Harnessing the evolved bacteria as cystine-addicted living biocatalyst for antitumor metabolic therapy

Bacteria-based metabolic therapy has been acknowledged as a promising strategy for tumor treatment. However, the insufficient efficiency of wild-type bacteria severely restricts their therapeutic efficacy. Here, we successfully develop a ʟ-cystine (CySS)-addicted bacterial biocatalyst for metabolic therapy through a dual-selection directed evolution strategy. Our evolved strain (namely SFEc+) exhibits a 36-fold increase in CySS uptake and a 23-fold improvement in total activity of cysteine desulfhydrases compared to the wild-type strain. By conjugating with DMXAA-loaded liposomes, the engineered biocatalyst not only prevents the influx of nutrients into the tumor microenvironment by blocking neovasculature but also achieves efficient and durable CySS catabolism locally. The absence of CySS disrupts redox homeostasis in tumor cells and strikingly increases intracellular ROS level, resulting in effective tumor elimination. In multiple murine tumor models, intravenous administration of the engineered living biocatalyst displays favorable therapeutic outcomes. Our work not only highlights the promise of directed evolution strategy in enhancing the stability and efficiency of bacteria-based living biocatalyst, but also provides the new opportunities in antitumor metabolic therapy. To underlie the mechanisms of the global reprogramming of SFEc+'s metabolic network , we performed a comprehensive transcriptomic analysis to compare the genetic expression profiles between SFEc+ and Ec.