Evaluation of the Enzymatic Arsenal Secreted by Myceliophthora thermophila During Growth on Sugarcane Bagasse With a Focus on LPMOs

Abstract After chemical pretreatment, improved amenability of agrowaste biomass for enzymatic saccharification needs an understanding of the effect exerted by pretreatments on biomass for enzymatic deconstruction. In present studies, NaOH, NH4OH and H2SO4 pretreatments effectively changed visible morphology imparting distinct fibrous appearance to sugarcane bagasse (SCB). Filtrate analysis after NaOH, NH4OH and H2SO4 pretreatments yielded release of soluble reducing sugars (SRS) in range of ~0.17–0.44%, ~0.38–0.75% and ~2.9–8.4% respectively. Gravimetric analysis of pretreated SCB (PSCB) biomass also revealed dry weight loss in range of ~25.8–44.8%, ~11.1–16.0% and ~28.3–38.0% by the three pretreatments in the same order. Release of soluble components other than SRS, majorly reported to be soluble lignins, were observed highest for NaOH followed by H2SO4 and NH4OH pretreatments. Decrease or absence of peaks attributed to lignin and loosened fibrous appearance of biomass during FTIR and SEM studies respectively further corroborated with our observations of lignin removal. Application of commercial cellulase increased raw SCB saccharification from 1.93% to 38.84%, 25.56% and 9.61% after NaOH, H2SO4 and NH4OH pretreatments. Structural changes brought by cell wall degrading enzymes were first time shown visually confirming the cell wall disintegration under brightfield, darkfield and fluorescence microscopy. The microscopic evidence and saccharification results proved that the chemical treatment valorized the SCB by making it amenable for enzymatic saccharification.

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Dissecting cellobiose metabolic pathway and its application in biorefinery through consolidated bioprocessing in Myceliophthora thermophila

Fungal Biology and Biotechnology ◽

10.1186/s40694-019-0083-8 ◽

2019 ◽

Vol 6 (1) ◽

Cited By ~ 1

Author(s):

Jingen Li ◽

Shuying Gu ◽

Zhen Zhao ◽

Bingchen Chen ◽

Qian Liu ◽

...

Keyword(s):

Malic Acid ◽

Plant Biomass ◽

Consolidated Bioprocessing ◽

Cellulase Activity ◽

Industrial Applications ◽

Efficient Production ◽

Myceliophthora Thermophila ◽

Final Strain ◽

Cellobiose Metabolism ◽

Cost Efficient

Abstract Background Lignocellulosic biomass has long been recognized as a potential sustainable source for industrial applications. The costs associated with conversion of plant biomass to fermentable sugar represent a significant barrier to the production of cost-competitive biochemicals. Consolidated bioprocessing (CBP) is considered a potential breakthrough for achieving cost-efficient production of biomass-based fuels and commodity chemicals. During the degradation of cellulose, cellobiose (major end-product of cellulase activity) is catabolized by hydrolytic and phosphorolytic pathways in cellulolytic organisms. However, the details of the two intracellular cellobiose metabolism pathways in cellulolytic fungi remain to be uncovered. Results Using the engineered malic acid production fungal strain JG207, we demonstrated that the hydrolytic pathway by β-glucosidase and the phosphorolytic pathway by phosphorylase are both used for intracellular cellobiose metabolism in Myceliophthora thermophila, and the yield of malic acid can benefit from the energy advantages of phosphorolytic cleavage. There were obvious differences in regulation of the two cellobiose catabolic pathways depending on whether M. thermophila JG207 was grown on cellobiose or Avicel. Disruption of Mtcpp in strain JG207 led to decreased production of malic acid under cellobiose conditions, while expression levels of all three intracellular β-glucosidase genes were significantly up-regulated to rescue the impairment of the phosphorolytic pathway under Avicel conditions. When the flux of the hydrolytic pathway was reduced, we found that β-glucosidase encoded by bgl1 was the dominant enzyme in the hydrolytic pathway and deletion of bgl1 resulted in significant enhancement of protein secretion but reduction of malate production. Combining comprehensive manipulation of both cellobiose utilization pathways and enhancement of cellobiose uptake by overexpression of a cellobiose transporter, the final strain JG412Δbgl2Δbgl3 produced up to 101.2 g/L and 77.4 g/L malic acid from cellobiose and Avicel, respectively, which corresponded to respective yields of 1.35 g/g and 1.03 g/g, representing significant improvement over the starting strain JG207. Conclusions This is the first report of detailed investigation of intracellular cellobiose catabolism in cellulolytic fungus M. thermophila. These results provide insights that can be applied to industrial fungi for production of biofuels and biochemicals from cellobiose and cellulose.

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