAAO) is a valuable flavoenzyme capable of being used in various practical applications, such as in determining D-amino acids and producing a material for semisynthetic cephalosporins, requiring higher thermal stability, higher catalytic activity, and broad substrate specificity. In this study, we isolated the thermophilic fungus Rasamsonia emersonii strain YA, which can grow on several D-amino acids as the sole nitrogen source, from a compost and characterized DAAO (ReDAAO ...[more]

PMID: 31420577

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Project description:BackgroundThe filamentous fungus Rasamsonia emersonii has immense potential to produce biorefinery relevant thermostable cellulase and hemicellulase enzymes using lignocellulosic biomass. Previously in our lab, a hyper-cellulase producing strain of R. emersonii was developed through classical breeding and system biology approaches. ACE1, a pivotal transcription factor in fungi, plays a crucial role in negatively regulating the expression of cellulase genes. In order to identify the role of ACE1 in cellulase production and to further improve the lignocellulolytic enzyme production in R. emersonii, CRISPR/Cas9 mediated disruption of ACE1 gene was employed.ResultsA gene-edited ∆ACE1 strain (GN11) was created, that showed 21.97, 20.70 and 24.63, 9.42, 18.12%, improved endoglucanase, cellobiohydrolase (CBHI), β-glucosidase, FPase, and xylanase, activities, respectively, as compared to parental strain M36. The transcriptional profiling showed that the expression of global regulator (XlnR) and different CAZymes genes including endoglucanases, cellobiohydrolase, β-xylosidase, xylanase, β-glucosidase and lytic polysaccharide mono-oxygenases (LPMOs) were significantly enhanced, suggesting critical roles of ACE1 in negatively regulating the expression of various key genes associated with cellulase production in R. emersonii. Whereas, the disruption of ACE1 significantly down-regulated the expression of CreA repressor gene as also evidenced by 2-deoxyglucose (2-DG) resistance phenotype exhibited by edited strain GN11 as well as appreciably higher constitutive production of cellulases in the presence of glucose and mixture of glucose and disaccharide (MGDs) both in batch and flask fed batch mode of culturing. Furthermore, ∆ACE1 strains were evaluated for the hydrolysis of biorefinery relevant steam/acid pretreated unwashed rice straw slurry (Praj Industries Ltd; 15% substrate loading rate) and were found to be significantly superior when compared to the benchmark enzymes produced by parent strain M36 and Cellic Ctec3.ConclusionsCurrent work uncovers the crucial role of ACE1 in regulating the expression of the various cellulase genes and carbon catabolite repression mechanism in R. emersonii. This study represents the first successful report of utilizing CRISPR/Cas9 genome editing technology to disrupt the ACE1 gene in the thermophlic fungus R. emersonii. The improved methodologies presented in this work might be applied to other commercially important fungal strains for which genetic manipulation tools are limited.

Dataset Information

A novel thermostable D-amino acid oxidase of the thermophilic fungus Rasamsonia emersonii strain YA.

Publications

A novel thermostable D-amino acid oxidase of the thermophilic fungus Rasamsonia emersonii strain YA.

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