Knock-out of genes of metabolic pathways is conventionally used in the metabolic engineering of microorganisms, but it is not applicable for genes of essential pathways. In order to avoid undesirable effects caused by gene deletion, it is attractive to develop riboswitches to dynamically control the metabolic pathways of microbial cell factories. In this regard, the aim of this study is to utilize the lysine riboswitch to control gene expressions of the biosynthetic pathways and by-pathways and thus improve lysine production in Corynebacterium glutamicum. To achieve this, a natural lysine riboswitch from Lactobacillus plantarum (LPRS) was first detected and then fused with RFP to test its functionality. After that, engineered lysine-activated (Lys-A) and lysine-repressed (Lys-R) riboswitches were successfully screened by dual genetic selection. Furthermore, the optimized A263 and R152 were applied to control the expression of aspartate kinase III and homoserine dehydrogenase in the lysine-producing strain C. glutamicum QW45, respectively. In contrast with QW45, the growth of the resulting A263-lysC mutant QW48 was similar to that of QW45; however, the growth of the resulting R357-hom mutant QW54 was slightly inhibited, indicating an inhibition of threonine biosynthesis caused by the riboswitch upon binding of intracellular lysine. Importantly, the lysine production of QW48 and QW54 was, respectively, 35% and 43% higher than that of the parent strain QW45, implying more metabolic flux directed into the lysine synthesis pathway. Finally, the engineered A263 and R357 were simultaneously applied to the same mutant QW55, which greatly improved lysine production. Thus, the approach demonstrated in this work could be principally used as a powerful tool to dynamically control any other undesired metabolic pathways.

Combining immunotherapy with photodynamic therapy (PDT) can optimize their synergistic therapeutic efficacy against triple-negative breast cancer (TNBC), but the limited accumulation of photosensitizers at the tumor site and the off-target binding of PD-L1 antibody with hematopoietic cells and normal tissues severely reduce the therapeutic efficacy and increase the systemic toxicity, leading to failure in the clinical treatment. Therefore, engineered Escherichia coli Rosetta(DE3) expressing PD-L1 nanobody (Rosetta(pPD-L1nb)) are constructed and covalently conjugated with a photosensitizer, chlorin e6 (Ce6), to develop hybrid bacteria (CeRosetta(pPD-L1nb)). As a facultative anaerobe, CeRosetta(pPD-L1nb) can targetedly accumulate and colonize at the hypoxic and hypernutrient microenvironment of tumor, consistently synthesize PD-L1nb and compete for nutrition consumption with tumor cells to inhibit tumor growth. Under light irradiation, CeRosetta(pPD-L1nb) can not only generate reactive oxygen species (ROS) to directly eliminate tumor cells and induce the infiltration of immune cells into tumors and the immunogenic cell death (ICD) to activate adaptive immune responses, but also spatiotemporally release PD-L1nb due to the PDT-induced lysis in the tumor site to reverse the immunosuppressive tumor microenvironment and activate the systemic antitumor immune response, therefore exert synergistic antitumor effects of anti-PD-L1 therapy and PDT. Altogether, the hybrid bacteria CeRosetta(pPD-L1nb) can serve as an all-in-one strategy for optimizing the synergistic therapeutic efficacy in the combination therapy of TNBC.