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 ß-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 ß-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.

Efficient evaluation of cellulose digestibility by Trichoderma reesei Rut-C30 cultures in online monitored shake flasks.

BACKGROUND: Pretreated lignocellulosic biomass is considered as a suitable feedstock for the sustainable production of chemicals. However, the recalcitrant nature of cellulose often results in very cost-intensive overall production processes. A promising concept to reduce the costs is consolidated bioprocessing, which integrates in a single step cellulase production, cellulose hydrolysis, and fermentative conversion of produced sugars into a valuable product. This approach, however, requires assessing the digestibility of the applied celluloses and, thus, the released sugar amount during the fermentation. Since the released sugars are completely taken up by Trichoderma reesei Rut-C30 and the sugar consumption is stoichiometrically coupled to oxygen uptake, the respiration activity was measured to evaluate the digestibility of cellulose. RESULTS: The method was successfully tested on commercial cellulosic substrates identifying a correlation between the respiration activity and the crystallinity of the substrate. Pulse experiments with cellulose and cellulases suggested that the respiration activity of T. reesei on cellulose can be divided into two distinct phases, one limited by enzyme activity and one by cellulose-binding-sites. The impact of known (cellobiose, sophorose, urea, tween 80, peptone) and new (miscanthus steepwater) compounds enhancing cellulase production was evaluated. Furthermore, the influence of two different pretreatment methods, the OrganoCat and OrganoSolv process, on the digestibility of beech wood saw dust was tested. CONCLUSIONS: The introduced method allows an online evaluation of cellulose digestibility in complex and non-complex cultivation media. As the measurements are performed under fermentation conditions, it is a valuable tool to test different types of cellulose for consolidated bioprocessing applications. Furthermore, the method can be applied to identify new compounds, which influence cellulase production.

Morphology and enzyme production of Trichoderma reesei Rut C-30 are affected by the physical and structural characteristics of cellulosic substrates.

The industrial production of cellulolytic enzymes is dominated by the filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina). In order to develop optimal enzymatic cocktail, it is of importance to understand the natural regulation of the enzyme profile as response to the growth substrate. The influence of the complexity of cellulose on enzyme production by the microorganisms is not understood. In the present study we attempted to understand how different physical and structural properties of cellulose-rich substrates affected the levels and profiles of extracellular enzymes produced by T. reesei. Enzyme production by T. reesei Rut C-30 was studied in submerged cultures on five different cellulose-rich substrates, namely, commercial cellulose Avicel® and industrial-like cellulosic pulp substrates which consist mainly of cellulose, but also contain residual hemicellulose and lignin. In order to evaluate the hydrolysis of the substrates by the fungal enzymes, the spatial polymer distributions were characterised by cross-polarisation magic angle spinning carbon-13 nuclear magnetic resonance (CP/MAS (13)C-NMR) in combination with spectral fitting. Proteins in culture supernatants at early and late stages of enzyme production were labeled by Tandem Mass Tags (TMT) and protein profiles were analysed by liquid chromatography-tandem mass spectrometry. The data have been deposited to the ProteomeXchange with identifier PXD001304. In total 124 proteins were identified and quantified in the culture supernatants, including cellulases, hemicellulases, other glycoside hydrolases, lignin-degrading enzymes, auxiliary activity 9 (AA9) family (formerly GH61), supporting activities of proteins and enzymes acting on cellulose, proteases, intracellular proteins and several hypothetical proteins. Surprisingly, substantial differences in the enzyme profiles were found even though there were minor differences in the chemical composition between the cellulose-rich substrates.

Investigating process parameters to enhance (hemi)cellulolytic enzymes activity produced by Trichoderma reesei RUT-C30 using deoiled oil palm mesocarp fiber in solid-state fermentation.

