Promotion of extracellular lignocellulolytic enzymes production by restraining the intracellular β-glucosidase in Penicillium decumbens

Chen, Mei; Qin, Yuqi; Qing, Cao; Liu, Guodong; Li, Jie; Li, Zhonghai; Zhao, Jian; Qu, Yinbo

doi:10.1016/j.biortech.2013.03.099

Cited by 52 publications

(33 citation statements)

References 30 publications

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“…In addition to cre - 1 , two genes involved in cellulase production were chosen as targets: an endoplasmic reticulum stress regulator res - 1 homolog [50] and the gh1 - 1 gene encoding β-glucosidase [10, 49, 51–53]. M2, a strain with constitutive Cas9 expression and inactive amdS (through a large insertion of 104 nucleotides) that was derived from M1 (a strain carrying the amdS gene under the WT background, see Fig.…”

Section: Resultsmentioning

confidence: 99%

Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering

Liu

Gao

et al. 2017

Biotechnol Biofuels

445

360

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BackgroundOver the past 3 years, the CRISPR/Cas9 system has revolutionized the field of genome engineering. However, its application has not yet been validated in thermophilic fungi. Myceliophthora thermophila, an important thermophilic biomass-degrading fungus, has attracted industrial interest for the production of efficient thermostable enzymes. Genetic manipulation of Myceliophthora is crucial for metabolic engineering and to unravel the mechanism of lignocellulose deconstruction. The lack of a powerful, versatile genome-editing tool has impeded the broader exploitation of M. thermophila in biotechnology.ResultsIn this study, a CRISPR/Cas9 system for efficient multiplexed genome engineering was successfully developed in the thermophilic species M. thermophila and M. heterothallica. This CRISPR/Cas9 system could efficiently mutate the imported amdS gene in the genome via NHEJ-mediated events. As a proof of principle, the genes of the cellulase production pathway, including cre-1, res-1, gh1-1, and alp-1, were chosen as editing targets. Simultaneous multigene disruptions of up to four of these different loci were accomplished with neomycin selection marker integration via a single transformation using the CRISPR/Cas9 system. Using this genome-engineering tool, multiple strains exhibiting pronounced hyper-cellulase production were generated, in which the extracellular secreted protein and lignocellulase activities were significantly increased (up to 5- and 13-fold, respectively) compared with the parental strain.ConclusionsA genome-wide engineering system for thermophilic fungi was established based on CRISPR/Cas9. Successful expansion of this system without modification to M. heterothallica indicates it has wide adaptability and flexibility for use in other Myceliophthora species. This system could greatly accelerate strain engineering of thermophilic fungi for production of industrial enzymes, such as cellulases as shown in this study and possibly bio-based fuels and chemicals in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0693-9) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering

Liu

Gao

et al. 2017

Biotechnol Biofuels

445

360

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“…3a, b). In contrast, the transcription of major β-glucosidase gene bgl1 [24, 25] showed decrease in both xlnR A871V and gpdA (p):: xlnR than those in wild-type strain (Fig. 3a, b).…”

Section: Resultsmentioning

confidence: 99%

Combining manipulation of transcription factors and overexpression of the target genes to enhance lignocellulolytic enzyme production in Penicillium oxalicum

Gao

Xia

et al. 2017

Biotechnol Biofuels

Self Cite

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BackgroundLignocellulolytic enzymes are the main enzymes to saccharify lignocellulose from renewable plant biomass in the bio-based economy. The production of these enzymes is transcriptionally regulated by multiple transcription factors. We previously engineered Penicillium oxalicum for improved cellulase production via manipulation of three genes in the cellulase expression regulatory network. However, the potential of combinational engineering of multiple regulators and their targets at protein abundance and activity levels has not been fully explored.ResultsHere, we verified that a point mutation XlnRA871V in transcription factor XlnR enhanced the expression of lignocellulolytic enzymes, particularly hemicellulases, in P. oxalicum. Then, overexpression of XlnRA871V with a constitutive PDE_02864 promoter was combined with the overexpression of cellulase transcriptional activator ClrB and deletion of carbon catabolite repressor CreA. The resulted strain RE-7 showed 8.9- and 51.5-fold increased production of cellulase and xylanase relative to the starting strain M12, respectively. Further overexpression of two major cellulase genes cbh1-2 and eg1 enabled an additional 13.0% improvement of cellulase production. In addition, XlnRA871V led to decreased production of β-glucosidase and amylase, which could be attributed to the reduced transcription of corresponding enzyme-encoding genes.ConclusionsThe results illustrated that combinational manipulation of the involved transcription factors and their target genes was a viable strategy for efficient production of lignocellulolytic enzymes in filamentous fungi. The striking negative effect of XlnRA871V mutation on amylase production was also highlighted.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0783-3) contains supplementary material, which is available to authorized users.

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“…Cytophaga hutchinsonii Cel5A ( Ch Cel5A) was heterologously expressed and purified using a previously described method . Penicillium decumbens β‐glucanase 2 ( Pd Bgl2) was expressed and purified as previously described , as was Caldicellulosiruptor saccharolyticus 0678 (Csac_0678) . Trichoderma reesei Cel7A ( Tr Cel7A) was purified as described .…”

Section: Methodsmentioning

confidence: 99%

Determination of the action modes of cellulases from hydrolytic profiles over a time course using fluorescence‐assisted carbohydrate electrophoresis

Zhang

Wang

et al. 2015

Electrophoresis

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Fluorescence-assisted carbohydrate electrophoresis (FACE) is a sensitive and simple method for the separation of oligosaccharides. It relies on labeling the reducing ends of oligosaccharides with a fluorophore, followed by PAGE. Concentration changes of oligosaccharides following hydrolysis of a carbohydrate polymer could be quantitatively measured continuously over time using the FACE method. Based on the quantitative analysis, we suggested that FACE was a relatively high-throughput, repeatable, and suitable method for the analysis of the action modes of cellulases. On account of the time courses of their hydrolytic profiles, the apparent processivity was used to show the different action modes of cellulases. Cellulases could be easily differentiated as exoglucanases, β-glucosidases, or endoglucanases. Moreover, endoglucanases from the same glycoside hydrolases family had a variety of apparent processivity, indicating the different modes of action. Endoglucanases with the same binding capacities and hydrolytic activities had similar oligosaccharide profiles, which aided in their classification. The hydrolytic profile of Trichoderma reesei Cel12A, an endoglucanases from T. reesei, contained glucose, cellobiose, and cellotriose, which revealed that it may have a new glucosidase activity, corresponding to that of EC 3.2.1.74. A hydrolysate study of a T. reesei Cel12A-N20A mutant demonstrated that the FACE method was sufficiently sensitive to detect the influence of a single-site mutation on enzymatic activity.

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Promotion of extracellular lignocellulolytic enzymes production by restraining the intracellular β-glucosidase in Penicillium decumbens

Cited by 52 publications

References 30 publications

Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering

Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering

Combining manipulation of transcription factors and overexpression of the target genes to enhance lignocellulolytic enzyme production in Penicillium oxalicum

Determination of the action modes of cellulases from hydrolytic profiles over a time course using fluorescence‐assisted carbohydrate electrophoresis

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