SignificanceLight–oxygen–voltage (LOV) domain photoreceptors are found ubiquitously in nature and possess highly diverse signaling roles and mechanisms. Here, we show that a class of fungal LOV proteins dynamically associates with anionic plasma membrane phospholipids by a blue light-switched electrostatic interaction. This reversible association is rapidly triggered by blue light and ceases within seconds when illumination ceases. Within the native host, we predict that these proteins regulate G-protein signaling by the controlled recruitment of fused regulator of G-protein signaling (RGS) domains; in applied contexts, we anticipate that engineered chimeric versions of such proteins will be useful for rapid optogenetic membrane localization of fused proteins through direct interaction with the membrane itself, without requiring additional components to direct subcellular localization.

Natural product biosynthetic pathways generate molecules of enormous structural complexity and exquisitely tuned biological activities. Studies of natural products have led to the discovery of many pharmaceutical agents, particularly antibiotics. Attempts to harness the catalytic prowess of biosynthetic enzyme systems, for both compound discovery and engineering, have been limited by a poor understanding of the evolution of the underlying gene clusters. We developed an approach to study the evolution of biosynthetic genes on a cluster-wide scale, integrating pairwise gene coevolution information with large-scale phylogenetic analysis. We used this method to infer the evolution of type II polyketide gene clusters, tracing the path of evolution from the single ancestor to those gene clusters surviving today. We identified 10 key gene types in these clusters, most of which were swapped in from existing cellular processes and subsequently specialized. The ancestral type II polyketide gene cluster likely comprised a core set of five genes, a roster that expanded and contracted throughout evolution. A key C24 ancestor diversified into major classes of longer and shorter chain length systems, from which a C20 ancestor gave rise to the majority of characterized type II polyketide antibiotics. Our findings reveal that (i) type II polyketide structure is predictable from its gene roster, (ii) only certain gene combinations are compatible, and (iii) gene swaps were likely a key to evolution of chemical diversity. The lessons learned about how natural selection drives polyketide chemical innovation can be applied to the rational design and guided discovery of chemicals with desired structures and properties.evolution | polyketide | natural products | gene cluster

Microorganisms produce a wide range of natural products (NPs) with clinically and agriculturally relevant biological activities. In bacteria and fungi, genes encoding successive steps in a biosynthetic pathway tend to be clustered on the chromosome as biosynthetic gene clusters (BGCs). Historically, “activity-guided” approaches to NP discovery have focused on bioactivity screening of NPs produced by culturable microbes. In contrast, recent “genome mining” approaches first identify candidate BGCs, express these biosynthetic genes using synthetic biology methods, and finally test for the production of NPs. Fungal genome mining efforts and the exploration of novel sequence and NP space are limited, however, by the lack of a comprehensive catalog of BGCs encoding experimentally-validated products. In this study, we generated a comprehensive reference set of fungal NPs whose biosynthetic gene clusters are described in the published literature. To generate this dataset, we first identified NCBI records that included both a peer-reviewed article and an associated nucleotide record. We filtered these records by text and homology criteria to identify putative NP-related articles and BGCs. Next, we manually curated the resulting articles, chemical structures, and protein sequences. The resulting catalog contains 197 unique NP compounds covering several major classes of fungal NPs, including polyketides, non-ribosomal peptides, terpenoids, and alkaloids. The distribution of articles published per compound shows a bias towards the study of certain popular compounds, such as the aflatoxins. Phylogenetic analysis of biosynthetic genes suggests that much chemical and enzymatic diversity remains to be discovered in fungi. Our catalog was incorporated into the recently launched Minimum Information about Biosynthetic Gene cluster (MIBiG) repository to create the largest known set of fungal BGCs and associated NPs, a resource that we anticipate will guide future genome mining and synthetic biology efforts toward discovering novel fungal enzymes and metabolites.

We report the construction of a single-component optogenetic Rac1 (opto-Rac1) to control actin polymerization by dynamic membrane recruitment.

We describe single-component optogenetic probes whose activation dynamics depend on both light and temperature. We used the BcLOV4 photoreceptor to stimulate Ras and PI3K signaling in mammalian cells, allowing activation over a large dynamic range with low basal levels. Surprisingly, we found that BcLOV4 membrane translocation dynamics could be tuned by both light and temperature such that membrane localization spontaneously decayed at elevated temperatures despite constant illumination. Quantitative modeling predicted BcLOV4 activation dynamics across a range of light and temperature inputs and thus provides an experimental roadmap for BcLOV4-based probes. BcLOV4 drove strong and stable signal activation in both zebrafish and fly cells, and thermal inactivation provided a means to multiplex distinct blue-light sensitive tools in individual mammalian cells. BcLOV4 is thus a versatile photosensor with unique light and temperature sensitivity that enables straightforward generation of broadly applicable optogenetic tools.

