Flavin-binding fluorescent proteins (FPs) are genetically encoded in vivo reporters, which are derived from microbial and plant LOV photoreceptors. In this study, we comparatively analyzed ROS formation and light-driven antimicrobial efficacy of eleven LOV-based FPs. In particular, we determined singlet oxygen (1O2) quantum yields and superoxide photosensitization activities via spectroscopic assays and performed cell toxicity experiments in E. coli. Besides miniSOG and SOPP, which have been engineered to generate 1O2, all of the other tested flavoproteins were able to produce singlet oxygen and/or hydrogen peroxide but exhibited remarkable differences in ROS selectivity and yield. Accordingly, most LOV-FPs are potent photosensitizers, which can be used for light-controlled killing of bacteria. Furthermore, the two variants Pp2FbFP and DsFbFP M49I, exhibiting preferential photosensitization of singlet oxygen or singlet oxygen and superoxide, respectively, were shown to be new tools for studying specific ROS-induced cell signaling processes. The tested LOV-FPs thus further expand the toolbox of optogenetic sensitizers usable for a broad spectrum of microbiological and biomedical applications.

Biotechnological production in bacteria enables access to numerous valuable chemical compounds. Nowadays, advanced molecular genetic toolsets, enzyme engineering as well as the combinatorial use of biocatalysts, pathways, and circuits even bring new-to-nature compounds within reach. However, the associated substrates and biosynthetic products often cause severe chemical stress to the bacterial hosts. Species of the Pseudomonas clade thus represent especially valuable chassis as they are endowed with multiple stress response mechanisms, which allow them to cope with a variety of harmful chemicals. A built-in cell envelope stress response enables fast adaptations that sustain membrane integrity under adverse conditions. Further, effective export machineries can prevent intracellular accumulation of diverse harmful compounds. Finally, toxic chemicals such as reactive aldehydes can be eliminated by oxidation and stress-induced damage can be recovered. Exploiting and engineering these features will be essential to support an effective production of natural compounds and new chemicals. In this article, we therefore discuss major resistance strategies of Pseudomonads along with approaches pursued for their targeted exploitation and engineering in a biotechnological context. We further highlight strategies for the identification of yet unknown tolerance-associated genes and their utilisation for engineering next-generation chassis and finally discuss effective measures for pathway fine-tuning to establish stable cell factories for the effective production of natural compounds and novel biochemicals.

Diseases caused by multi-drug resistant pathogens have become a global concern. Therefore, new approaches suitable for treating these bacteria are urgently needed. In this study, we analyzed genetically encoded photosensitizers (PS) related to the green fluorescent protein (GFP) or light-oxygen-voltage (LOV) photoreceptors for their exogenous applicability as light-triggered antimicrobial agents. Depending on their specific photophysical properties and photochemistry, these PSs can produce different toxic ROS (reactive oxygen species) such as O2•− and H2O2 via type-I, as well as 1O2 via type-II reaction in response to light. By using cell viability assays and microfluidics, we could demonstrate differences in the intracellular and extracellular phototoxicity of the applied PS. While intracellular expression and exogenous supply of GFP-related PSs resulted in a slow inactivation of E. coli and pathogenic Gram-negative and Gram-positive bacteria, illumination of LOV-based PSs such as the singlet oxygen photosensitizing protein SOPP3 resulted in a fast and homogeneous killing of these microbes. Furthermore, our data indicate that the ROS type and yield as well as the localization of the applied PS protein can strongly influence the antibacterial spectrum and efficacy. These findings open up new opportunities for photodynamic inactivation of pathogenic bacteria.