Osipova, Zinaida M

Makhin A.P., Miturich V.S., Vavilov M.V., Lyakhovich M.S., Andrianova A.A., Zagitova R.I., Shmygarev V.I., Fadeeva A.A., Yatskin O.N., Belozerova O.A., Tsatsakis A., Yampolsky I.V., Kaskova Z.M.

In the light of recent progress in the development of SARS-CoV-2 main protease inhibitors, the synthesis of their key fragment, heterocyclic amino acids, is of great interest. Here, we report a method for the preparation of two new quisqualic acid analogs containing hydantoin and imidazolidinone moieties. The hydantoin analog was obtained using an amide ester cyclization, while the imidazolidinone unit was constructed by reductive amination and subsequent cyclization of a substituted ethylenediamine with carbonyldiimidazole. The presented approach provides the convergent synthesis of target analogs in 8 and 5 steps respectively.

Stevani C.V., Zamuner C.K., Bastos E.L., de Nóbrega B.B., Soares D.M., Oliveira A.G., Bechara E.J., Shakhova E.S., Sarkisyan K.S., Yampolsky I.V., Kaskova Z.M.

More than 125 known species of fungi, all part of the Agaricales order, can spontaneously emit light. This bioluminescence results from the oxidation of a luciferin derived from caffeic acid by oxygen under the action of the enzyme luciferase. The production and regeneration of caffeic acid tie together the Krebs cycle and the Shikimic Acid pathway in both fungi and plants. Therefore, successful genetic manipulation of luciferase has led to the development of bioluminescent reporters and eukaryotic organisms that exhibit self-sustained glow. This review aims to discuss the underlying mechanisms of fungal bioluminescence, with a focus on the biochemical and chemical processes that lead to light emission, along with an elaboration on its extensive biotechnological applications.

Kotlobay A.A., Dubinnyi M.A., Kovalchuk S.I., Makhin A.P., Miturich V.S., Lyakhovich M.S., Fontaine D.M., Southworth T.L., Shmygarev V.I., Yatskin O.N., Branchini B.R., Yampolsky I.V., Kaskova Z.M.

Bioluminescence of insects is a well-known natural phenomenon in the focus of interest of scientific research. While the mechanisms of bioluminescence in Coleoptera have been extensively studied, there is a lack of information about the chemistry of light emission in Diptera species. Here we report the Keroplatus spp. oxyluciferin structure elucidation and identification as 3-hydroxykynurenic acid. Additionally, the present study provides the first direct evidence of the relationship between the bioluminescent systems of Orfelia and Keroplatus. However, the properties of the putative Orfelia oxyluciferin suggest that the light emission mechanisms are not identical.

Bolt Y.V., Dubinnyi M.A., Litvinenko V.V., Kotlobay A.A., Belozerova O.A., Zagitova R.I., Shmygarev V.I., Yatskin O.N., Guglya E.B., Kublitski V.S., Baranov M.S., Yampolsky I.V., Kaskova Z.M., Tsarkova A.S.

Zagitova R.I., Purtov K.V., Shcheglov A.S., Mineev K.S., Dubinnyi M.A., Myasnyanko I.N., Belozerova O.A., Pakhomova V.G., Petushkov V.N., Rodionova N.S., Lushpa V.A., Guglya E.B., Kovalchuk S., Kozhemyako V.B., Mirza J.D., et. al.

Biochemistry of bioluminescence of the marine parchment tubeworm Chaetopterus has been in research focus for over a century; however, the results obtained by various groups contradict each other. Here, we report the isolation and structural elucidation of three compounds from Chaetomorpha linum algae, which demonstrate bioluminescence activity with Chaetopterus luciferase in the presence of Fe2+ ions. These compounds are derivatives of polyunsaturated fatty acid peroxides. We have also obtained their structural analogues and demonstrated their activity in the bioluminescence reaction, thus confirming the broad substrate specificity of the luciferase.

Kotlobay A.A., Dubinnyi M.A., Polevoi A.V., Kovalchuk S.I., Kaskova Z.M.

