Enhancing the E. coli Cell Density Expressing the Recombinant Cellulolytic Chimeric Enzyme from Acetivibrio...

In this study, optimization of medium components for high cell density culturing of Escherichia coli BL21(DE3) cells expressing the gene coding for the recombinant cellulolytic chimera, CtGH1-L1-CtGH5-F194A was carried out. The chimera comprised an N-terminal β-glucosidase, of glycoside hydrolase family 1, CtGH1 and a C-terminal mutant of an β-1,4-endoglucanase of family GH5, CtGH5-F194A both from Clostridium thermocellum (now known as Acetivibrio thermocellus), fused together with natural linker L1. The growth dynamics of E. coli BL21(DE3) cells was analysed in minimal salt medium (M9) supplemented with glycerol or glucose. The glycerol-supplemented medium provided higher growth (OD₆₀₀ ~5) and lower acetic acid accumulation (0.3 g/L) than that of glucose supplemented medium giving OD₆₀₀ ~4 and acetic acid accumulation of 2.1 g/L. The optimum inoculum size of 5% (v/v) and initial medium pH 7.0 gave the maximum cell growth. The screening of medium components performed by Plackett-Burman design, revealed KH₂PO₄, (NH₄)₂HPO₄ and glycerol as significant variables influencing the cell growth. Response surface methodology (RSM) and central composite design (CCD) utilized for optimization of medium component composition gave final concentrations, KH₂PO₄ (11.22 g/L), (NH₄)₂HPO₄ (5.19 g/L) and glycerol (18.11 g/L) resulting in model predicted value of cell, OD₆₀₀ 7.7. The experimental validation of optimized concentration of medium components displayed cell, OD₆₀₀ of 7.0, fairly matching with the model predicted value. The optimised M9 medium gave 5.3 g_DCW/L with a total protein yield of 12.5 mg/g_DCW displaying 59 U/mg and 44 U/mg β-1,4-endoglucanase and β-glucosidase activity, respectively. Statistical optimization of a defined minimal medium enhanced recombinant protein yield and enzyme activity while reducing the substrate input and acetate accumulation, thereby providing a reproducible and scalable platform for enzyme production. This study establishes the first medium‑optimization framework tailored to E. coli producing a recombinant chimeric enzyme, enabling controlled growth and supporting fed‑batch strategies for high‑cell‑density culturing.