Glutamine involvement in nitrogen regulation of cellulase production in fungi

Background Cellulase synthesized by fungi can environment-friendly and sustainably degrades cellulose to fermentable sugars for producing cellulosic biofuels, biobased medicine and fine chemicals. Great efforts have been made to study the regulation mechanism of cellulase biosynthesis in fungi with the focus on the carbon sources, while little attention has been paid to the impact and regulation mechanism of nitrogen sources on cellulase production. Results Glutamine displayed the strongest inhibition effect on cellulase biosynthesis in Trichoderma reesei, followed by yeast extract, urea, tryptone, ammonium sulfate and l-glutamate. Cellulase production, cell growth and sporulation in T. reesei RUT-C30 grown on cellulose were all inhibited with the addition of glutamine (a preferred nitrogen source) with no change for mycelium morphology. This inhibition effect was attributed to both l-glutamine itself and the nitrogen excess induced by its presence. In agreement with the reduced cellulase production, the mRNA levels of 44 genes related to the cellulase production were decreased severely in the presence of glutamine. The transcriptional levels of genes involved in other nitrogen transport, ribosomal biogenesis and glutamine biosynthesis were decreased notably by glutamine, while the expression of genes relevant to glutamate biosynthesis, amino acid catabolism, and glutamine catabolism were increased noticeably. Moreover, the transcriptional level of cellulose signaling related proteins ooc1 and ooc2, and the cellular receptor of rapamycin trFKBP12 was increased remarkably, whose deletion exacerbated the cellulase depression influence of glutamine. Conclusion Glutamine may well be the metabolite effector in nitrogen repression of cellulase synthesis, like the role of glucose plays in carbon catabolite repression. Glutamine under excess nitrogen condition repressed cellulase biosynthesis significantly as well as cell growth and sporulation in T. reesei RUT-C30. More importantly, the presence of glutamine notably impacted the transport and metabolism of nitrogen. Genes ooc1, ooc2, and trFKBP12 are associated with the cellulase repression impact of glutamine. These findings advance our understanding of nitrogen regulation of cellulase production in filamentous fungi, which would aid in the rational design of strains and fermentation strategies for cellulase production in industry.

Comparative Secretomics Analysis Reveals the Major Components of Penicillium oxalicum 16 and Trichoderma reesei RUT-C30

In this study, the major secretome components of Penicillium oxalicum 16 and Trichoderma reesei RUT-C30 under wheat bran (WB) and rice straw (RS) solid-state fermentation were systematically analyzed. The activities of the major components, e.g., cellulase, hemicellulase, and amylase, were consistent with their abundance in the secretomes. P. oxalicum 16 secreted more abundant glycoside hydrolases than T. reesei RUT-C30. The main up-regulated proteins from the induction of WB, compared with that from RS, were amylase, pectinase, and protease, whereas the main down-regulated enzymes were cellulase, hemicellulase, swollenin, and lytic polysaccharide monooxygenase (LPMO). Specifically, WB induced more β-1,4-glucosidases, namely, S8B0F3 (UniProt ID), and A0A024RWA5 than RS, but RS induced more β-1,4-exoglucanases and β-1,4-endoglucanases, namely, A0A024RXP8, A024SH76, S7B6D6, S7ZP52, A024SH20, A024S2H5, S8BGM3, S7ZX22, and S8AIJ2. The P. oxalicum 16 xylanases S8AH74 and S7ZA57 were the major components responsible for degrading soluble xylan, and S8BDN2 probably acted on solid-state hemicellulose instead of soluble xylan. The main hemicellulase component of T. reesei RUT-C30 in RS was the xyloglucanase A0A024S9Z6 with an abundance of 16%, but T. reesei RUT-C30 lacked the hemicellulase mannanase and had a small amount of the hemicellulase xylanase. P. oxalicum 16 produced more amylase than T. reesei RUT-C30, and the results suggest amylase S7Z6T2 may degrade soluble starch. The percentage of the glucoamylase S8B6D7 did not significantly change, and reached an average abundance of 5.5%. The major auxiliary degradation enzymes of P. oxalicum 16 were LPMOs S7Z716 and S7ZPW1, whereas those of T. reesei RUT-C30 were swollenin and LPMOs A0A024SM10, A0A024SFJ2, and A0A024RZP7.

