Ronglin He

Trpac1, a pH response transcription regulator, is involved in cellulase gene expression in Trichoderma reesei

Fungi grow over a relatively wide pH range and adapt to extracellular pH through a genetic regulatory system mediated by a key component PacC, which is a pH transcription regulator. The cellulase production of the filamentous fungi Trichoderma reesei is sensitive to ambient pH. To investigate the connection between cellulase expression regulation and ambient pH, an ortholog of Aspergillus nidulans pacC, Trpac1, was identified and functionally characterized using a target gene deletion strategy. Deleting Trpac1 dramatically increased the cellulase production and the transcription levels of the major cellulase genes at neutral pH, which suggested Trpac1 is involved in the regulation of cellulase production. It was further observed that the expression levels of transcription factors xyr1 and ace2 also increased in the ΔTrpac1 mutant at neutral pH. In addition, the ΔTrpac1 mutant exhibited conidiation defects under neutral and alkaline pH. These results implied that Trpac1 in involved in growth and development process and cellulase gene expression in T. reesei.

Strain improvement and process optimization of Trichderma reesei Rut C30 for enhanced cellulase production.

Background: Cellulase activity and production cost are the main limiting factors for the conversion of lignocellulosic biomass into biofuels and bioproductions. In particular, the high cost of cellulase production in the utilization of lignocellulosic biomass led to a poorer competiveness than starch utilization for biofuels and bioproducts. High cost of cellulase production is still a key barrier. Increasing the cellulase activity and reducing its cost have been targets for numerous research and development efforts. Results: After two rounds of mutagenesis, the mutant 30s-3-13 was obtained from Trichoderma reesei Rut C30 and exhibited more than doubled filter paper activity and carboxymethyl cellulase activity and 0.45-fold cotton activity separately. The cellulase activity and productivity reached 15.6 FPU ml-1 and 144 FPU l-1h-1 in a 5 l fermentor using the optimized process parameters. The C/N ratio, temperature and pH control were the significant factors affecting the cellulase activity. Conclusion: The results showed that the mutant 30s-3-13 was capable of high cellulase production at lower cost. The combination of strain improvement and process optimization was a promising approach for the cost-effective production of cellulase in the future.

Effects of butanol on high value product production in Schizochytrium limacinum B4D1

Schizochytrium is a microalgae-like fungus and is widely used for producing docosahexaenoic acid (DHA). It is also a promising source of squalene and carotenoids. However, few fermentation strategies are available in enhancing squalene and carotenoid content in Schizochytrium. This study showed that butanol addition had multiple effects on Schizochytrium limacinum B4D1. First, butanol addition altered the lipid content of cells. Second, 6g/L of butanol decreased the proportion of DHA by nearly 40%. Third, the squalene content increased 31-fold in the presence of 6g/L butanol. Finally, cells accumulated more carotenoids upon butanol addition. Specifically, when cells were treated with 8g/L butanol, the astaxanthin content increased to 245 times than that of the untreated control. These results are helpful for the commercial exploitation of Schizochytrium in producing squalene and carotenoids.

Construction of an efficient RNAi system in the cellulolytic fungus Trichoderma reesei

An improved RNA interference method was developed in Trichoderma reesei, using convergent dual promoters for efficient and high-throughput RNA silencing. This new vector allowed for the silencing of the eGFP gene and target genes to occur simultaneously, significantly facilitating the rapid screening of the transformants using eGFP as a reporter.

Genome sequence of Talaromyces piceus 9-3 provides insights into lignocellulose degradation

Many species of Penicillium have exhibited great potential for lignocellulose hydrolysis. The filamentous fungus Talaromyces piceus 9-3 (anamorph: Penicillium piceum), which was isolated from compost wastes in China, was sequenced in this study. Compared with the cellulase producer T. reesei, T. piceus 9-3 processes a lignocellulolytic enzyme system comprising more diverse enzymatic components, especially hemicellulases. This report will facilitate the use of this strain for biomass degradation.

Protein disulfide isomerase homolog TrPDI2 contributing to cellobiohydrolase production in Trichoderma reesei.

The majority of the cysteine residues in the secreted proteins form disulfide bonds via protein disulfide isomerase (PDI)-mediated catalysis, stabilizing the enzyme activity. The role of PDI in cellulase production is speculative, as well as the possibility of PDI as a target for improving enzyme production efficiency of Trichoderma reesei, a widely used producer of enzyme for the production of lignocellulose-based biofuels and biochemicals. Here, we report that a PDI homolog, TrPDI2 in T. reesei exhibited a 36.94% and an 11.81% similarity to Aspergillus niger TIGA and T. reesei PDI1, respectively. The capability of TrPDI2 to recover the activity of reduced and denatured RNase by promoting refolding verified its protein disulfide isomerase activity. The overexpression of Trpdi2 increased the secretion and the activity of CBH1 at the early stage of cellulase induction. In addition, both the expression level and redox state of TrPDI2 responded to cellulase induction in T. reesei, providing sustainable oxidative power to ensure cellobiohydrolase maturation and production. The results suggest that TrPDI2 may contribute to cellobiohydrolase secretion by enhancing the capability of disulfide bond formation, which is essential for protein folding and maturation.

