Dataset Information

Enhancing cellulase and hemicellulase production by genetic modification of the carbon catabolite repressor gene, creA, in Acremonium cellulolyticus.

ABSTRACT: Acremonium cellulolyticus is one of several fungi that offer promise as an alternative to Trichoderma reesei for use in industrial cellulase production. However, the mechanism of cellulase production has not been studied at the molecular level because adequate genetic engineering tools for use in A. cellulolyticus are lacking. In the present study, we developed a gene disruption method for A. cellulolyticus, which needs a longer homologous region length. We cloned a putative A. cellulolyticus creA gene that is highly similar to the creA genes derived from other filamentous fungi, and isolated a creA disruptant strain by using the disruption method. Growth of the creA disruptant on agar plates was slower than that of the control strain. In the wild-type strain, the CreA protein was localized in the nucleus, suggesting that the cloned gene encodes the CreA transcription factor. Cellulase and xylanase production by the creA disruptant were higher than that of the control strain at the enzyme and transcription levels. Furthermore, the creA disruptant produced cellulase and xylanase in the presence of glucose. These data suggest both that the CreA protein functions as a catabolite repressor protein, and that disruption of creA is effective for enhancing enzyme production by A. cellulolyticus.

SUBMITTER: Fujii T

PROVIDER: S-EPMC3917621 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Enhancing cellulase and hemicellulase production by genetic modification of the carbon catabolite repressor gene, creA, in Acremonium cellulolyticus.

Fujii Tatsuya T Inoue Hiroyuki H Ishikawa Kazuhiko K

AMB Express 20131220 1

Acremonium cellulolyticus is one of several fungi that offer promise as an alternative to Trichoderma reesei for use in industrial cellulase production. However, the mechanism of cellulase production has not been studied at the molecular level because adequate genetic engineering tools for use in A. cellulolyticus are lacking. In the present study, we developed a gene disruption method for A. cellulolyticus, which needs a longer homologous region length. We cloned a putative A. cellulolyticus cr ...[more]

PMID: 24360128

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Project description:Filamentous fungi of the genus Aspergillus are of particular interest for biotechnological applications due to their natural capacity to secrete carbohydrate-active enzymes (CAZy) that target plant biomass. The presence of easily metabolizable sugars such as glucose, whose concentrations increase during plant biomass hydrolysis, results in the repression of CAZy-encoding genes in a process known as carbon catabolite repression (CCR), which is undesired for the purpose of large-scale enzyme production. To date, the C2H2 transcription factor CreA has been described as the major CC repressor in Aspergillus spp., although little is known about the role of posttranslational modifications in this process. In this work, phosphorylation sites were identified by mass spectrometry on Aspergillus nidulans CreA, and subsequently, the previously identified but uncharacterized site S262, the characterized site S319, and the newly identified sites S268 and T308 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was investigated. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 was not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. All sites were shown to be important for glycogen and trehalose metabolism. This study highlights the importance of CreA phosphorylation sites for the regulation of CCR. These sites are interesting targets for biotechnological strain engineering without the need to delete essential genes, which could result in undesired side effects.IMPORTANCE In filamentous fungi, the transcription factor CreA controls carbohydrate metabolism through the regulation of genes encoding enzymes required for the use of alternative carbon sources. In this work, phosphorylation sites were identified on Aspergillus nidulans CreA, and subsequently, the two newly identified sites S268 and T308, the previously identified but uncharacterized site S262, and the previously characterized site S319 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was characterized. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 is not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. This work characterized novel CreA phosphorylation sites under carbon catabolite-repressing conditions and showed that they are crucial for CreA protein turnover, control of carbohydrate utilization, and biotechnologically relevant enzyme production.

Dataset Information

Enhancing cellulase and hemicellulase production by genetic modification of the carbon catabolite repressor gene, creA, in Acremonium cellulolyticus.

Publications

Enhancing cellulase and hemicellulase production by genetic modification of the carbon catabolite repressor gene, creA, in Acremonium cellulolyticus.

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