Bioreactor optimization of heterologous protein co-expression in a recombinant Lactobacillus spp. strain

Abstract

The development of high-performance probiotic strains capable of synthesizing therapeutic proteins requires an integrated approach combining genetic engineering with optimization of fermentation conditions. In this study, an improved three-stage cultivation protocol for a recombinant Lactobacillus spp. strain co-expressing interleukins IL-10 and IL-22 was developed and validated in a bioreactor with automatic control of critical parameters, including pH, temperature, and dissolved oxygen. Recombinant strains transformed with the pLacDualIL10/IL22 plasmid were used. Cultivation was performed in a glass bioreactor (working volume 600 mL) equipped with automated monitoring and regulation of pH, temperature, and dissolved oxygen. A three-stage режим was implemented, consisting of a growth phase (37°C, pH 6.8), an induction phase (1 mM IPTG, 30°C), and a production phase. Protein purification was carried out by tangential flow ultrafiltration followed by affinity chromatography on Ni-NTA agarose. Quantitative determination of IL-10 and IL-22 was performed using ELISA; purity was assessed by SDS-PAGE, and biological activity was evaluated using a PBMC lymphocyte proliferation assay. Application of the three-stage strategy resulted in a significant increase in target protein yield, reaching 120±15 mg/L for IL-10 and 95±10 mg/L for IL-22, which is 2—3 times higher than that achieved in flask cultivation. Subsequent purification provided high-purity proteins (>95 % and >93 %, respectively) with preserved biological activity of 1,2×106 U/mg for IL-10 and 8,5 × 105 U/mg for IL-22. The proposed methodology demonstrates high reproducibility and efficiency, confirming its potential for scalable production of functional recombinant cytokines using a probiotic platform.