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Review
. 2025 Jun 27;47(4):68.
doi: 10.1007/s10529-025-03602-7.

Expression of cellulase in Candida glycerogenes and strengthen the expression level for application in residue-containing fermentation to enhance glycerol production

Yuxin Gao  1   2 , Dongqi Jiang  1   2 , Mengying Wang  1   2 , Xueqing Du  1   2 , Hong Zong  1   2 , Bin Zhuge  3   4
Affiliations
Review

Expression of cellulase in Candida glycerogenes and strengthen the expression level for application in residue-containing fermentation to enhance glycerol production

Yuxin Gao et al. Biotechnol Lett. .

Abstract

Objectives: Expressing cellulase systems in C. glycerinogenes with extracellular secretion ability, and using fermentation with residue, enabled the recombinant strain to degrade lignocellulosic waste for efficient glycerol production, offering new option for agricultural waste transformation.

Results: Candida glycerinogenes is employed as the host strain, various cellulases were screened. Signal peptide mining and screening, and semi-rational design strategy was adopted in the host strain. The recombinant strain Cg4 had a total cellulase activity of 13.6 U/mL. Cg4 was applied to 110 g/L sugarcane bagasse hydrolysate with 40 g/L of pretreated bagasse as the substrate. The glycerol yield reached 50 g/L, with a 43.3% bagasse utilization rate, the amount of cellulase used in the degradation of lignocellulose was reduced by 12.1%.

Conclusion: Opened up a route for efficient degradation of agricultural waste of lignocellulose by C. glycerinogenes for glycerol production, the emission of lignocellulose waste had been reduced, and decreasing the need for cellulases in hydrolysis.

Keywords: Candida glycerinogenes; Cellulases; Glycerol; Lignocellulose; Semi-rational design; Signal peptide.

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Conflict of interest statement

Declarations. Competing interests: The authors have no relevant financial or non-financial interests to disclose. Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors. Support information: Table S1. β-Glucosidase, endoglucanase and exoglucanase derived from Trichoderma reesei and Trichoderma viride. Table S2.The strains and plasmids used in this study. Table S3. In molecular docking, the frequencies of amino acid residue sites within 5 Å of CEL3A with ligand cellobiose, EG1 with ligand CMC, and CBH2 with ligand β-glucan are determined. Table S4. PCR primer of EG1,CBH2,CEL3A and its mutants. Table S5. The signal peptides from Saccharomyces cerevisiae which are used for the expression of exogenous proteins in Pichia pastoris and the intrinsic signal peptides of C. glycerinogenes. Fig. S1. Ramachandran plot assessment analysis of the modeling of CEL3A,EG1,CBH2. The (a), (c), (e) analyses were performed by PyMOL, the (b), (d), (f) were performed by AlphaFold 2 Fig. S2. The protein concentration, total cellulase enzymatic activity, and specific enzymatic activity of the recombinant strains of the C.glycerinogenes complex cellulase after multi-strategy modification;Cg1 serves as the initial strain of the complex cellulase; Cg2 is the recombinant strain following signal peptide mining; Cg3 is the recombinant strain after semi-rational design modification of each cellulase; Cg4 is the recombinant strain integrating the signal peptide mining and semi-rational design strategy Fig. S3. Determination of extracellular CEL3A-WT and Q91, R156 and D267 mutant residues activity; Determination of extracellular EG1-WT and T272 and C385 mutant residues activity; Determination of extracellular CBH-WT and V251, G289 and L294 mutant residues activity Fig. S4. Superposition effect of CEL3A,EG1,CBH2. (a), (c), (e): Preliminary screening of mutants and superimposition schemes. (b), (d), (f): Catalytic activity of key mutants in the superposition process Fig. S5. 10% SDS-PAGE analysis of composite cellulase and its engineered strain. Lane M, the molecular mass standard; Lanes 1 to 4, crude enzyme solution of wild composite cellulase; signal peptide-engineered composite cellulase; composite cellulase modified by site-directed mutagenesis; composite cellulase engineered based on signal peptide engineering and site-directed mutagenesis

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