Diabetes, a metabolic disorder, ranks among the top 10 leading causes of death globally (Lin et al., 2020). Blood glucose level soar in this medical condition, which often leads to severe cardiovascular and neurological complications over time, potentially resulting in death if untreated (Dal Canto et al., 2019, Luna et al., 2021). In diabetes, there is either insufficient insulin production or insulin resistance, necessitating external intervention to regulate blood sugar levels (Sahoo et al., 2024a). Along with dietary and lifestyle modifications, the use of recombinant human insulin is recommended for managing diabetes (Dal Canto et al., 2019). However, the rising number of diabetic patients, the preference for oral insulin (which requires higher doses), and strict intellectual property regulations have made insulin increasingly unaffordable. Recent studies suggest that a novel expression system is needed to overcome the shortcomings of current expression systems employed for insulin production (Bhoria et al., 2022, Sahoo et al., 2024a).

Pseudomonas fluorescens, a BSL-1-gram negative bacterium, has been reported to produce multiple heterologous proteins in their soluble state. It can produce heterologous proteins that account for more than 50 % of the total cell protein. It has the capability of industrial-scale fermentation, such as ∼10 kl culture volume consisting of mineral media supplemented with a carbon source. Notably, P. fluorescens does not accumulate acetate, a byproduct that typically impairs the fermentation efficiency in microbes (Das et al., 2022; Retallack et al., 2012). However, proteases of the host cell have a detrimental effect on recombinant proteins, especially small-sized proteins (<∼15 kDa) such as insulin and proinsulin etc. (Sakhel et al., 2021). Strategies like deleting or inhibiting host proteases through strain engineering or protease inhibitors often reduce the host's robustness, leading to lower yields and titers of the target protein during fermentation (Öktem et al., 2023). To mitigate these challenges, the protein is often expressed in conjunction with a fusion partner, ideally a native protein from the host organism. In our previous study, we successfully expressed human insulin fused with native Glutathione-S-transferase (GST) and DsbA in P. fluorescens. However, only cytoplasmic expression was achieved majorly in the form of inclusion bodies, and higher soluble protein was achieved by lowering the culture temperature at the expense of loss in the final titer (Sahoo et al., 2024b).

Chaperones, also known as heat shock proteins, assist nascent proteins in proper folding. Although host cells naturally possess chaperones, the rapid synthesis of foreign proteins often necessitates the overexpression of native chaperones or the introduction of heterologous chaperones (Fatima et al., 2021, Mamipour et al., 2017). Similarly, signal peptides are critical for the correct cellular localization and secretion of target proteins. They also play a role in protein folding, such as facilitating disulfide bond formation by directing the synthesized protein to the periplasmic oxidizing environment. Native signal peptides offer an additional benefit by protecting recombinant proteins from host proteases (Freudl, 2018, Owji et al., 2018). The synergistic effect of chaperone overexpression and signal peptide expression can enhance the production of soluble proteins and their transportation to either a desired cellular location or the fermentation supernatant. Moreover, it has been observed that properly folded proteins are less susceptible to proteolytic degradation compared to their unfolded counterparts (Wingfield, 2015).

Recombinant proteins are often expressed with peptide tags such as the 6xHis-tag or GST tag to facilitate purification. The 6xHis-tag is commonly used for purification via Ni-NTA affinity chromatography (Silva et al., 2021). However, the position of the His-tag (whether at the N- or C-terminal of the target protein) can significantly impact the final titer, solubility and quality of the protein (Köppl et al., 2022). When expressing the target protein as a fusion construct, placing the His-tag between the fusion partner and the target protein allows for the easy removal of both the fusion partner and the His-tag after purification. It is crucial that the His-tag remains sufficiently exposed after folding to ensure effective interaction with the Ni-NTA resin during purification. While vectors with a low-copy replication origin (ori) often result in higher yields of soluble protein, vectors with a high-copy ori can sometimes produce higher titers of the target protein, either in a soluble form or as inclusion bodies (Lozano Terol et al., 2021). Additionally, using antibiotic resistance encoding genes as selectable markers in vectors introduces metabolic pressure on the host. Different antibiotics, each with distinct mechanisms of action, may influence the final titer of the target protein (Peubez et al., 2010). Therefore, optimizing the location of the His-tag, ori type, and selection markers is essential for maximizing protein yield and quality.

The concentration of antibiotics is crucial for maintaining plasmid stability (adequate plasmid copies) without adversely affecting cell growth and protein titer (Feizollahzadeh et al., 2017, Grijalva-Hernández et al., 2019). A suboptimal inoculum size leads to a prolonged lag phase where delayed biomass build-up and slower metabolic activation are observed. Conversely, a superoptimal inoculum size causes rapid nutrient depletion and accumulation of metabolic waste. Ultimately, improper inoculum size leads to lower titer and productivity in the recombinant protein production process, which necessitates inoculum size optimization (ZHANG et al., 2015). The optical density (OD) at the time of induction must be carefully optimized to ensure that the cell culture is in a metabolically active state to support high-level protein synthesis (Rosano and Ceccarelli, 2014). It is also essential that the gene expression is induced before the late logarithmic growth phase, as induction in the late logarithmic or stationary phase can result in a reduced yield of growth-associated products (Mühlmann et al., 2017). When tac promoter is used, Isopropyl β-d-1-thiogalactopyranoside (IPTG) is used as an inducer. Enough IPTG is required to initiate transcription, but it is expensive and cytotoxic at higher concentrations. Also, the amount of IPTG and OD of the culture at the time of induction affects the solubility of the expressed protein. Hence, physicochemical parameters such as antibiotic concentration, incoculum percentage, OD for induction and IPTG concentration require optimization (Rouhani et al., 2020, Singha et al., 2021).

In this study, optimal protein titer was achieved by systematically screening and selecting the appropriate plasmid vector ori, His-tag placement and suitable antibiotic resistance encoding gene for selection. We examined both the individual and combined effects of chaperones and signal peptides, with further process parameter optimization to enhance protein expression. A bioreactor study was conducted to assess host cell behavior and protein solubility at a larger fermentation scale. The secondary structure of the purified protein was characterized and validated through circular dichroism (CD) spectroscopy and compared with commercial insulin to ensure similarity.