to the extracellular matrix (ECM) via focal adhesions. [3] Thus, new tools for inducible control over RhoA activity may greatly enhance understanding of cytoskeletal dynamics and mechanotransduction. [4,5] Optogenetics is highly attractive for this purpose owing to its high spatiotemporal precision versus pharmacological and genetic techniques that can be encumbered by slow uptake/washout kinetics and pleotropic effects. Because small GTPases and their activating guanine nucleotide exchange factors (GEFs) signal at the plasma membrane, optogenetic membrane localization techniques are effective for inducible control over their function, where cytosol-sequestered proteins are dynamically recruited to the cytosol-facing inner leaflet of the plasma membrane to upregulate their signaling. [6] Based on earlier reported chemically inducible dimerization (CID)-based approaches for RhoA membrane recruitment, [7,8] optogenetic heterodimerization and photoactivatable chemically induced dimerization between a photo-responsive protein and a protein-binding partner (one of which is membrane localized) have been widely used to control upstream RhoA-activating GEFs [9][10][11][12][13][14] and phosphatases. [15] The heterodimerization strategy is sensitive to the stoichiometry of the two components, and thus may require expression leveltuning by clonal cell line selection and/or multiple fluorescent tags (i.e., separate for each component) at the expense of optical bandwidth otherwise useful for visualizing other fluorescent probes. [16][17][18] Previously, we reported the direct optogenetic control over RhoA GTPase itself by another mechanism of inducible clustering of RhoA fused to an oligomerizing form of plant cryptochrome, which presumably increases the binding avidity of the GTPase to membrane GEFs. However, this system has limited spatial resolution due to extensive cytosolic diffusion beyond the optical stimulation field prior to stable membrane localization post-oligomerization. [19] Recently, we reported that BcLOV4, a light-oxygen-voltage (LOV) flavoprotein from Botrytis cinerea, rapidly translocates to the plasma membrane in mammalian cells via a blue lightregulated electrostatic protein-lipid interaction (PLI) with the inner leaflet. [20,21] This direct interaction with the membrane itself is powerful for creating "single-component" tools for dynamic membrane recruitment of fused peripheral membrane proteins, without the obligate heterodimerization-or Optogenetic tools are created to control RhoA GTPase, a central regulator of actin organization and actomyosin contractility. RhoA GTPase, or its upstream activator ARHGEF11, is fused to BcLOV4, a photoreceptor that can be dynamically recruited to the plasma membrane by a light-regulated protein-lipid electrostatic interaction with the inner leaflet. Direct membrane recruitment of these proteins induces potent contractile signaling sufficient to separate adherens junctions with as little as one pulse of blue light. Induced cytoskeletal morphology changes are dependent o...

We created optogenetic tools to control RhoA GTPase, a central regulator of actin organization and actomyosin contractility. RhoA GTPase, or its upstream activating GEF effectors, were fused to BcLOV4, a photoreceptor that can be dynamically recruited to the plasma membrane by a light-regulated protein-lipid electrostatic interaction with the inner leaflet. Direct membrane recruitment of these effectors induced potent contractile signaling sufficient to separate adherens junctions in response to as little as one pulse of blue light. Cytoskeletal morphology changes were dependent on the alignment of the spatially patterned stimulation with the underlying cell polarization. RhoA-mediated cytoskeletal activation induced YAP nuclear localization within minutes and subsequent mechanotransduction, verified by YAP-TEAD transcriptional activity. These single-component tools, which do not require protein binding partners, offer spatiotemporally precise control over RhoA signaling that will advance the study of its diverse regulatory roles in cell migration, morphogenesis, and cell cycle maintenance.

We describe the efficient creation of single-component optogenetic tools for membrane recruitment-based signaling perturbation using BcLOV4 technology. The workflow requires two plasmids to create six different domain arrangements of the dynamic membrane binder BcLOV4, a fluorescent reporter, and the fused signaling protein of interest. Screening of this limited set of genetic constructs for expression characteristics and dynamic translocation in response to one pulse of light is sufficient to identify viable signaling control tools. The reliability of this streamlined approach is demonstrated by the creation of an optogenetic Cdc42 GTPase and Rac1-activating Tiam1 GEF protein, which together with our other recently reported technologies, completes a toolbox for spatiotemporally precise induction of Rho-family GTPase signaling at the GEF or GTPase level, for driving filopodial protrusions, lamellipodial protrusions, and cell contractility, respectively mediated by Cdc42, Rac1, and RhoA.