Whole Genome Sequence and CAZyme distribution of the cellulase hyper producing filamentous fungus Penicillium janthinellum NCIM 1366

Penicillium janthinellum NCIM 1366, capable of secreting cellulases that are highly efficient in the hydrolysis of lignocellulosic biomass, was sequenced to understand its cellulolytic machinery. De novo sequencing and assembly revealed a 37.6 Mb genome encoding 11,848 putative proteins, 93% of which had significant BLAST-P hits. The majority of the top hits (those with over 60% UniProt identity) belonged to P. brasilianum. Carbohydrate active enzymes (CAZymes) and other enzymes involved in lignocellulose degradation were also predicted from this strain and compared with those of the industrial workhorse of cellulase production- Trichoderma reesei RUT-C30. The comparison showed that the fungus encodes a far higher number of CAZYmes (422) as compared to T. reesei RUT-C30 (244), which gives a plausible explanation for its overall effectiveness in biomass hydrolysis. An analysis of the secreted CAZymes and annotated ligninases identified 216 predicted proteins which may be directly involved in the breakdown of lignocellulose

Consolidated bioprocessing of lignocellulose for production of glucaric acid by an artificial microbial consortium

Background The biomanufacturing of d-glucaric acid has attracted increasing interest because it is one of the top value-added chemicals produced from biomass. Saccharomyces cerevisiae is regarded as an excellent host for d-glucaric acid production. Results The opi1 gene was knocked out because of its negative regulation on myo-inositol synthesis, which is the limiting step of d-glucaric acid production by S. cerevisiae. We then constructed the biosynthesis pathway of d-glucaric acid in S. cerevisiae INVSc1 opi1Δ and obtained two engineered strains, LGA-1 and LGA-C, producing record-breaking titers of d-glucaric acid: 9.53 ± 0.46 g/L and 11.21 ± 0.63 g/L d-glucaric acid from 30 g/L glucose and 10.8 g/L myo-inositol in fed-batch fermentation mode, respectively. However, LGA-1 was preferable because of its genetic stability and its superior performance in practical applications. There have been no reports on d-glucaric acid production from lignocellulose. Therefore, the biorefinery processes, including separated hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF) and consolidated bioprocessing (CBP) were investigated and compared. CBP using an artificial microbial consortium composed of Trichoderma reesei (T. reesei) Rut-C30 and S. cerevisiae LGA-1 was found to have relatively high d-glucaric acid titers and yields after 7 d of fermentation, 0.54 ± 0.12 g/L d-glucaric acid from 15 g/L Avicel and 0.45 ± 0.06 g/L d-glucaric acid from 15 g/L steam-exploded corn stover (SECS), respectively. In an attempt to design the microbial consortium for more efficient CBP, the team consisting of T. reesei Rut-C30 and S. cerevisiae LGA-1 was found to be the best, with excellent work distribution and collaboration. Conclusions Two engineered S. cerevisiae strains, LGA-1 and LGA-C, with high titers of d-glucaric acid were obtained. This indicated that S. cerevisiae INVSc1 is an excellent host for d-glucaric acid production. Lignocellulose is a preferable substrate over myo-inositol. SHF, SSF, and CBP were studied, and CBP using an artificial microbial consortium of T. reesei Rut-C30 and S. cerevisiae LGA-1 was found to be promising because of its relatively high titer and yield. T. reesei Rut-C30 and S. cerevisiae LGA-1were proven to be the best teammates for CBP. Further work should be done to improve the efficiency of this microbial consortium for d-glucaric acid production from lignocellulose.

High-dose rapamycin exerts a temporary impact on T. reesei RUT-C30 through gene trFKBP12

