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BackgroundCellulase is the one of the largest contributors to the high production costs of the lignocellulose-based biorefineries. As the most widely used cellulase producer, Trichoderma reesei has two weaknesses, deficiencies in β-glucosidase and cellobiohydrolase II. This work aimed at solving this problem by simultaneous enhancement of the beta–exo synergism and exo–exo synergism in T. reesei cellulase to increase the cellulose degrading capability, i.e. enhanced co-expression of the β-glucosidase gene the cellobiohydrolase II gene of T. reesei.ResultsEnhanced co-expression of the β-glucosidase gene and the cellobiohydrolase II gene in T. reesei using the strong promoter Pcbh1 was found successful in overcoming the two weaknesses. Filter paper activities of T. reesei cellulase were greatly elevated, which were 7.21 ± 0.45 (E7, Aabgl1 and Trcbh2) and 7.69 ± 0.42 (F6, Anbgl1 and Trcbh2) FPIU/mL. They were much higher than that of the parental strain Rut-C30, 2.45 ± 0.36 FPIU/mL. Enzymatic hydrolysis yields were also improved, from 67.22 ± 1.61% by Rut-C30 cellulase to 87.98 ± 0.65% by E7 cellulase and 86.50 ± 1.01% by F6 cellulase. The substrate loading for 1 g glucose release from SECS were decreased, from 2.9637 g SECS using Rut-C30 cellulase to 2.0291 g SECS using E7 cellulase and 2.0573 g SECS using F6 cellulase. As a result, the efficiency of the process from SECS to glucose was substantially improved.ConclusionsEnhanced co-expression of the β-glucosidase gene and the cellobiohydrolase II gene in T. reesei using the strong promoter Pcbh1 in T. reesei was proven triumphal in the simultaneous enhancement of the beta–exo synergism and exo–exo synergism in T. reesei cellulase. This strategy also improved the cellulase production, enzymatic hydrolysis yield and the efficiency of the process from SECS to glucose in the context of on-site cellulase production. This work is a commendable attempt in the cellulase composition optimization at the transcriptional level.

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.

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Neurospora crassa is a filamentous fungus potent in secreting cellulase and degrading lignocellulosic materials. Here, heterologous cis-aconitic acid decarboxylase was expressed in N. crassa to synthesize itaconic acid which is a high potential platform chemical with applications as an alternative for petroleum-based products. The present study demonstrated that itaconic acid can be produced directly from cellulose and other lignocellulosic materials by the engineered strain, with the highest production of 20.414 ± 0.674 mg/L. The multivariate data analysis methods were used in the parameter analysis of the conversion process. It was found by the hot map analysis that itaconic acid production can promote the secretion of cellulase in N. crassa. Principal component analysis suggested that itaconic acid production was closely related to the concentration of the glucose degraded from lignocelluloses, indicating that the secretion of cellulase is key to the direct conversion of cellulose to itaconic acid in the engineered N. crassa. This work demonstrates that N. crassa could be considered as a new platform in the application of cellulose conversion to itaconic acid.

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