Production of recombinant human insulin using constitutive and non-IPTG inducible promoters in Pseudomonas fluorescens and scale-up study

Abstract

Diabetes mellitus, a complex metabolic disorder, ranks among the most widespread diseases globally. Recombinant human insulin is crucial for diabetes treatment, although lifestyle modifications are also recommended. Insulin is majorly produced in bacterial host systems as it does not require any complex PTMs to be functionally active. Traditionally, IPTG-inducible promoters drive expression in most of these systems; however, IPTG is both cytotoxic and cost-intensive. This study explored alternative regulatory elements, including constitutive and native inducible promoters, in the Pseudomonas fluorescens expression platform. The proinsulin fusion protein was expressed at titers up to 54.5 mg/L in a 2 L bioreactor, yielding a ∼29 % increase compared to shake-flask using a constitutive promoter (P_PsbA)-driven system. Anthranilate (5 mM) and m-toluate (7.5 mM) were identified as optimal inducers for anthranilate (P_Ant) and benzoate (P_Ben) promoter-based expression systems, respectively, with systematic screening and optimization of various inducers and their concentrations. Scaling up to a 2 L bioreactor further enhanced production, with P_Ant-mediated expression achieving a ∼7% titer increase over flask-scale cultures. The fusion protein and insulin produced through these systems were validated by Western blotting, and insulin integrity was confirmed via MALDI-TOF. Both P_Ant- and P_Ben-based expression systems can be used for differential expression of recombinant proteins such as insulin chains, as well as the heavy and light chains of monoclonal antibodies. The present study demonstrates a process for the IPTG-free expression of recombinant human insulin in a P. fluorescens-based host system.