Background Knowledge with respect to regulatory systems for cellulase production is prerequisite for exploitation of such regulatory networks to increase cellulase production, improve fermentation efficiency and reduce the relevant production cost. The target of rapamycin (TOR) signaling pathway is considered as a central signaling hub coordinating eukaryotic cell growth and metabolism with environmental inputs. However, how and to what extent the TOR signaling pathway and rapamycin are involved in cellulase production remain elusive. Result At the early fermentation stage, high-dose rapamycin (100 μM) caused a temporary inhibition effect on cellulase production, cell growth and sporulation of Trichoderma reesei RUT-C30 independently of the carbon sources, and specifically caused a tentative morphology defect in RUT-C30 grown on cellulose. On the contrary, the lipid content of T. reesei RUT-C30 was not affected by rapamycin. Accordingly, the transcriptional levels of genes involved in the cellulase production were downregulated notably with the addition of rapamycin. Although the mRNA levels of the putative rapamycin receptor trFKBP12 was upregulated significantly by rapamycin, gene trTOR (the downstream effector of the rapamycin–FKBP12 complex) and genes associated with the TOR signaling pathways were not changed markedly. With the deletion of gene trFKBP12, there is no impact of rapamycin on cellulase production, indicating that trFKBP12 mediates the observed temporary inhibition effect of rapamycin. Conclusion Our study shows for the first time that only high-concentration rapamycin induced a transient impact on T. reesei RUT-C30 at its early cultivation stage, demonstrating T. reesei RUT-C30 is highly resistant to rapamycin, probably due to that trTOR and its related signaling pathways were not that sensitive to rapamycin. This temporary influence of rapamycin was facilitated by gene trFKBP12. These findings add to our knowledge on the roles of rapamycin and the TOR signaling pathways play in T. reesei.

Consolidated bioprocessing of lignocellulose for production of glucaric acid by an artificial microbial consortium

Background: The biomanufacturing of D-glucaric acid has been attracted increasing interest because it is one of the top value-added chemicals produced from biomass. Saccharomyces cerevisiae is regarded as an excellent host for D-glucaric acid production. Results: The opi1 gene was knocked out because of its negative regulation on myo-inositol synthesis, which is the limiting step of D-glucaric acid production by S. cerevisiae. Then we constructed the biosynthetic pathway of D-glucaric acid in S. cerevisiae INVSc1 opi1Δ and obtained two engineered strains, LGA-1 and LGA-C, producing record breaking titers of D-glucaric acid, 9.53 ± 0.46 g/L and 11.21 ± 0.63 g/L D-glucaric acid from 30 g/L glucose and 10.8 g/L myo-inositol in the mode of fed-batch fermentation, respectively. However, LGA-1 was more preferable because of the genetic stability and the outperformance in applications. So far, there have been no reports on D-glucaric acid production from lignocellulose. Therefore, the biorefinery processes including separated hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF) and consolidated bioprocessing (CBP) were investigated and compared. CBP by an artificial microbial consortium composed of Trichoderma reesei Rut-C30 and S. cerevisiae LGA-1 was found to have relatively high D-glucaric acid titers and yields after 7 d fermentation, 0.54 ± 0.12 g/L D-glucaric acid from 15 g/L Avicel, and 0.45 ± 0.06 g/L D-glucaric acid from 15 g/L steam exploded corn stover (SECS), respectively. In attempts to design the microbial consortium for more efficient CBP the team consisted of the two members, T. reesei Rut-C30 and S. cerevisiae LGA-1, was found to be the best with excellent work distribution and collaboration.Conclusions: Two engineered S. cerevisiae strains, LGA-1 and LGA-C, with high titers of D-glucaric acid were obtained. This indicates that S. cerevisiae INVSc1 was an excellent host. Lignocellulose is a more preferable substrate than myo-inositol. SHF, SSF and CBP were studied and CBP by an artificial microbial consortium of T. reesei Rut-C30 and S. cerevisiae LGA-1 was found to be promising because of the relatively high titer and yield. T. reesei Rut-C30 and S. cerevisiae LGA-1were proved to be the best teammates for CBP. Further work should be done to improve the efficiency of this microbial consortium for D-glucaric acid production from lignocellulose.

Glucose-lactose mixture feeds in industry-like conditions: a gene regulatory network analysis on the hyperproducing Trichoderma reesei strain Rut-C30