Abstract Keroplatus is a genus of fungus gnats family Keroplatidae (Diptera, Bibionomorpha). Larvae of some species emit a constant blue light from the body. The bioluminescence of Keroplatidae is one of the least studied of all terrestrial insects and very few facts are known to date of its biology and biochemistry. Here we report the high level of riboflavin in Keroplatus testaceus larvae, a fluorescent compound that might be relative to its bioluminescent system. We suppose that riboflavin may play a role in Keroplatus spp. bioluminescence.

Kaskova Z.M., Bolt Y.V., Baleeva N.S., Nelyubina Y.V., Andrianova A.A., Tsarkova A.S.

AbstractWe report a simple two-step method for the synthesis of a novel highly fluorescent benzothiophene-based dye comprising five fused rings and exhibiting a large Stokes shift (Δλ = 152 nm or Δν = 5482 cm–1 in ethanol). Structural features of the obtained compound allow easy functionalization of the carbon core and open new possibility for the development of a series of new classes of fluorescent dyes.

Burakova L.P., Lyakhovich M.S., Mineev K.S., Petushkov V.N., Zagitova R.I., Tsarkova A.S., Kovalchuk S.I., Yampolsky I.V., Vysotski E.S., Kaskova Z.M.

Ca2+-regulated photoproteins of ctenophores lose bioluminescence activity when exposed to visible light. Little is known about the chemical nature of chromophore photoinactivation. Using a total synthesis strategy, we have established the structures of two unusual coelenterazine products, isolated from recombinant berovin of the ctenophore Beroe abyssicola, which are Z/E isomers. We propose that during light irradiation, these derivatives are formed from 2-hydroperoxycoelenterazine via the intermediate 8a-peroxide by a mechanism reminiscent of that previously described for the auto-oxidation of green-fluorescent-protein-like chromophores.

Dubinnyi M.A., Ivanov I.A., Rodionova N.S., Kovalchuk S.I., Kaskova Z.M., Petushkov V.N.

AbstractCold extract from bioluminescent earthworm Henlea sp. was studied by HPLC, 1D and 2D NMR and LC‐HRMS analysis. An abundant structural analog of the luciferin was isolated and identified as α‐C‐mannosyltryptophan (ManTrp), the product of unusual C2‐glycosylation found earlier in humans, ascidians and other animals. Two compounds in cold extract (P300b, P300c) were characterized as C2‐substituted derivatives of tryptophan. We hypothesize that a series of tryptophan‐containing compounds are possible participants of bioluminescence‐related metabolism in Henlea sp.

Kotlobay A.A., Kaskova Z.M., Yampolsky I.V.

Optoanalytical methods based on using genetically encoded bioluminescent enzymes,luciferases, allow one to obtain highly sensitive signals, are non-invasive, and require no external irradiation. Bioluminescence is based on the chemical reaction of oxidation of a low-molecular-weight substrate (luciferin) by atmospheric oxygen, which is catalyzed by an enzyme (luciferase). Relaxation of the luciferin oxidation product from its excited state is accompanied by a release of a quantum of light, which can be detected as an analytical signal.The ability to express luciferase genes in various heterological systems and high quantum yields of luminescence reactions have made these tools rather popular in biology and medicine. Amongseveral naturally available luciferases, a few have been found to be useful for practicalapplication. Luciferase size, the wavelength of its luminescence maximum, enzyme thermostability, optimal pH of the reaction, and the need for cofactors areparameters that may differ for luciferases from different groups of organisms, and this fact directly affects the choice of the application area for each enzyme. It is quite important to overview the whole range of currently available luciferases based ontheir biochemical properties before choosing one bioluminescent probe suitable for a specific application.

Osipova Z.M., Shcheglov A.S., Yampolsky I.V.

Bioluminescent systems are increasingly being used for the development of highly sensitive optical imaging techniques in vivo. However, it is necessary to inject expensive and unstable synthetic substrates (luciferins) before each analysis for most of the systems applied. Autonomous bacterial and fungal bioluminescent systems, that recently have become available for implementation in eukaryotic cells, in our opinion, may be developed into an effective tool in new technologies of bioluminescent imaging.

