Recombinant collagen-like protein batch production in stirred tank bioreactors

Abstract

The collagen family of proteins represents an abundant biopolymer that serves indispensable functions in maintaining the human body. Concerns over the halal/kosher status, sustainability, quality, and immunogenicity of animal-derived collagens have driven efforts to explore alternatives. Among these, recombinant bacterial collagen-like proteins emerged as prospective sustainable sources for various industries. This study investigates the scalability of a recombinant Rhodopseudomonas palustris collagen-like protein (RPCLP) production in E. coli. Before scale-up, the fermentation medium and process must be optimized to boost the growth and maximize RPCLP concentration. Hence, RPCLP was expressed in E. coli BL21(DE3) using the pColdII vector, and induction was accomplished using IPTG and temperature reduction. The optimal M9-casamino acid medium components were determined using a one-factor-at-a-time (OFAT) optimization. The screening of factors (using Taguchi OA) and, subsequently, optimization of selected factor levels (using FCCCD) to maximize RPCLP concentration were conducted in a 2 L bioreactor. Kinetic modeling was investigated by using the Monod and logistic growth models, and Luedeking-Piret models for the glucose consumption and RPCLP production. Nonlinear regression was used to determine kinetic parameters, and ANOVA analyses were conducted to evaluate the models. The fermentation was then upscaled into a 7.5 L stirred tank reactor (STR) via two scale-up criteria, namely constant oxygen mass transfer coefficient (constant kLa) and constant impeller tip speed. The soluble protein, obtained via ultrasonication, was characterized through trypsin digestion and SDS-PAGE, and purified using metal affinity chromatography. MALDI-TOF was also conducted to determine its molecular weight. The designs of the screening and optimization experiments were made by using Design-Expert software. The factors that significantly govern the growth of recombinant E. coli expressing R. palustris collagen-like proteins in the 2 L bioreactor were identified. The optimum conditions for the growth of the recombinant host cells and the concentration of recombinant collagen-like protein in the 2 L bioreactor were established. Optimum conditions yielded 2 g/L RPCLP, which is moderate compared to typical yields for recombinant collagen-like proteins. In the kinetics study, the Monod and logistic growth models fitted well, with significant R2 values (0.96 and 0.99, respectively). In contrast, the Luedeking-Piret models generated were not in accord with experimental data for glucose consumption and RPCLP production (R2 values ? 0.7). The kinetic parameters were determined, and the findings suggest that the production is partially growth-associated. The constant impeller tip speed criterion was effective as the maximum tip speed of 0.73 m/s prevents shear damage of the cells and the protein product. Contrarily, the constant kLa criterion was inadequate for scale-up, possibly due to heterogeneous environments caused by improper mixing and mass transfer during kLa measurement, leading to inaccurate estimates. Concentrations of approximately 2.1 to 2.5 g/L RPCLP were obtained in both scales using the constant impeller tip criterion. These concentrations were comparable to a previous study that obtained approximately 1 g/L recombinant Scl2 protein from a 2 L batch culture. A protein band that corresponds to the estimated size of the recombinant RPCLP from the literature was detected via SDS-PAGE. This study is the first to demonstrate successful medium optimization, kinetic modeling, and scale-up of recombinant RPCLP production to a 7.5 L bioreactor. The findings establish a protocol for an enhanced bioprocess to cater to the expanding market demands for sustainable, halal, or vegan collagens.