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 been attracted increasing interest and the industrial yeast Saccharomyces cerevisiae is regarded as an excellent host for D-glucaric acid production. Results Here we constructed the biosynthetic pathway of D-glucaric acid in S. cerevisiae INVSc1 whose opi1 was knocked out 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. Due to the genetic stability and the outperformance in subsequent applications, however, LGA-1 was a preferable strain. As one of the top chemicals from biomass, there have been no reports on D-glucaric acid production from lignocellulose, which is the most abundant renewable on earth. Therefore, the biorefinery processes of lignocellulose for D-glucaric acid production including separated hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF) and consolidated bioprocessing (CBP) were investigated in this work and 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. Conclusions 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. This desirable and promising approach for direction production of D-glucaric acid from lignocellulose deserves extensive and in-depth research.

Improvement of Saccharification and Delignification Efficiency of Trichoderma reesei Rut-C30 by Genetic Bioengineering

Trichoderma reesei produces various saccharification enzymes required for biomass degradation. However, the lack of an effective lignin-degrading enzyme system reduces the species’ efficiency in producing fermentable sugars and increases the pre-treatment costs for biofuel production. In this study, we heterologously expressed the Ganoderma lucidum RMK1 versatile peroxidase gene (vp1) in the Rut-C30 strain of T. reesei. The expression of purified 6×His-tag–containing recombinant G. lucidum-derived protein (rVP1) was confirmed through western blot, which exhibited a single band with a relative molecular weight of 39 kDa. In saccharification and delignification studies using rice straw, the transformant (tVP7, T. reesei Rut-C30 expressing G. lucidum-derived rVP1) showed significant improvement in the yield of total reducing sugar and delignification, compared with that of the parent T. reesei Rut-C30 strain. Scanning electron microscopy (SEM) of tVP7-treated paddy straw showed extensive degradation of several layers of its surface compared with the parent strain due to the presence of G. lucidum-derived rVP1. Our results suggest that the expression of ligninolytic enzymes in cellulase hyperproducing systems helps to integrate the pre-treatment and saccharification steps that may ultimately reduce the costs of bioethanol production.