Background: The degradation of cellulose and hemicellulose molecules into simpler sugars such as glucose is part of the second generation biofuel production process. Hydrolysis of lignocellulosic substrates is usually performed by enzymes produced and secreted by the fungus Trichoderma reesei. Studies identifying transcription factors involved in the regulation of cellulase production have been conducted but no overview of the whole regulation network is available. A transcriptomic approach with mixtures of glucose and lactose, used as a substrate for cellulase induction, was used to help us decipher missing parts in the network of T. reesei Rut-C30. Results: Experimental results on the Rut-C30 hyperproducing strain confirmed the impact of sugar mixtures on the enzymatic cocktail composition. The transcriptomic study shows a temporal regulation of the main transcription factors and a lactose concentration impact on the transcriptional profile. A gene regulatory network built using BRANE Cut software reveals three sub-networks related to i) a positive correlation between lactose concentration and cellulase production, ii) a particular dependence of the lactose onto the β-glucosidase regulation and iii) a negative regulation of the development process and growth. Conclusions: This work is the first investigating a transcriptomic study regarding the effects of pure and mixed carbon sources in a fed-batch mode. Our study expose a co-orchestration of xyr1, clr2 and ace3 for cellulase and hemicellulase induction and production, a fine regulation of the β-glucosidase and a decrease of growth in favor of cellulase production. These conclusions provide us with potential targets for further genetic engineering leading to better cellulase-producing strains in industry-like conditions.

Penicillium janthinellum NCIM1366 shows improved biomass hydrolysis and a larger number of CAZymes with higher induction levels over Trichoderma reesei RUT-C30

Background Major cost of bioethanol is attributed to enzymes employed in biomass hydrolysis. Biomass hydrolyzing enzymes are predominantly produced from the hyper-cellulolytic mutant filamentous fungus Trichoderma reesei RUT-C30. Several decades of research have failed to provide an industrial grade organism other than T. reesei, capable of producing higher titers of an effective synergistic biomass hydrolyzing enzyme cocktail. Penicillium janthinellum NCIM1366 was reported as a cellulase hyper producer and a potential alternative to T. reesei, but a comparison of their hydrolytic performance was seldom attempted. Results Hydrolysis of acid or alkali-pretreated rice straw using cellulase enzyme preparations from P. janthinellum and T. reesei indicated 37 and 43% higher glucose release, respectively, with P. janthinellum enzymes. A comparison of these fungi with respect to their secreted enzymes indicated that the crude enzyme preparation from P. janthinellum showed 28% higher overall cellulase activity. It also had an exceptional tenfold higher beta-glucosidase activity compared to that of T. reesei, leading to a lower cellobiose accumulation and thus alleviating the feedback inhibition. P. janthinellum secreted more number of proteins to the extracellular medium whose total concentration was 1.8-fold higher than T. reesei. Secretome analyses of the two fungi revealed higher number of CAZymes and a higher relative abundance of cellulases upon cellulose induction in the fungus. Conclusions The results revealed the ability of P. janthinellum for efficient biomass degradation through hyper cellulase production, and it outperformed the established industrial cellulase producer T. reesei in the hydrolysis experiments. A higher level of induction, larger number of secreted CAZymes and a high relative proportion of BGL to cellulases indicate the possible reasons for its performance advantage in biomass hydrolysis.

Glucose-lactose mixture feeds in industry-like conditions: a gene regulatory network analysis on the hyperproducing Trichoderma reesei strain Rut-C30

Background The degradation of cellulose and hemicellulose molecules into simpler sugars such as glucose is part of the second generation biofuel production process. Hydrolysis of lignocellulosic substrates is usually performed by enzymes produced and secreted by the fungus Trichoderma reesei. Studies identifying transcription factors involved in the regulation of cellulase production have been conducted but no overview of the whole regulation network is available. A transcriptomic approach with mixtures of glucose and lactose, used as a substrate for cellulase induction, was used to help us decipher missing parts in the network of T. reesei Rut-C30. Results Experimental results on the Rut-C30 hyperproducing strain confirmed the impact of sugar mixtures on the enzymatic cocktail composition. The transcriptomic study shows a temporal regulation of the main transcription factors and a lactose concentration impact on the transcriptional profile. A gene regulatory network built using BRANE Cut software reveals three sub-networks related to i) a positive correlation between lactose concentration and cellulase production, ii) a particular dependence of the lactose onto the β-glucosidase regulation and iii) a negative regulation of the development process and growth. Conclusions This work is the first investigating a transcriptomic study regarding the effects of pure and mixed carbon sources in a fed-batch mode. Our study expose a co-orchestration of xyr1, clr2 and ace3 for cellulase and hemicellulase induction and production, a fine regulation of the β-glucosidase and a decrease of growth in favor of cellulase production. These conclusions provide us with potential targets for further genetic engineering leading to better cellulase-producing strains in industry-like conditions.