Kotlobay A.A., Dubinnyi M.A., Purtov K.V., Guglya E.B., Rodionova N.S., Petushkov V.N., Bolt Y.V., Kublitski V.S., Kaskova Z.M., Ziganshin R.H., Nelyubina Y.V., Dorovatovskii P.V., Eliseev I.E., Branchini B.R., Bourenkov G., et. al.

Significance We report the identification and characterization of the small molecule aspects of the Odontosyllis undecimdonta bioluminescence system. The chemical structures of the 4 best-known marine luciferins are as diverse as their phylogenetic distribution. The unique structure of Odontosyllis luciferin provides a key insight into a completely novel chemical basis of bioluminescence. Odontosyllis oxyluciferin is the only green primary emitter described for any known bioluminescent marine organism. Together with Odontosyllis luciferase, our recent findings provide insight into the biochemistry and photochemistry of a new light-emitting system. Our studies represent a crucial step in the development of orthogonal luminescence-based analytical methods for a variety of applications, including live animal imaging and pharmaceutical development. Marine polychaetes Odontosyllis undecimdonta, commonly known as fireworms, emit bright blue-green bioluminescence. Until the recent identification of the Odontosyllis luciferase enzyme, little progress had been made toward characterizing the key components of this bioluminescence system. Here we present the biomolecular mechanisms of enzymatic (leading to light emission) and nonenzymatic (dark) oxidation pathways of newly described O. undecimdonta luciferin. Spectral studies, including 1D and 2D NMR spectroscopy, mass spectrometry, and X-ray diffraction, of isolated substances allowed us to characterize the luciferin as an unusual tricyclic sulfur-containing heterocycle. Odontosyllis luciferin does not share structural similarity with any other known luciferins. The structures of the Odontosyllis bioluminescent system’s low molecular weight components have enabled us to propose chemical transformation pathways for the enzymatic and nonspecific oxidation of luciferin.

Shimomura O., Oba Y., Stevani C.V., Tsarkova A.S., Kaskova Z.M.

Shimomura O., Stevani C.V., Kaskova Z.M., Tsarkova A.S., Yampolsky I.V.

Bubyrev A.I., Tsarkova A.S., Kaskova Z.M.

Current studies of fungal bioluminescent systems, including the purification of luciferase, require large quantities of the synthetic substrate luciferin available. The existing synthetic method for the luciferin produces low yield of the target molecule. In the current work, the synthesis of fungal luciferin was scaled up to gram quantities and an effective method for its final purification was found. The total yield in comparison with the previous method was increased by 3.5 times; the overall cost of synthesis fell by 3 times.

Xu Y., Bai Y.

Dorozhkin S.V., Kharissova O.V., Kharisov B.I.

Qu C., Du H.

Hu W., Li M., Feng Y., Wang X., Yang S., Gao Y., Jiang D., Lan X.

Zhang W., Zheng R., Geng W., Wu X., Gao X., Zhou L., An Z., Liu C., Song Z., Ji H., Yang H., Wu X.

AbstractHispidin (1) is a polyphenolic compound with a wide range of pharmacological activities that is distributed in both plants and fungi. In addition to natural extraction, hispidin can be obtained by chemical or enzymatic synthesis. In this study, the identification and characterization of an undescribed enzyme, PheG, from Phellinus igniarius (P. igniarius), which catalyzes the construction of a key C─C bond in the enzymatic synthesis of hispidin are reported. It is demonstrated in vitro that PheG generates hispidin by catalyzing C─C bond formation in the aldol condensation reaction. Based on these results, a plausible pathway for hispidin biosynthesis is proposed by utilizing the primary triacetic acid lactone (TAL, 2) and 3,4‐dihydroxybenzaldehyde (3). The mechanisms for the aldol condensation reaction of PheG are investigated using molecular dynamics (MD) simulations, molecular mechanics/generalized Born surface area (MM/GBSA) binding free energy calculations, density functional theory, and site‐specific mutations. The locations of the key amino acid residues that catalyze the conversion of substrates 2 and 3 to hispidin at the active site of PheG‐1 are identified. This study provides a new method for preparing hispidin with high efficiency and low cost.

Hattori M., Wazawa T., Orioka M., Hiruta Y., Nagai T.