This study investigates the synthesis of (hemi)cellulolytic enzymes, including endoglucanase (CMCase), xylanase, and ß-glucosidase, employing Trichoderma reesei RUT-C30 and deoiled oil palm mesocarp fiber (OPMF) through solid-state fermentation (SSF). The objective was to determine the optimal process conditions for achieving high enzyme activities through a one-factor-at-a-time approach. The study primarily focused on the impact of the solid-to-liquid ratio, incubation period, initial pH, and temperature on enzyme activity. The effects of OPMF pretreatment, particularly deoiling and fortification, were explored. This approach significantly improved enzyme activity levels compared to the initial conditions, with CMCase increasing by 111.6 %, xylanase by 665.2 %, and ß-Glucosidase by 1678.1 %. Xylanase and ß-glucosidase activities, peaking at 1346.75 and 9.89 IU per gram dry substrate (GDS), respectively, under optimized conditions (1:4 ratio, pH 7.5, 20 °C, 9-day incubation). With lower moisture levels, CMCase reached its maximum activity of 227.84 IU/GDS. The study highlights how important it is for agro-industrial byproducts to support environmentally sustainable practices in the palm oil industry. It also emphasizes how differently each enzyme reacts to changes in process parameters.

Development of an economically competitive Trichoderma-based platform for enzyme production: Bioprocess optimization, pilot plant scale-up, techno-economic analysis and life cycle assessment.

Despite decades of research and industrial applications of Trichoderma reesei, the development of industrially relevant strains for enzyme production including a low-cost and scalable bioprocess remains elusive. Herein, bioprocess optimization, pilot plant scale-up, techno-economic analysis and life-cycle assessment for enzyme production by an engineered T. reesei strain are reported. The developed bioprocess increased in âˆ¼ 2-fold protein productivity (0.39 g.L-1.h-1) and 1.6-fold FPase activity (196 FPU.L-1.h-1), reducing the fermentation in 4 days. Cultivation in a 65-L pilot plant bioreactor resulted in 54 g.L-1 protein in 7 days, highlighting the robustness and scalability of this bioprocess. Techno-economic analysis indicates an enzyme cost of âˆ¼ 3.2 USD.kg-1, which is below to the target proposed (4.24 USD.kg-1) in the NREL/TP-5100-47764 report, while life-cycle assessment shows a carbon footprint reduction of approximately 50% compared to a typical commercial enzyme. This study provides the fundamental knowledge for the design of economically competitive Trichoderma technologies for industrial use.

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 ß-1,4-glucosidases, namely, S8B0F3 (UniProt ID), and A0A024RWA5 than RS, but RS induced more ß-1,4-exoglucanases and ß-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.

Enhanced cellulase production from Trichoderma reesei Rut-C30 by engineering with an artificial zinc finger protein library.

Trichoderma reesei Rut-C30 is a well-known cellulase producer, and improvement of its cellulase production is of great interest. An artificial zinc finger protein (AZFP) library is constructed for expression in T. reesei Rut-C30, and a mutant strain T. reesei U3 is selected based on its enhanced cellulase production. The U3 mutant shows a 55% rise in filter paper activity and 8.1-fold increased ß-glucosidase activity, when compared to the native strain T. reesei Rut-C30. It is demonstrated that enhanced ß-glucosidase activity was due to elevated transcription level of ß-glucosidase gene in the U3 mutant. Moreover, significant elevation in transcription levels of several putative Azfp-U3 target genes is detected in the U3 mutant, including genes encoding hypothetical transcription factors and a putative glycoside hydrolase. Furthermore, U3 cellulase shows 115% higher glucose yield from pretreated corn stover, when compared to the cellulase of T. reesei Rut-C30. These results demonstrate that AZFP can be used to improve cellulase production in T. reesei Rut-C30. Our current work offers the establishment of an alternative strategy to develop fungal cell factories for improved production of high value industrial products.

Influence of buffer systems on Trichoderma reesei Rut C-30 morphology and cellulase production

The cellulase enzyme production is a key issue in the enzymatic hydrolysis of lignocellulosic materials. Since fungal morphology influences the productivity of fungal fermentations, it is of major importance to well know the fungal behavior during culture for cellulase production. In this work, the influence of medium supplementation, with different buffer systems at two different concentrations and pH conditions, on the morphology of T. reesei Rut C-30 and cellulase production, was investigated. A medium without buffer was used as control. The results suggest that fungal morphology is significantly dependent on the addition of different buffer systems to the nutrient broth. The mycelial morphology shows a clear transition from clumped to pelleted forms in cultures with variation of buffer systems and concentration. The higher filter paper activity was obtained using 100 mM succinate buffer, at pH 4.8, in the medium supplementation, corresponding to a dispersed mycelial morphology.