Functional expression and secretion of basic Fibroblast Growth Factor in Lactococcus lactis

Pooi Leng Ho¹Yu Feng Chua¹Jun Ping Quek¹Say Kong Ng¹Fong Tian Wong^2*Dave Siak-Wei Ow^1*

• ¹Bioprocessing Technology Institute (A*STAR), Singapore, Singapore
• ²Institute of Molecular and Cell Biology (A*STAR), Singapore, Singapore

Cultivated meat, produced by in vitro cell culture in bioreactors, offers a sustainable alternative to traditional meat sources. A significant challenge in its production is the high cost of mitogenic growth factors, which are essential supplements in serum-free media for cultivating meat cells. One strategy to reduce cost involves minimizing purification cost by using a foodgrade host to secrete growth factors. In this study, we investigate the recombinant production of FGF2 (Fibroblast Growth Factor 2) through secretion in Lactococcus lactis, a Generally Recognized As Safe (GRAS) organism. We have generated a recombinant L. lactis strain and an optimal expression strategy to enable the production of secreted bioactive growth factors.Our results demonstrate that this system can produce FGF2 which were able to promote the proliferation of fish Anguilla japonica pre-adipocytic cells. To enhance the secretion in L. lactis, we employed the USP45 secretory peptide and a secretion propeptide (PP1). We also optimized various culture parameters that influence protein expression, including media formulation, nisin concentration, induction timing, temperature, and culture duration. Despite minimal purification beyond affinity purification and buffer exchange, we were able to obtain comparable specific activity to commercial FGF2. The final yields can be derived at 1.97 mg/L and through simple protein purification and buffer exchange. Finally, this study highlights the potential use of L. lactis secretion as an endotoxin-free alternative, compared to E. coli, for production of growth factors for use in cultivated meat production.