Abstract:Filamentous fungi are remarkable producers of enzymes dedicated to the degradation of sugar polymers found in the plant cell wall. Here, we integrated transcriptomic data to identify novel transcription factors (TFs) related to the control of gene expression of lignocellulosic hydrolases in Trichoderma reesei and Aspergillus nidulans. Using various sets of differentially expressed genes, we identified some putative cis-regulatory elements that were related to known binding sites for Saccharomyces cerevisiae TF… Show more
“…Because its Neurospora crassa orthologue COL-26 was found to regulate both glucose sensing and glucose metabolism, BglR might have a broader function in regulation [44]. A loss of AZF1 resulted in strongly reduced expression levels of cellulases [45]. VIB1, another regulator of cellulases, was found to be a functional homologue of the N. crassa vib - 1 .…”
Biorefineries, designed for the production of lignocellulose-based chemicals and fuels, are receiving increasing attention from the public, governments, and industries. A major obstacle for biorefineries to advance to commercial scale is the high cost of the enzymes required to derive the fermentable sugars from the feedstock used. As summarized in this review, techno-economic studies suggest co-localization and integration of enzyme manufacturing with the cellulosic biorefinery as the most promising alternative to alleviate this problem. Thus, cultivation of Trichoderma reesei, the principal producer of lignocellulolytic enzymes, on the lignocellulosic biomass processed on-site can reduce the cost of enzyme manufacturing. Further, due to a complex gene regulation machinery, the fungus can adjust the gene expression of the lignocellulolytic enzymes towards the characteristics of the feedstock, increasing the hydrolytic efficiency of the produced enzyme cocktail. Despite extensive research over decades, the underlying regulatory mechanisms are not fully elucidated. One aspect that has received relatively little attention in literature is the influence the characteristics of a lignocellulosic substrate, i.e., its chemical and physical composition, has on the produced enzyme mixture. Considering that the fungus is dependent on efficient enzymatic degradation of the lignocellulose for continuous supply of carbon and energy, a relationship between feedstock characteristics and secretome composition can be expected. The aim of this review was to systematically collect, appraise, and aggregate data and integrate results from studies analyzing enzyme production by T. reesei on insoluble cellulosic model substrates and lignocellulosic biomass. The results show that there is a direct effect of the substrate’s complexity (rated by structure, composition of the lignin–carbohydrate complex, and recalcitrance in enzymatic saccharification) on enzyme titers and the composition of specific activities in the secretome. It further shows that process-related factors, such as substrate loading and cultivation set-up, are direct targets for increasing enzyme yields. The literature on transcriptome and secretome composition further supports the proposed influence of substrate-related factors on the expression of lignocellulolytic enzymes. This review provides insights into the interrelation between the characteristics of the substrate and the enzyme production by T. reesei, which may help to advance integrated enzyme manufacturing of substrate-specific enzymes cocktails at scale.
“…Because its Neurospora crassa orthologue COL-26 was found to regulate both glucose sensing and glucose metabolism, BglR might have a broader function in regulation [44]. A loss of AZF1 resulted in strongly reduced expression levels of cellulases [45]. VIB1, another regulator of cellulases, was found to be a functional homologue of the N. crassa vib - 1 .…”
Biorefineries, designed for the production of lignocellulose-based chemicals and fuels, are receiving increasing attention from the public, governments, and industries. A major obstacle for biorefineries to advance to commercial scale is the high cost of the enzymes required to derive the fermentable sugars from the feedstock used. As summarized in this review, techno-economic studies suggest co-localization and integration of enzyme manufacturing with the cellulosic biorefinery as the most promising alternative to alleviate this problem. Thus, cultivation of Trichoderma reesei, the principal producer of lignocellulolytic enzymes, on the lignocellulosic biomass processed on-site can reduce the cost of enzyme manufacturing. Further, due to a complex gene regulation machinery, the fungus can adjust the gene expression of the lignocellulolytic enzymes towards the characteristics of the feedstock, increasing the hydrolytic efficiency of the produced enzyme cocktail. Despite extensive research over decades, the underlying regulatory mechanisms are not fully elucidated. One aspect that has received relatively little attention in literature is the influence the characteristics of a lignocellulosic substrate, i.e., its chemical and physical composition, has on the produced enzyme mixture. Considering that the fungus is dependent on efficient enzymatic degradation of the lignocellulose for continuous supply of carbon and energy, a relationship between feedstock characteristics and secretome composition can be expected. The aim of this review was to systematically collect, appraise, and aggregate data and integrate results from studies analyzing enzyme production by T. reesei on insoluble cellulosic model substrates and lignocellulosic biomass. The results show that there is a direct effect of the substrate’s complexity (rated by structure, composition of the lignin–carbohydrate complex, and recalcitrance in enzymatic saccharification) on enzyme titers and the composition of specific activities in the secretome. It further shows that process-related factors, such as substrate loading and cultivation set-up, are direct targets for increasing enzyme yields. The literature on transcriptome and secretome composition further supports the proposed influence of substrate-related factors on the expression of lignocellulolytic enzymes. This review provides insights into the interrelation between the characteristics of the substrate and the enzyme production by T. reesei, which may help to advance integrated enzyme manufacturing of substrate-specific enzymes cocktails at scale.