Synthesizing value-added products from methane by a new Methylomonas

Methane and methanol are potential carbon sources of industrial micro‐organisms in addition to crop‐derived bio‐carbon sources. Methanotrophs that can utilize these simple, stable and large amounts chemicals are expected to be developed into ‘cell factories’ for the production of specific chemicals. In this study, a methanotroph that can synthesize lycopene, C30 carotenoid and exopolysaccharides (EPS) with relative better performances from C1 substrates was isolated, and its performances were evaluated.

Pretreating cellulases with hydrophobins for improving bioconversion of cellulose: an experimental and computational study

Lignocellulosic biomass sugars bring benefits to both the economy and the environment, but their application is limited by their high process cost. In the present contribution, the class II hydrophobin (HFBII) was first reported to be used as an additive to pretreat cellulases prior to hydrolysis, leading to a remarkable improvement of the biodegradation of corn stover and microcrystalline cellulose. 2 mg g−1 HFBII resulted in 37.1% and 55.4% conversion of the substrates, increased by 32.5% and 40.6%, respectively, compared with the control. In particular, HFBII was shown to have a better effect than polyethylene glycol 6000 on the conversion of corn stover, increasing the degradation by 24.5%. 73.1% enzymatic activity was retained after 48 h, whereas the control was 65.3%. Furthermore, the binding of HFBII to cellulase was investigated by molecular dynamics simulations and free-energy calculations. The tunnel-forming loops of the cellulase exhibit a high binding affinity for HFBII. In the formed complex, about 70% hydrophobic patches of the HFBII were found to be exposed, which could compete with the cellulase for hydrophobic adsorption sites of lignin residues. The decrease of the adsorption between the latter two would benefit the enzymatic hydrolysis. Structural analysis indicates that the flexibility of enzymatic tunnel loops was significantly enhanced, and the active area was enlarged, thereby promoting the enzymatic activity. The experimental results and the underlying mechanism provided herein are envisioned to help understand the potential application of HFBII in the practical green hydrolysis of cellulose by reducing the enzyme load.

An ethanolamine kinase Eki1 affects radial growth and cell wall integrity in Trichoderma reesei

Ethanolamine kinase (ATP:ethanolamine O-phosphotransferase, EC 2.7.1.82) catalyzes the committed step of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. The functions of eki genes that encode ethanolamine kinase have been intensively studied in mammalian cells, fruit flies and yeast. However, the role of the eki gene has not yet been characterized in filamentous fungi. In this study, Treki1, an ortholog of Saccharomyces cerevisiae EKI1, was identified and functionally characterized using a target gene deletion strategy in Trichoderma reesei. A Treki deletion mutant was less sensitive to cell wall stressors calcofluor white and Congo red and released fewer protoplasts during cell wall digestion than the parent strain QM9414. Further transcription analysis showed that the expression levels of five genes that encode chitin synthases were drastically increased in the ΔTreki1 mutant. The chitin content was also increased in the null mutant of Treki1 comparing to the parent strain. In addition, the ΔTreki1 mutant exhibited defects in radial growth, conidiation and the accumulation of ethanolamine. The results indicate that Treki1 plays a key role in growth and development and in the maintenance of cell wall integrity in T. reesei.

A β-glucosidase Hyperproducing Strain, Pencillium piceum: Novel Characterization of Lignocellulolytic Enzyme Systems and Its Application in Biomass Bioconversion

In this chapter, the β-glucosidase over producing strain Pencillium piceum and the β-glucosidase characteristics will be introduced. Through several rounds of dimethyl sulfate mutagenesis, the β-glucosidase activity of P. piceum reached 53.12 IU/ml. Two new β-glucosidases, promising bifunctional enzymes for lignocellulosic bioconversion, have been found in the extracellular protein of P. piceum. The two new β-glucosidases played an important role in forming multiple soluble cellulose inducers via high transglycosylation activity and novel enzymatic activity. Further, the two new β-glucosidases showed the strong synergism with different cellulases by removing multiple inhibitors for cellulase. The rational computer-aided strategies were devised to enhance the thermostability of the main β-glucosidase (Cel3A) from Penicillium piceum H16. Pencillium piceum, high-yielding β-glucosidase with high enzymatic activity and good thermostability, may provide a good synergetic effect on Trichoderma reesei for improving cellulose hydrolysis of different substrates.