[P006] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P03-396] Engineering of probiotic Escherichia coli Nissle 1917 for 3-hydroxy-3-methylbutyrate production in an antibiotic-and inducer-free expression system

○Alice P. Du¹, Jenny J. Hung¹, Ethan I. Lan¹ (1. National Yang Ming Chiao Tung University (Taiwan))

Keywords:

Escherichia coli Nissle 1917,HMB,Antibiotic-free,Inducer-free,metabolic engineering

The global population aged 65 and older is projected to exceed 1.6 billion by 2050. Given that muscle mass and function decline progressively and accelerate with advancing age, this demographic shift highlights the urgent need for strategies to manage sarcopenia—the age-related loss of muscle mass and strength. 3-Hydroxy-3-methylbutyrate (HMB), a leucine-derived metabolite, is recognized for its potential in promoting muscle protein synthesis and reducing oxidative stress. In this study, we developed a high-value, probiotic-based production platform designed for enhanced biosafety and versatile delivery of HMB. Using the clinically established probiotic Escherichia coli Nissle 1917 (EcN) as a host, we engineered a biosynthetic pathway for the de novo production of HMB from glucose. While initial strains yielded 0.3 g/L, overcoming a key metabolic bottleneck allowed us to significantly increase the HMB concentration to 2 g/L. To enhance the biosafety and technical feasibility for future applications, we established an antibiotic-free and inducer-free expression system. This strategy addresses the inherent incompatibility between antibiotic selection and the gastrointestinal environment, maintaining plasmid stability under antibiotic-free conditions. Furthermore, by eliminating the need for chemical inducers—which present biocompatibility concerns and are difficult to maintain at optimal concentrations in vivo—this system allows for HMB production without external intervention. Our results demonstrate that we established an inducer-free and antibiotic-free expression system for HMB production in EcN, enabling a safer and more stable production platform. This strategy enhances the feasibility of in vivo applications and represents a step toward developing a sustainable probiotic-based solution for supporting muscle health in aging populations.