Calcium signaling positively regulates cellulase translation and secretion in a Clr-2-overexpressing, catabolically derepressed strain of Penicillium funiculosum
Abstract Background Low-cost cellulase production is vital to sustainable second-generation biorefineries. The catabolically derepressed strain of Penicillium funiculosum NCIM1228 (PfMig188 or ∆Mig1) secretes a superior set of cellulolytic enzymes, that are most suitable for 2G biorefineries. At a 3...
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| Format: | Article |
| Language: | English |
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BMC
2024-02-01
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| Series: | Biotechnology for Biofuels and Bioproducts |
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| Online Access: | https://doi.org/10.1186/s13068-023-02448-3 |
| _version_ | 1797275518009081856 |
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| author | Anmoldeep Randhawa Olusola A. Ogunyewo Kamran Jawed Syed Shams Yazdani |
| author_facet | Anmoldeep Randhawa Olusola A. Ogunyewo Kamran Jawed Syed Shams Yazdani |
| author_sort | Anmoldeep Randhawa |
| collection | DOAJ |
| description | Abstract Background Low-cost cellulase production is vital to sustainable second-generation biorefineries. The catabolically derepressed strain of Penicillium funiculosum NCIM1228 (PfMig188 or ∆Mig1) secretes a superior set of cellulolytic enzymes, that are most suitable for 2G biorefineries. At a 3% (w/w) load, the ∆Mig1 secretome can release > 80% of fermentable sugars from lignocellulose at a 15% (w/v) biomass load, irrespective of the type of biomass and pretreatment. The robustness of the secretome can be further increased by improving the cellulase production capacity of the fungal strain. Results We began by identifying the transcription factor responsible for cellulase production in NCIM1228. An advanced RNA-seq screen identified three genes, clr-2, ctf1a and ctf1b; the genes were cloned under their native promoters and transformed into NCIM1228. Of the three, clr-2 overexpression led to twofold higher cellulase production than the parent strain and was thus identified as the transcriptional activator of cellulase in NCIM1228. Next, we overexpressed clr-2 in ∆Mig1 and expected an exponential increase in cellulolytic attributes accredited to the reinforced activation mechanisms, conjoint with diminished negative regulation. Although clr-2 overexpression increased the transcript levels of cellulase genes in ∆Mig1, there was no increase in cellulase yield. Even a further increase in the transcript levels of clr-2 via a stronger promoter was ineffective. However, when the CaCO3 concentration was increased to 5 g/l in the growth medium, we achieved a 1.5-fold higher activity of 6.4 FPU/ml in the ∆Mig1 strain with clr-2 overexpression. Enthused by the calcium effect, a transcriptomic screen for genes encoding Ca2+-activated kinase identified ssp1, whose overexpression could further increase cellulase yield to ~ 7.5 FPU/ml. Investigation of the mechanism revealed that calcium signaling exclusively enhances the translation and secretion of cellulase in Penicillium funiculosum. Conclusions Our study identifies for the first time that cellulose activates two discrete signaling events to govern cellulase transcription and posttranscriptional processes (translation, processing and secretion) in P. funiculosum NCIM1228. Whereas Clr-2, the transcriptional activator of cellulase, governs transcription, calcium signaling specifically activates cellulase translation and secretion. |
| first_indexed | 2024-03-07T15:15:36Z |
| format | Article |
| id | doaj.art-e964c529b7ef44a5a72a4660f396045f |
| institution | Directory Open Access Journal |
| issn | 2731-3654 |
| language | English |
| last_indexed | 2024-03-07T15:15:36Z |
| publishDate | 2024-02-01 |
| publisher | BMC |
| record_format | Article |
| series | Biotechnology for Biofuels and Bioproducts |
