Development of a lactose-complemented antibiotic-free host-vector platform for stable enzyme expression in Limosilactobacillus fermentum 217 - 82.

Food-grade microbial bioprocessing requires genetically stable and antibiotic-free host-vector systems that are compatible with food-related applications. However, the use of Limosilactobacillus fermentum as a biomanufacturing chassis is limited by inefficient DNA delivery and the lack of strain-adapted food-grade selection strategies. In this study, a proof-of-concept, lactose-complemented antibiotic-free host-vector framework was established in L. fermentum 217 - 82. An efficient electrotransformation protocol was first established as an enabling step for host engineering, yielding a transformation efficiency of 7.50 × 10⁵ CFU/µg DNA. The genes lacM and lacLM were individually targeted to validate their involvement in lactose utilization, and subsequent gene inactivation experiments confirmed their utility as effective markers for metabolic complementation. Antibiotic-free shuttle vectors were constructed by combining lacZα-assisted plasmid screening in E. coli with host-specific lacM/lacLM-mediated lactose complementation in L. fermentum. The resulting recombinant strains maintained plasmid stability during serial passage and supported functional heterologous expression of cis-proline 4-hydroxylase. Functional validation using cis-proline 4-hydroxylase confirmed that the platform supported whole-cell L-proline-to-hydroxyproline bioconversion, with hydroxyproline production reaching 147.62 µg/mL. These findings demonstrate the feasibility of integrating lactose-dependent selection, stable antibiotic-free plasmid maintenance, functional heterologous enzyme expression, and whole-cell bioconversion within a strain-adapted host-vector framework in L. fermentum 217 - 82. Although broader applicability remains to be validated using additional heterologous proteins and process conditions, the established framework provides a methodological basis for the future development of more generally applicable antibiotic-free expression platforms in L. fermentum.