Bioluminescence, an optical marker that does not require excitation by light, allows researchers to simultaneously observe multiple targets, each exhibiting a different color. Notably, the colors of the bioluminescent proteins must sufficiently vary to enable simultaneous detection. Here, we aimed to introduce a method that can be used to expand the color variation by tuning dual-acceptor bioluminescence resonance energy transfer. Using this approach, we could visualize multiple targets with up to 20 colors through single-shot acquisition using a color complementary metal-oxide semiconductor camera. Overall, this method enables simple and simultaneous observation of multiple biological targets and phenomena.

Diaz R., Bermudes D.

The fungal genus Omphalotus is noted for its bioluminescence and the production of biologically active secondary metabolites. We isolated 47 fungal strains of Omphalotus olivascens germinated from spores of a single mushroom. We first noted a high degree of variation in the outward appearances in radial growth and pigmentation among the cultures. Radial growth rates fell into at least five distinct categories, with only slower-growing isolates obtained compared with the parental dikaryon. Scanning UV-vis spectroscopy of liquid-grown cultures showed variation in pigmentation in both the absorption intensity and peak absorption wavelengths, indicating that some isolates vary from the parental strain in both pigment concentration and composition. Bioluminescence intensity was observed to have isolates with both greater and lesser intensities, while the increased emission in response to caffeic acid was inversely proportional to the unstimulated output. Under UV illumination, the media of the parental strain was observed to be brightly fluorescent, which was not due to the pigment, while the isolates also varied from greater to lesser intensity and in their peak emission. At least three separate fluorescent bands were observed by gel electrophoresis from one of the cultures, while only one was observed in others. In a subset of the cultures, fluorescence intensity varied significantly in response to casamino acids. None of this subset produced an antibiotic effective against Staphylococcus aureus, and only the haploids, but not the parental heterokaryon, produced an antibiotic consistent with illudin M effective against Mycobacterium smegmatis. This same subset produced an anticancer agent that was highly potent against MDA-MB-468 breast cancer tumor cells. We interpret these variations in haploids as significant in altering Omphalotus physiology and its production of secondary metabolites, which may in turn alter their ecology and life cycle, and could be further applied to studying fungal physiologies and facilitate linking them to their genetic underpinnings.

Zhang J., Du H., Lu M.

Fan J., Wei P., Li Y., Zhang S., Ren Z., Li W., Yin W.

Sun S., Ni Q., Luo D., Hong X., Li W., Wang T., Lang X., Du H.

Yuan Z., Jiang Q., Liang G.

Bioluminescence is a natural process where biological organisms produce light through chemical reactions. These reactions predominantly occur between small-molecule substrates and luciferase within bioluminescent organisms. Bioluminescence imaging (BLI) has shown significant potential in biomedical research owing to its non-invasive, real-time observation and quantification. In this review, we introduced the chemical mechanism of bioluminescent systems and categorized several strategies that successfully addressed the native limitations, including improvements on the chemical structures of luciferase-luciferin bioluminescence system and bioluminescence resonance energy transfer (BRET) methods. In addition, we also reviewed and summarized recent advances in bioimaging applications. We hope that this review can provide effective guidance for the development and application of bioluminescent systems in the field of bioimaging.

Dmitrieva D.A., Belozerova O.A., Mishin A.V., Yampolsky I.V., Kotlobay A.A.

Among nearly a hundred known bioluminescent systems, only about a dozen have been studied to some extent, and the structures of only a few luciferins have been established. Moreover, the biosynthesis pathway is known only for two of them - the fungal and bacterial ones. Marine polychaetes of the Odontosyllis genus possess bright bioluminescence. The structures of its bioluminescence system key components were recently elucidated, and a possible pathway of the luciferin biosynthesis was proposed. Here we report the transaminase enzyme from Odontosyllis undecimdonta, the first potential participant of the cascade. We demonstrate that the discovered ferment catalyzes the transamination of the cys2DOPA, one of the potential luciferin biosynthetic precursors. The results of the experiments support the hypothesis that the discovered enzyme might be the part of the Odontosyllis luciferin biosynthesis pathway.

Perry B.A., Desjardin D.E., Stevani C.V.

