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

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.

1. He, Y. Probiotic potential of lactic acid bacteria isolated from yaks / Y. He, F. Li, M. Xu, Z. Wang, H. Li // Animal Diseases. — 2024. — Vol. 4, No. 1. — Art. 17. DOI: 10.1186/s44149-024-00124-z.

2. Wei, H. X. IL 10 and IL 22 in mucosal immunity: driving protection and pathology / H. X. Wei, B. Wang, B. Li // Frontiers in Immunology. — 2020. — Vol. 11. — Art. 1315. DOI: 10.3389/fimmu.2020.01315.

3. Zhang, J. Characterization of Lactobacillus spp. Isolated from layer hens as probiotic candidates / J. Zhang, A. R. McWhorter, S. Khan, Y. Sun, J. Wang // BMC Veterinary Research. — 2025. — Vol. 21, No. 1. — Art. 416. DOI: 10.1186/s12917-025-04847-0.

4. Vandermies, M. Bioreactor scale strategies for the production of recombinant protein in the yeast Yarrowia lipolytica / M. Vandermies, P. Fickers // Microorganisms. — 2019. — Vol. 7, No. 2. — Art. 40. DOI: 10.3390/microorganisms7020040.

5. Wu, J. Genetic tools for the development of recombinant lactic acid bacteria / J. Wu, Y. Xin, J. Kong, T. Guo // Microbial Cell Factories. — 2021. — Vol. 20, No. 1. — Art. 118. DOI: 10.1186/s12934-021-01607-1.

6. Yang, Y. Metabolic engineering of Lactobacilli spp. For disease treatment / Y. Yang, P. Yu, Y. Huang, W. Zhang, Y. Nie, C. Gao // Microbial Cell Factories. — 2025. — Vol. 24, No. 1. — Art. 53. DOI: 10.1186/s12934-025-02682-4.

7. Rudolph, S. E. Set up and utilization of a three dimensional in vitro bioreactor system for human intestinal studies and microbial co cultures / S. E. Rudolph, E. Bethi, L. Iglesias Ledon, H. U. Kumarasinghe, O. Da’darah, G. K. Salgam, K. C. Vieira, Y. Chen, D. L. Kaplan // Current Protocols. — 2024. — Vol. 4, No. 9. — Art. e70013. DOI: 10.1002/cpz1.70013.

8. Tian, X. Application of 8 parallel micro bioreactor system with non invasive optical pH and DO biosensor in high throughput screening of L lactic acid producing strain / X. Tian, G. Zhou, W. Wang, J. Zhang, Y. Cai // Bioresources and Bioprocessing. — 2018. — Vol. 5, No. 1. — Art. 20. DOI: 10.1186/s40643-018-0207-7.