Dataset Information

Comparative proteomics of Rhizopus delemar ATCC 20344 unravels the role of amino acid catabolism in fumarate accumulation.

ABSTRACT: The filamentous fungus Rhizopus delemar naturally accumulates relatively high amounts of fumarate. Although the culture conditions that increase fumarate yields are well established, the network underlying the accumulation of fumarate is not yet fully understood. We set out to increase the knowledge about fumarate accumulation in R. delemar. To this end, we combined a transcriptomics and proteomics approach to identify key metabolic pathways involved in fumarate production in R. delemar, and propose that a substantial part of the fumarate accumulated in R. delemar during nitrogen starvation results from the urea cycle due to amino acid catabolism.

SUBMITTER: Odoni DI

PROVIDER: S-EPMC5376114 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Comparative proteomics of Rhizopus delemar ATCC 20344 unravels the role of amino acid catabolism in fumarate accumulation.

Odoni Dorett I DI Tamayo-Ramos Juan A JA Sloothaak Jasper J van Heck Ruben G A RGA Martins Dos Santos Vitor A P VAP de Graaff Leo H LH Suarez-Diez Maria M Schaap Peter J PJ

PeerJ 20170330

The filamentous fungus Rhizopus delemar naturally accumulates relatively high amounts of fumarate. Although the culture conditions that increase fumarate yields are well established, the network underlying the accumulation of fumarate is not yet fully understood. We set out to increase the knowledge about fumarate accumulation in R. delemar. To this end, we combined a transcriptomics and proteomics approach to identify key metabolic pathways involved in fumarate production in R. ...[more]

PMID: 28382234

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Project description:Rhizopus delemar causes devastating mucormycosis in immunodeficient individuals. Despite its medical importance, R. delemar remains understudied largely due to the lack of available genetic markers, the presence of multiple gene copies due to genome duplication, and mitotically unstable transformants resulting from conventional and limited genetic approaches. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) system induces efficient homologous and non-homologous break points and generates individual and multiple mutant alleles without requiring selective marker genes in a wide variety of organisms including fungi. Here, we have successfully adapted this technology for inducing gene-specific single nucleotide (nt) deletions in two clinical strains of R. delemar: FGSC-9543 and CDC-8219. For comparative reasons, we first screened for spontaneous uracil auxotrophic mutants resistant to 5-fluoroorotic acid (5-FOA) and obtained one substitution (f1) mutationin the FGSC-9543 strain and one deletion (f2) mutation in the CDC-8219 strain. The f2 mutant was then successfully complemented with a pyrF-dpl200 marker gene. We then introduced a vector pmCas9:tRNA-gRNA that expresses both Cas9 endonuclease and pyrF-specific gRNA into FGSC-9543 and CDC-8219 strains and obtained 34 and 42 5-FOA resistant isolates, respectively. Candidate transformants were successively transferred eight times by propagating hyphal tips prior to genotype characterization. Sequencing of the amplified pyrF allele in all transformants tested revealed a single nucleotide (nt) deletion at the 4th nucleotide before the protospacer adjacent motif (PAM) sequence, which is consistent with CRISPR-Cas9 induced gene mutation through non-homologous end joining (NHEJ). Our study provides a new research tool for investigating molecular pathogenesis mechanisms of R. delemar while also highlighting the utilization of CRISPR-Cas9 technology for generating specific mutants of Mucorales fungi.

Dataset Information

Comparative proteomics of Rhizopus delemar ATCC 20344 unravels the role of amino acid catabolism in fumarate accumulation.

Publications

Comparative proteomics of <i>Rhizopus delemar</i> ATCC 20344 unravels the role of amino acid catabolism in fumarate accumulation.

Similar Datasets

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