Development of Novel Expression System and Bioprocess for the Production of Recombinant Human Insulin using Pseudomonas Fluorescens

Abstract

The increasing global burden of diabetes, coupled with the rising demand for oral insulin formulations, necessitates alternative expression systems to meet escalating production requirements. In this study, we report heterologous expression of human insulin in Pseudomonas fluorescens, a biosafety level-1 (BSL-1) unconventional host bacterium. Insulin was produced as a GST-proinsulin (GPI) fusion protein and purified by Ni-NTA affinity chromatography, which was subsequently converted to active insulin. Structural characterization through MALDI-TOF, RP-HPLC, and circular dichroism established similarity to the insulin standard. Shake flask cultures produced 145.35 mg/l of proinsulin fusion protein, and it was enhanced to 214.7 mg/l in batch bioreactors. Inclusion body formation was a significant bottleneck; however, combinatorial co-expression of DsbA and Pbp chaperones, in combination with customized signal peptides, greatly improved solubility (~60%). Optimization of vector components and induction parameters enhanced soluble protein yields. The GPI titer reached a maximum of 234.58 mg/l with 71.6% solubility and was partially secreted (~15%) into the culture supernatant in the bioreactor. Kinetic modeling (logistic, Luedeking-Piret, Pirt) elucidated growth, substrate utilization, and product formation dynamics, providing quantitative parameters for process scale-up. Medium optimization via Plackett-Burman and Box-Behnken designs, and ANN-GA hybrid modeling revealed that glucose, NaCl, NH₄Cl, and MgSO₄ significantly influence titer and their optimum levels. This strategy enabled a fed-batch titer of 1145 mg/l - representing a 19-fold improvement over unoptimized flask conditions. Alternative regulatory systems were created to overcome IPTG-inducible expression limitations, such as cytotoxicity and high costs. Constitutive (PPsbA) and native inducible promoters (PAnt, PBen) were used, reaching titers of up to 54.5 mg/l with improved scalability in 2 l bioreactors. This study establishes P. fluorescens as a promising and scalable host system for recombinant insulin, demonstrating improved solubility, reduced downstream burden, and an industrially relevant process, while highlighting its potential for broader therapeutic protein production.