All known bioluminescent fungi are basidiomycetes belonging to the Agaricales. They emit 520–530 nm wavelength light 24 h per day in a circadian rhythm. The number of known bioluminescent fungi has more than doubled in the past 15 years from 64 to 132 species. We currently recognize five distinct lineages of bioluminescent Agaricales belonging to the Omphalotaceae (18 species), Physalacriaceae (14), Mycenaceae (96), Lucentipes lineage (3), and Cyphellopsidaceae (1). They are distributed across the globe with the highest diversity occurring on woody or leafy substrates in subtropical closed canopy forests with high plant diversity. With the caveat that most regions of the world have not been extensively sampled for bioluminescent fungi, the areas with the most known species are Japan (36), South America (30), North America (27), Malesia, South Asia, and Southeast Asia (26), Europe (23), Central America (21), China (13), Africa (10), Australasia, Papua New Guinea, and New Caledonia (11), and the Pacific Islands (5). Recent studies have elucidated the biochemical and genetic pathways of fungal bioluminescence and suggest the phenomenon originated a single time early in the evolution of the Agaricales. Multiple independent evolutionary losses explain the absence of luminescence in many species found within the five lineages and in the majority of Agaricales.

Coubris C., Mirzaei K., Duchatelet L., Mallefet J.

The bioluminescent European brittle star Amphiura filiformis produces blue light at the arm-spine level thanks to a biochemical reaction involving coelenterazine as substrate and a Renilla-like luciferase as an enzyme. This echinoderm light production depends on a trophic acquisition of the coelenterazine substrate. Without an exogenous supply of coelenterazine, this species loses its luminous capabilities. Moreover, this species was recently shown not to produce coelenterazine storage forms. As an infaunal suspensive feeder, A. filiformis is assumed to find enough substrate to maintain its bioluminescence capabilities efficiently. To date, no studies have investigated the putative source of coelenterazine in the brittle star diet. A combined analysis using listing based on visual observations and metabarcoding on the planktonic communities highlights planktonic species known as light emitters using coelenterazine. Besides, the A. filiformis stomach content was analyzed seasonally via metabarcoding technique, and coelenterazine-related preys were underlined. Results provide evidence of the presence of preys containing coelenterazine in the fjord environment and within the stomach content of the ophiuroid throughout the year. The results are consistent with the demonstration of the trophic acquisition of luminous capabilities in A. filiformis and give a new step by underlying the constant presence of coelenterazine suppliers throughout the year for the luminescence reaction occurring within this species.

Sangeetha B., Leroy K.I., Udaya Kumar B.

ABSTRACTThe technique of using naturally occurring light‐emitting reactants (photoproteins and luciferases] that have been extracted from a wide range of animals is known as bioluminescence imaging, or BLI. This imaging offers important details on the location and functional state of regenerative cells inserted into various disease‐modeling animals. Reports on gene expression patterns, cell motions, and even the actions of individual biomolecules in whole tissues and live animals have all been made possible by bioluminescence. Generally speaking, bioluminescent light in animals may be found down to a few centimetres, while the precise limit depends on the signal's brightness and the detector's sensitivity. We can now spatiotemporally visualize cell behaviors in any body region of a living animal in a time frame process, including proliferation, apoptosis, migration, and immunological responses, thanks to BLI. The biological applications of in vivo BLI in nondestructively monitoring biological processes in intact small animal models are reviewed in this work, along with some of the advancements that will make BLI a more versatile molecular imaging tool.

Silva-Filho A.G., Mombert A., Nascimento C.C., Nóbrega B.B., Soares D.M., Martins A.G., Domingos A.H., Santos I., Della-Torre O.H., Perry B.A., Desjardin D.E., Stevani C.V., Menolli N.

During nocturnal field expeditions in the Brazilian Atlantic Rainforest, an unexpected bioluminescent fungus with reduced form was found. Based on morphological data, the taxon was first identified as belonging to the cyphelloid genus Maireina, but in our phylogenetic analyses, Maireina was recovered and confirmed as a paraphyletic group related to genera Merismodes and Cyphellopsis. Maireina filipendula, Ma. monacha, and Ma. subsphaerospora are herein transferred to Merismodes. Based upon morphological and molecular characters, the bioluminescent cyphelloid taxon is described as the new genus Eoscyphella, characterized by a vasiform to urceolate basidiomata, subglobose to broadly ellipsoid basidiospores, being pigmented, weakly to densely encrusted external hyphae, regularly bi-spored basidia, unclamped hyphae, and an absence of both conspicuous long external hairs and hymenial cystidia. Phylogenetic analyses based on ITS rDNA and LSU rDNA support the proposal of the new genus and confirm its position in Cyphellopsidaceae. Eoscyphella luciurceolata represents a new lineage of bioluminescent basidiomycetes with reduced forms.