“…At neutral pH, the abolition of the pac1 gene increases Xyr1 activity [ 5 , 55 ] PoxMBF1 In Penicillium oxalicum , it binds directly to the promoter regions of principal cellulase and xylanase genes to induce cellulase production [ 111 ] PoxFlbC In Penicillium oxalicum (FlbC orthologue in Aspergillus ), it upregulates most of the cellulase genes [ 112 ] PoxAtf1 In filamentous fungi, it controls the expression of cellulase and xylanase genes during the solid-state fermentation [ 113 ] Blr1, Blr2, and Env1 Photoreceptor proteins (Blr1, Blr2, and Env1) in both light and dark regulate the cellulase gene from T. reesei . The photoreceptor regulative mechanism of carbon metabolism in T. reesei is dependent on Blr1 and Blr2 controlled by the Env1 photoreceptor [ 114 ] Seb1 In Talaromyces pinophilus EMU, Seb1 transcription factor binds to the stress response element (STRE) and CRISPR-Cas9 mediated degradation of seb1 gene led to the 20–40% increment in FPase activity [ 123 ] TrAZF1 In T. reesei and A. nidulans positively regulates the activity of BGL and CBH by interacting with the cel7a, cel45a , and sow promoter region [ 115 ] PoSet1/PoSet2 In Penicillium oxalicum , PoSet1 and PoSet2 are responsible for methylation of histone (H3) lysine 4 and lysine 36, respectively. They are involved in the upregulation (PoSet1) or down regulation (PoSet2) of cellulase production [ 116 ] MAT1-2-1 It is a mating-type locus protein of T. reesei modulates cellulase gene expression in daylight by interacting with the Xyr1 and binds to the cbh1 promoter [ 117 ] Fig.…”
Section: Molecular Mechanisms Behind the Cellulase Expressionmentioning
Cellulose is the utmost plenteous source of biopolymer in our earth, and fungi are the most efficient and ubiquitous organism in degrading the cellulosic biomass by synthesizing cellulases. Tailoring through genetic manipulation has played a substantial role in constructing novel fungal strains towards improved cellulase production of desired traits. However, the traditional methods of genetic manipulation of fungi are time-consuming and tedious. With the availability of the full-genome sequences of several industrially relevant filamentous fungi, CRISPR-CAS (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) technology has come into the focus for the proficient development of manipulated strains of filamentous fungi. This review summarizes the mode of action of cellulases, transcription level regulation for cellulase expression, various traditional strategies of genetic manipulation with CRISPR-CAS technology to develop modified fungal strains for a preferred level of cellulase production, and the futuristic trend in this arena of research.
“…Lignocellulosic biomass is one of the most abundant, renewable, and relatively inexpensive raw materials for biorefineries. This biomass is used for the production of value-added chemicals and fuels [ 1 – 3 ]. Filamentous fungi are the principal producers of hydrolytic enzymes dedicated to the degradation of lignocellulosic biomass [ 4 – 6 ].…”
Background
The filamentous fungus Trichoderma reesei is one of the best producers of cellulase and has been widely studied for the production of cellulosic ethanol and bio-based products. We previously reported that Mn2+ and N,N-dimethylformamide (DMF) can stimulate cellulase overexpression via Ca2+ bursts and calcium signalling in T. reesei under cellulase-inducing conditions. To further understand the regulatory networks involved in cellulase overexpression in T. reesei, we characterised the Mn2+/DMF-induced calcium signalling pathway involved in the stimulation of cellulase overexpression.
Results
We found that Mn2+/DMF stimulation significantly increased the intracellular levels of cAMP in an adenylate cyclase (ACY1)-dependent manner. Deletion of acy1 confirmed that cAMP is crucial for the Mn2+/DMF-stimulated cellulase overexpression in T. reesei. We further revealed that cAMP elevation induces a cytosolic Ca2+ burst, thereby initiating the Ca2+ signal transduction pathway in T. reesei, and that cAMP signalling causes the Ca2+ signalling pathway to regulate cellulase production in T. reesei. Furthermore, using a phospholipase C encoding gene plc-e deletion strain, we showed that the plc-e gene is vital for cellulase overexpression in response to stimulation by both Mn2+ and DMF, and that cAMP induces a Ca2+ burst through PLC-E.
Conclusions
The findings of this study reveal the presence of a signal transduction pathway in which Mn2+/DMF stimulation produces cAMP. Increase in the levels of cAMP activates the calcium signalling pathway via phospholipase C to regulate cellulase overexpression under cellulase-inducing conditions. These findings provide insights into the molecular mechanism of the cAMP–PLC–calcium signalling pathway underlying cellulase expression in T. reesei and highlight the potential applications of signal transduction in the regulation of gene expression in fungi.
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