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Deletion of the middle region of the transcription factor ClrB in Penicillium oxalicum enables cellulase production in the presence of glucose.

ABSTRACT: Enzymes that degrade lignocellulose to simple sugars are of great interest in research and for biotechnology because of their role in converting plant biomass to fuels and chemicals. The synthesis of cellulolytic enzymes in filamentous fungi is tightly regulated at the transcriptional level, with the transcriptional activator ClrB/CLR-2 playing a critical role in many species. In Penicillium oxalicum, clrB overexpression could not relieve the dependence of cellulase expression on cellulose as an inducer, suggesting that clrB is controlled post-transcriptionally. In this study, using a reporter gene system in yeast, we identified the C-terminal region of ClrB/CLR-2 as a transcriptional activation domain. Expression of clrB^ID , encoding a ClrB derivative in which the DNA-binding and transcriptional activation domains are fused together to remove the middle region, led to cellulase production in the absence of cellulose in P. oxalicum Strikingly, the clrB^ID -expressing strain produced cellulase on carbon sources that normally repress cellulase expression, including glucose and glycerol. Results from deletion of the carbon catabolite repressor gene creA in the clrB^ID -expressing strain suggested that the effect of clrB^ID is independent of CreA's repressive function. A similar modification of clrB in Aspergillus niger resulted in the production of a mannanase in glucose medium. Taken together, these results indicate that ClrB suppression under noninducing conditions involves its middle region, suggesting a potential strategy to engineer fungal strains for improved cellulase production on commonly used carbon sources.

SUBMITTER: Gao L

PROVIDER: S-EPMC6901314 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Deletion of the middle region of the transcription factor ClrB in Penicillium oxalicum enables cellulase production in the presence of glucose.

Gao Liwei L Xu Yanning Y Song Xin X Li Shiying S Xia Chengqiang C Xu Jiadi J Qin Yuqi Y Liu Guodong G Qu Yinbo Y

The Journal of biological chemistry 20191028 49

Enzymes that degrade lignocellulose to simple sugars are of great interest in research and for biotechnology because of their role in converting plant biomass to fuels and chemicals. The synthesis of cellulolytic enzymes in filamentous fungi is tightly regulated at the transcriptional level, with the transcriptional activator ClrB/CLR-2 playing a critical role in many species. In Penicillium oxalicum, clrB overexpression could not relieve the dependence of cellulase expression on c ...[more]

PMID: 31659120

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Project description:BackgroundIn cellulolytic fungi, induction and repression mechanisms synchronously regulate the synthesis of cellulolytic enzymes for accurate responses to carbon sources in the environment. Many proteins, particularly transcription regulatory factors involved in these processes, were identified and genetically engineered in Penicillium oxalicum and other cellulolytic fungi. Despite such great efforts, its effect of modifying a single target to improve the production of cellulase is highly limited.ResultsIn this study, we developed a systematic strategy for the genetic engineering of P. oxalicum to enhance cellulase yields, by enhancing induction (by blocking intracellular inducer hydrolysis and increasing the activator level) and relieving the repression. We obtained a trigenic recombinant strain named 'RE-10' by deleting bgl2 and creA, along with over-expressing the gene clrB. The cellulolytic ability of RE-10 was significantly improved; the filter paper activity and extracellular protein concentration increased by up to over 20- and 10-fold, respectively, higher than those of the wild-type (WT) strain 114-2 both on pure cellulose and complex wheat bran media. Most strikingly, the cellulolytic ability of RE-10 was comparable with that of the industrial P. oxalicum strain JU-A10-T obtained by random mutagenesis. Comparative proteomics analysis provided further insights into the differential secretomes between RE-10 and WT strains. In particular, the enzymes and accessory proteins involved in lignocellulose degradation were elevated specifically and dramatically in the recombinant, thereby confirming the importance of them in biomass deconstruction and implying a possible co-regulatory mechanism.ConclusionsWe established a novel route to substantially improve cellulolytic enzyme production up to the industrial level in P. oxalicum by combinational manipulation of three key genes to amplify the induction along with derepression, representing a milestone in strain engineering of filamentous fungi. Given the conservation in the mode of cellulose expression regulation among filamentous fungi, this strategy could be compatible with other cellulase-producing fungi.