Bastos E.L., Quina F.H., Baptista M.S.

Roca-Sanjuán D.

Cabello M.C., Bartoloni F.H., Bastos E.L., Baader W.J.

Bioluminescence (BL) and chemiluminescence (CL) are interesting and intriguing phenomena that involve the emission of visible light as a consequence of chemical reactions. The mechanistic basis of BL and CL has been investigated in detail since the 1960s, when the synthesis of several models of cyclic peroxides enabled mechanistic studies on the CL transformations, which led to the formulation of general chemiexcitation mechanisms operating in BL and CL. This review describes these general chemiexcitation mechanisms—the unimolecular decomposition of cyclic peroxides and peroxide decomposition catalyzed by electron/charge transfer from an external (intermolecular) or an internal (intramolecular) electron donor—and discusses recent insights from experimental and theoretical investigation. Additionally, some recent representative examples of chemiluminescence assays are given.

Liu X., Wang M., Liu Y.

Fungal bioluminescence is widely distributed in the terrestrial environment. At a specific stage of growth, luminescent fungi shine green light at the fruiting body or mycelium. From the viewpoint of chemistry, fungal bioluminescence involves an in vivo cycle of caffeic acid. The complete cycle is composed of three stages: biosynthesis of luciferin from caffeic acid, luminescence process from luciferin to oxidized luciferin, and regeneration of caffeic acid from oxidized luciferin. Experimental studies roughly proposed this cycle but not the detailed reaction process and mechanism. Our previous theoretical study clearly described the mechanism of the middle stage. The present article attempts to describe the reaction processes and mechanisms of the other two stages by theoretical calculations. A complete theoretical study on the chemistry in the entire process of fungal bioluminescence is helpful to deeply understand fungal bioluminescence.

Garcia A.G., Steinbrenner A.D.

Plants rely on innate immune systems to defend against a wide variety of biotic attackers. Key components of innate immunity include cell-surface pattern-recognition receptors (PRRs), which recognize pest- and pathogen-associated molecular patterns (PAMPs). Unlike other classes of receptors that often have visible cell-death immune outputs upon activation, PRRs generally lack rapid methods for assessing function. Here, we describe a genetically encoded bioluminescent reporter of immune activation by heterologously expressed PRRs in the model organism Nicotiana benthamiana. We characterized N. benthamiana transcriptome changes in response to Agrobacterium tumefaciens and subsequent PAMP treatment to identify pattern-triggered immunity (PTI)-associated marker genes, which were then used to generate promoter-luciferase fusion fungal bioluminescence pathway (FBP) constructs. A reporter construct termed pFBP_2xNbLYS1::LUZ allows for robust detection of PTI activation by heterologously expressed PRRs. Consistent with known PTI signaling pathways, reporter activation by receptor-like protein (RLP) PRRs is dependent on the known adaptor of RLP PRRs, i.e., SOBIR1. The FBP reporter minimizes the amount of labor, reagents, and time needed to assay function of PRRs and displays robust sensitivity at biologically relevant PAMP concentrations, making it ideal for high throughput screens. The tools described in this paper will be powerful for investigations of PRR function and characterization of the structure-function of plant cell-surface receptors. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

Calvache C., Vazquez-Vilar M., Moreno-Giménez E., Orzaez D.

