Construction and evaluation of recombinant Lactobacillus plantarum expressing Micropterus salmoides hepcidin

Hepcidin is an antimicrobial peptide involved in innate immunity, and the addition of hepcidin or probiotics expressing hepcidin to feed can significantly enhance the disease resistance of farmed animals. Lactobacillus plantarum has the advantages of stable colonization in the animal gut, promotion of intestinal health, and efficient expression of foreign proteins. Previously, we have used L. plantarum (LP37) as a feed additive to promote the growth of Micropterus salmoides and Oreochromis mossambicus. The aim of this study is to construct a recombinant LP37 expressing hepcidin and evaluate its role in improving antibacterial ability and growth promotion in M. salmoides.

Based on the expression vector pSIP403, we used green fluorescent protein (GFP) as a reporter gene to compare the effects of four promoters on the expression levels of foreign proteins and selected the promoter TUFA with the highest expression level. By introducing the ribosomal binding site (RBS) and secretory signal peptide sequence (SP-PGH), we constructed a novel vector capable of efficiently expressing M. salmoides hepcidin (MsHep) extracellularly. Transformation of the novel vector into LP37 to obtain the recombinant strain: LP37-MsHep. This recombinant LP37-MsHep demonstrated considerable antibacterial activity against Aeromonas hydrophila and several other microbes. The dietary supplementation of recombinant LP37-MsHep for 60 days significantly enhanced the survival rates of M. salmoides and reduced tissue bacterial load in M. salmoides post infection with A. hydrophila, and regulated gene expression of FPN1, IL-10, TNF- $\alpha$ , and MHC-II involved in iron metabolism and immunity. Similar to LP37, dietary supplementation of recombinant LP37-MsHep also significantly improved the final body weight (FBW), weight gain ratio (WGR), and specific growth rate (SGR) and reduced the feed coefficient rate (FCR).

Collectively, the recombinant LP37-MsHep is capable of significantly improving growth performance and disease resistance of M. salmoides, presenting it as a promising green feed additive in aquaculture with broad application potential.