Project description:Cellulolytic enzyme hydrolysis of lignocellulose biomass to release fermentable sugars is one of the key steps in biofuel refining. Gene expression of fungal cellulolytic enzymes is tightly controlled at the transcriptional level. Key transcription factors such as activator ClrB/CLR2 and XlnR/XYR1, as well as repressor CreA/CRE1 play crucial roles in this process. The putative protein methyltransferase LaeA/LAE1 has also been reported to regulate the gene expression of the cellulolytic enzyme. The formation and gene expression of the cellulolytic enzyme was compared among Penicillium oxalicum wild type (WT) and seven mutants, including ?laeA (deletion of laeA), OEclrB (clrB overexpression), OEclrB?laeA (clrB overexpression with deletion of laeA), OExlnR (xlnR overexpression), OExlnR?laeA (xlnR overexpression with deletion of laeA), ?creA (deletion of creA), and ?creA?laeA (double deletion of creA and laeA). Results revealed that LaeA extensively affected the expression of glycoside hydrolase genes. The expression of genes that encoded the top 10 glycoside hydrolases assayed in secretome was remarkably downregulated especially in later phases of prolonged batch cultures by the deletion of laeA. Cellulase synthesis of four mutants ?laeA, OEclrB?laeA, OExlnR?laeA, and ?creA?laeA was repressed remarkably compared with their parent strains WT, OEclrB, OExlnR, and ?creA, respectively. The overexpression of clrB or xlnR could not rescue the impairment of cellulolytic enzyme gene expression and cellulase synthesis when LaeA was absent, suggesting that LaeA was necessary for the expression of cellulolytic enzyme gene activated by ClrB or XlnR. In contrast to LaeA positive roles in regulating prominent cellulase and hemicellulase, the extracellular ?-xylosidase formation was negatively regulated by LaeA. The extracellular ?-xylosidase activities improved over 5-fold in the OExlnR?laeA mutant compared with that of WT, and the expression of prominent ?-xylosidase gene xyl3A was activated remarkably. The cumulative effect of LaeA and transcription factor XlnR has potential applications in the production of more ?-xylosidase.

Project description:Background:The transition to a more environmentally friendly economy has prompted studies of modern biorefineries, including the utilization of low-value lignocellulose. The major challenge facing the widespread application of biorefineries is the high cost of enzymes that can efficiently hydrolyze recalcitrant cellulose to sugars. Penicillium oxalicum produces large amounts of plant-cell-wall-degrading enzymes, but their production is tightly controlled by complex regulatory networks, resulting in low yields of the native enzymes. Regulatory genes have been the targets of genetic engineering to improve enzyme production in microorganisms. In this study, we used transcriptomic profiling and genetic analyses to screen for and identify novel key regulators of cellulase and xylanase gene expression in P. oxalicum. Results:A comparative analysis of the transcriptomes of P. oxalicum HP7-1 on different carbon sources, including glucose, wheat bran, and wheat bran plus Avicel, identified 40 candidate genes regulating the expression of cellulolytic enzyme genes. Deletion mutants of 31 candidate genes were constructed in P. oxalicum ∆PoxKu70 and 11 resultant mutants showed significant changes in their filter-paper cellulase production compared with the parental strain ∆PoxKu70. Among these 11 mutants, ΔPoxCxrA, ΔPoxCxrB, and ΔPoxNsdD showed the most significant reduction in the enzyme production (96.8, 75.9, and 58.5%, respectively). Ten of these 11 genes are here reported to be involved in cellulase production for the first time. Further tests revealed that ΔPoxCxrA, ΔPoxCxrB, and ΔPoxNsdD displayed significantly reduced xylanase production, whereas ΔPoxCxrA produced negligible xylanase. Interestingly, ΔPoxCxrB and ΔPoxNsdD showed significantly increased β-glucosidase production. Real-time quantitative reverse transcription-PCR and an electrophoretic mobility shift assay (EMSA) showed that PoxCxrA, PoxCxrB, and PoxNsdD regulate the expression of one another, but the mode of regulation changes dynamically during the growth of fungal cells in the presence of cellulose. EMSA showed that PoxCxrA, PoxCxrB, and PoxNsdD directly bind the putative promoters of major cellulase and xylanase genes. Conclusions:We have detected and identified three key new regulatory genes, PoxCxrA, PoxCxrB, and PoxNsdD, that directly and indirectly regulate the expression of cellulase and xylanase genes in P. oxalicum. This study provides novel insights into the regulatory mechanisms of fungal cellulase and xylanase gene expression.

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Deletion of the middle region of the transcription factor ClrB in Penicillium oxalicum enables cellulase production in the presence of glucose.

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Deletion of the middle region of the transcription factor ClrB in <i>Penicillium oxalicum</i> enables cellulase production in the presence of glucose.

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