| spelling | doaj.art-e964c529b7ef44a5a72a4660f396045f2024-03-05T17:57:51ZengBMCBiotechnology for Biofuels and Bioproducts2731-36542024-02-0117111710.1186/s13068-023-02448-3Calcium signaling positively regulates cellulase translation and secretion in a Clr-2-overexpressing, catabolically derepressed strain of Penicillium funiculosumAnmoldeep Randhawa0Olusola A. Ogunyewo1Kamran Jawed2Syed Shams Yazdani3Microbial Engineering Group, International Centre for Genetic Engineering and BiotechnologyMicrobial Engineering Group, International Centre for Genetic Engineering and BiotechnologyMicrobial Engineering Group, International Centre for Genetic Engineering and BiotechnologyMicrobial Engineering Group, International Centre for Genetic Engineering and BiotechnologyAbstract Background Low-cost cellulase production is vital to sustainable second-generation biorefineries. The catabolically derepressed strain of Penicillium funiculosum NCIM1228 (PfMig188 or ∆Mig1) secretes a superior set of cellulolytic enzymes, that are most suitable for 2G biorefineries. At a 3% (w/w) load, the ∆Mig1 secretome can release > 80% of fermentable sugars from lignocellulose at a 15% (w/v) biomass load, irrespective of the type of biomass and pretreatment. The robustness of the secretome can be further increased by improving the cellulase production capacity of the fungal strain. Results We began by identifying the transcription factor responsible for cellulase production in NCIM1228. An advanced RNA-seq screen identified three genes, clr-2, ctf1a and ctf1b; the genes were cloned under their native promoters and transformed into NCIM1228. Of the three, clr-2 overexpression led to twofold higher cellulase production than the parent strain and was thus identified as the transcriptional activator of cellulase in NCIM1228. Next, we overexpressed clr-2 in ∆Mig1 and expected an exponential increase in cellulolytic attributes accredited to the reinforced activation mechanisms, conjoint with diminished negative regulation. Although clr-2 overexpression increased the transcript levels of cellulase genes in ∆Mig1, there was no increase in cellulase yield. Even a further increase in the transcript levels of clr-2 via a stronger promoter was ineffective. However, when the CaCO3 concentration was increased to 5 g/l in the growth medium, we achieved a 1.5-fold higher activity of 6.4 FPU/ml in the ∆Mig1 strain with clr-2 overexpression. Enthused by the calcium effect, a transcriptomic screen for genes encoding Ca2+-activated kinase identified ssp1, whose overexpression could further increase cellulase yield to ~ 7.5 FPU/ml. Investigation of the mechanism revealed that calcium signaling exclusively enhances the translation and secretion of cellulase in Penicillium funiculosum. Conclusions Our study identifies for the first time that cellulose activates two discrete signaling events to govern cellulase transcription and posttranscriptional processes (translation, processing and secretion) in P. funiculosum NCIM1228. Whereas Clr-2, the transcriptional activator of cellulase, governs transcription, calcium signaling specifically activates cellulase translation and secretion.https://doi.org/10.1186/s13068-023-02448-3CellulaseClr-2Ssp1 CaMKKCalciumPenicillium funiculosum |
| spellingShingle | Anmoldeep Randhawa Olusola A. Ogunyewo Kamran Jawed Syed Shams Yazdani Calcium signaling positively regulates cellulase translation and secretion in a Clr-2-overexpressing, catabolically derepressed strain of Penicillium funiculosum Biotechnology for Biofuels and Bioproducts Cellulase Clr-2 Ssp1 CaMKK Calcium Penicillium funiculosum |
| title | Calcium signaling positively regulates cellulase translation and secretion in a Clr-2-overexpressing, catabolically derepressed strain of Penicillium funiculosum |
| title_full | Calcium signaling positively regulates cellulase translation and secretion in a Clr-2-overexpressing, catabolically derepressed strain of Penicillium funiculosum |
| title_fullStr | Calcium signaling positively regulates cellulase translation and secretion in a Clr-2-overexpressing, catabolically derepressed strain of Penicillium funiculosum |
| title_full_unstemmed | Calcium signaling positively regulates cellulase translation and secretion in a Clr-2-overexpressing, catabolically derepressed strain of Penicillium funiculosum |
| title_short | Calcium signaling positively regulates cellulase translation and secretion in a Clr-2-overexpressing, catabolically derepressed strain of Penicillium funiculosum |
| title_sort | calcium signaling positively regulates cellulase translation and secretion in a clr 2 overexpressing catabolically derepressed strain of penicillium funiculosum |
| topic | Cellulase Clr-2 Ssp1 CaMKK Calcium Penicillium funiculosum |
| url | https://doi.org/10.1186/s13068-023-02448-3 |
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