ABSTRACTEngineered autonomous bioluminescence (EAB) offers many potential applications in Plant Synthetic Biology, notably asin vivoreporter system. Current EAB reporter configurations are limited for quantitative applications due to low dynamic range. We reconfigured theNeonothopanus nambifungal bioluminescence (NeoLuc) pathway to serve as a high-throughput and inexpensive reporter for quantitative analysis of gene expression. We showed that by configuring the first committed step in the pathway (HispS) as the transcriptional entry point instead of the fungal luciferase, the dynamic range of the output increased dramatically, equaling that of the FLuc/RLuc reporter, and outperforming it in high throughput capacity. Furthermore, the inclusion of an enhanced GFP as normalizer allowed transient ratiometric measurements inN. benthamiana. Fast and rich datasets generated by the NeoLuc/eGFP system enabled us to undertake the optimization of new challenging synthetic gene circuits, including a complex agrochemical/optogenetic dual input switch for tight control of engineered metabolic pathways.

Palkina K.A., Balakireva A.V., Belozerova O.A., Chepurnykh T.V., Markina N.M., Kovalchuk S.I., Tsarkova A.S., Mishin A.S., Yampolsky I.V., Sarkisyan K.S.

Hispidin is a polyketide found in plants and fungi. In bioluminescent fungi, hispidin serves as a precursor of luciferin and is produced by hispidin synthases. Previous studies revealed that hispidin synthases differ in orthologous polyketide synthases from non-bioluminescent fungi by the absence of two domains with predicted ketoreductase and dehydratase activities. Here, we investigated the hypothesis that the loss of these domains in evolution led to the production of hispidin and the emergence of bioluminescence. We cloned three orthologous polyketide synthases from non-bioluminescent fungi, as well as their truncated variants, and assessed their ability to produce hispidin in a bioluminescence assay in yeast. Interestingly, expression of the full-length enzyme hsPKS resulted in dim luminescence, indicating that small amounts of hispidin are likely being produced as side products of the main reaction. Deletion of the ketoreductase and dehydratase domains resulted in no luminescence. Thus, domain truncation by itself does not appear to be a sufficient step for the emergence of efficient hispidin synthases from orthologous polyketide synthases. At the same time, the production of small amounts of hispidin or related compounds by full-length enzymes suggests that ancestral fungal species were well-positioned for the evolution of bioluminescence.

Petushkov V., Vavilov M.V., Ivanov I., Ziganshin R.H., Rodionova N.S., Yampolsky I.V., Tsarkova A.S., Dubinnyi M.A.

The unexpected structure of the Henlea deazaflavine bioluminescence activator and Michaelis–Menten plot of its activation factor superimposed over a bioluminescent Henlea sp. worm.

Kotlobay A.A., Dubinnyi M.A., Polevoi A.V., Kovalchuk S.I., Kaskova Z.M.

Abstract Keroplatus is a genus of fungus gnats family Keroplatidae (Diptera, Bibionomorpha). Larvae of some species emit a constant blue light from the body. The bioluminescence of Keroplatidae is one of the least studied of all terrestrial insects and very few facts are known to date of its biology and biochemistry. Here we report the high level of riboflavin in Keroplatus testaceus larvae, a fluorescent compound that might be relative to its bioluminescent system. We suppose that riboflavin may play a role in Keroplatus spp. bioluminescence.

Liu Z., Li M., Wang S., Huang H., Zhang W.

Organosulfur natural products (NPs) refer to the different kinds of small molecular-containing sulfur (S) elements. Sulfur-containing NPs tightly link to the biochemical processes and play an important role in the pharmaceutical industry. The majority of S-containing NPs are generally isolated from Alliaceae plants or bacteria, and those from fungi are still relatively rare. In recent years, an increasing number of S-containing metabolites have been discovered in marine and terrestrial fungi, but there is no comprehensive and targeted review to summarize the studies. In order to make it more straightforward to better grasp the fungal-derived S-containing NPs and understand the particularity of marine S-containing NPs compared to those from terrestrial fungi, we summarized the chemical structures and biological activities of 89 new fungal-derived S-containing metabolites from 1929 when the penicillin was discovered to the present in this current review. The structural and bioactive diversity of these S-containing metabolites were concluded in detail, and the preliminary mechanism for C-S bond formation in fungi was also discussed briefly.

Dhanya T.M., Anjali Krishna G., Savitha D.P., Shanty A.A., Divya K.M., Priya S.K., Mohanan P.V.

Liu Y.

Bioluminescence (BL) is an amazing natural phenomenon whose visible light is produced by living organisms. BL phenomenon is quite pervasive and has been observed in 17 phyla of 4 kingdoms. This fascinating natural phenomenon has unceasingly attracted people’s curiosity from ancient era to today. For a very long time, we can only receive some sporadic and static information from experimental observations, the mechanism of most BL remains is unclear. How the chemical reaction of BL process is initiated? Where the energy for light emission comes from? How does the light emitter produce? What is the light emitter for a wild bioluminescent organism? How to regain luciferin for next bioluminescence when it is used up? The luciferin is utilized forthwith or stored and release for subsequent light emission? What factors affect the color and strength of a bioluminescence? How to artificially tune the bioluminescence for special application? Computational BL plays unreplaceable role in answering these mechanistic questions. In contrast with experimental BL, computational BL came very late. In the past two decades, computational BL has touched nearly all the bioluminescent systems with chemical bases via the method of multiscale simulation. In this review, the author firstly introduced the history, types and general chemical process of BL. Then, the computational scheme on BL was briefly epitomized. Using firefly BL as a paradigmatic case, the author summarized theoretical investigation on the six stages of general chemical process in a BL cycle: luciferin oxidation, peroxide thermolysis, light emission, luciferin regeneration, luciferin storage and luciferin release. At each stage, the available theoretical studies of other bioluminescent organisms are briefly introduced and compared with the firefly system. Basing on the mechanistic understanding, the author reviewed the up-to-date theoretical design on bioluminescent systems. Again, the firefly was mainly focused on, and the other possible systems were just briefly introduced. This review summarized the theoretical studies to date on BL and addressed the status, critical challenges and future prospects of computational BL. • The first review to summarize the up-to-date theoretical studies on bioluminescence. • A complete set of computational schemes on bioluminescence is epitomized. • The critical challenges in computational bioluminescence are summarized. • The future prospects of computational bioluminescence are systemically addressed.

Moreno-Giménez E., Selma S., Calvache C., Orzáez D.

Programmable transcriptional factors based on the CRISPR architecture are becoming commonly used in plants for endogenous gene regulation. In plants, a potent CRISPR tool for gene induction is the so-called dCasEV2.1 activation system, which has shown remarkable genome-wide specificity combined with a strong activation capacity. To explore the ability of dCasEV2.1 to act as a transactivator for orthogonal synthetic promoters, a collection of DNA parts was created (GB_SynP) for combinatorial synthetic promoter building. The collection includes (i) minimal promoter parts with the TATA box and 5'UTR regions, (ii) proximal parts containing single or multiple copies of the target sequence for the gRNA, thus functioning as regulatory cis boxes, and (iii) sequence-randomized distal parts that ensure the adequate length of the resulting promoter. A total of 35 promoters were assembled using the GB_SynP collection, showing in all cases minimal background and predictable activation levels depending on the proximal parts used. GB_SynP was also employed in a combinatorial expression analysis of an autoluminescence pathway in Nicotiana benthamiana, showing the value of this tool in extracting important biological information such as the determination of the limiting steps in an enzymatic pathway.

Soares D.M., Procópio D.P., Zamuner C.K., Nóbrega B.B., Bettim M.R., de Rezende G., Lopes P.M., Pereira A.B., Bechara E.J., Oliveira A.G., Freire R.S., Stevani C.V.

Environmental pollutants are today a major concern and an intensely discussed topic on the global agenda for sustainable development. They include a wide range of organic compounds, such as pharmaceutical waste, pesticides, plastics, and volatile organic compounds that can be found in air, soil, water bodies, sewage, and industrial wastewater. In addition to impacting fauna, flora, and fungi, skin absorption, inhalation, and ingestion of some pollutants can also negatively affect human health. Fungi play a crucial role in the decomposition and cycle of natural and synthetic substances. They exhibit a variety of growth, metabolic, morphological, and reproductive strategies and can be found in association with animals, plants, algae, and cyanobacteria. There are fungal strains that occur naturally in soil, sediment, and water that have inherent abilities to survive with contaminants, making the organism important for bioassay applications. In this context, we reviewed the applications of fungal-based bioassays as a versatile tool for environmental monitoring.