Discovery of a Fungal P450 with an Unusual Two-Step Mechanism for Constructing a Bicyclo[3.2.2]nonane Skeleton.

The successful biomimetic or chemoenzymatic synthesis of target natural products (NPs) and their derivatives relies on enzyme discovery. Herein, we discover a fungal P450 BTG5 that can catalyze the formation of a bicyclo[3.2.2]nonane structure through an unusual two-step mechanism of dimerization and cyclization in the biosynthesis of beticolin 1, whose bicyclo[3.2.2]nonane skeleton connects an anthraquinone moiety and a xanthone moiety. Further investigation reveals that BTG5-T318 not only determines the substrate selectivity but also alters the catalytic reactions, which allows the separation of the reaction to two individual steps, thereby understanding its catalytic mechanism. It reveals that the first heterodimerization undergoes the common oxidation process for P450s, while the second uncommon formal redox-neutral cyclization step is proved as a redox-mediated reaction, which has never been reported. Therefore, this work advances our understanding of P450-catalyzed reactions and paves the way for expansion of the diversity of this class of NPs through synthetic biology.

Synthetic Biology-based Construction of Unnatural Perylenequinones with Improved Photodynamic Anticancer Activities.

The construction of structural complexity and diversity of natural products is crucial for drug discovery and development. To overcome high dark toxicity and poor photostability of natural photosensitizer perylenequinones (PQs) for photodynamic therapy, herein, we aim to introduce the structural complexity and diversity to biosynthesize the desired unnatural PQs in fungus Cercospora through synthetic biology-based strategy. Thus, we first elucidate the intricate biosynthetic pathways of class B PQs and reveal how the branching enzymes create their structural complexity and diversity from a common ancestor. This enables the rational reprogramming of cercosporin biosynthetic pathway in Cercospora to generate diverse unnatural PQs without chemical modification. Among them, unnatural cercosporin A displays remarkably low dark toxicity and high photostability with retention of great photodynamic anticancer and antimicrobial activities. Moreover, it is found that, unlike cercosporin, unnatural cercosporin A could be selectively accumulated in cancer cells, providing potential targets for drug development. Therefore, this work provides a comprehensive foundation for preparing unnatural products with customized functions through synthetic biology-based strategies, thus facilitating drug discovery pipelines from nature.

Modular assembly of an artificially concise biocatalytic cascade for the manufacture of phenethylisoquinoline alkaloids.

Plant-derived alkaloids are an important class of pharmaceuticals. However, they still rely on phytoextraction to meet their diverse market demands. Since multistep biocatalytic cascades have begun to revolutionize the manufacture of natural or unnatural products, to address the synthetic challenges of alkaloids, herein we establish an artificially concise four-enzyme biocatalytic cascade with avoiding plant-derived P450 modification for synthesizing phenethylisoquinoline alkaloids (PEIAs) after enzyme discovery and enzyme engineering. Efficient biosynthesis of diverse natural and unnatural PEIAs is realized from readily available substrates. Most importantly, the scale-up preparation of the colchicine precursor (S)-autumnaline with a high titer is achieved after replacing the rate-limiting O-methylation by the plug-and-play strategy. This study not only streamlines future engineering endeavors for colchicine biosynthesis, but also provides a paradigm for constructing more artificial biocatalytic cascades for the manufacture of diverse alkaloids through synthetic biology.

Photoenzymatic Enantioselective Synthesis of Oxygen-Containing Benzo-Fused Heterocycles.

New-to-nature biocatalysis in organic synthesis has recently emerged as a green and powerful strategy for the preparation of valuable chiral products, among which chiral oxygen-containing benzo-fused heterocycles are important structural motifs in pharmaceutical industry. However, the asymmetric synthesis of these compounds through radical-mediated methods is challenging. Herein, a novel asymmetric radical-mediated photoenzymatic synthesis strategy is developed to realize the efficient enantioselective synthesis of oxygen-containing benzo-fused heterocycles through structure-guided engineering of a flavin-dependent 'ene'-reductase GluER. It shows that variant GluER-W100H could efficiently produce various benzoxepinones, chromanone and indanone with different benzo-fused rings in high yields with great stereoselectivities under visible light. Moreover, these results are well supported by mechanistic experiments, revealing that this photoenzymatic process involves electron donor-acceptor complex formation, single electron transfer and hydrogen atom transfer. Therefore, we provide an alternative green approach for efficient chemoenzymatic synthesis of important chiral skeletons of bioactive pharmaceuticals.

Designing a cercosporin-bioinspired bifunctional algicide with flocculation and photocatalysis for efficiently controlling harmful cyanobacterial blooms.

Harmful cyanobacterial blooms (HCBs) are spreading in freshwater ecosystems worldwide, adversely affecting drinking water supplies, aquatic production, recreational and tourism activities. Therefore, the efficient and environmentally friendly method is still of interest to be developed to effectively control HCBs. Inspired by the excellent algicidal activity of cercosporin (CP), a novel metal-free algaecide SiO2@EDU@CP (EDU, N-ethyl-N'-(3-dimethylaminopropyl)urea) with flocculation and photoremoval functions, was successfully designed and prepared in one-step to simultaneously introduce CP and EDU on SiO2 nanoparticles. It could rapidly form algae flocs in 20 min with 97.1% flocculation rate, and remove Microcystis aeruginosa within 12 h with 91.0% algicidal rate under 23 W compact fluorescent light irradiation without any leaked CP detected. Additionally, odorant ß-cyclocitral and toxin microcystin-LR were both photodegraded after treatment of SiO2@EDU@CP. Further mechanistic studies showed that the introduction of EDU significantly reversed the zeta potential of SiO2-COOH to achieve the flocculation through neutral charge, and the photophysical characterization of SiO2@EDU@CP revealed the improved charge separation ability to generate reactive oxygen species. More importantly, the utility of SiO2@EDU@CP was well demonstrated by its effectiveness for algae from Taihu Lake under natural sunlight and inability to regrow after treatment. This study not only establishes a bifunctional algicide SiO2@EDU@CP to efficiently control HCBs, but also provides design possibilities to develop more novel and efficient algicides for the better control of practical HCBs.

Enhanced production of aspochalasin D through genetic engineering of Aspergillus flavipes.

Aspochalasin D (AD) belongs to the polyketide-amino acid hybrid natural products with anti-cancer, anti-bacterial, and anti-fouling bioactivities. However, the low production limits its further application. In this study, AD was separated and identified from Aspergillus flavipes 3.17641. Next, besides the optimization of culture conditions using a single-factor experiment and response surface methodology, metabolic engineering was employed to increase the AD production. It shows that the deletion of the shunt gene aspoA and overexpression of the pathway-specific regulator aspoG significantly improve the AD production. Its production reached to 812.1 mg/L under the optimized conditions, with 18.5-fold increase. Therefore, this study not only provides a general method for improving the production of similar natural products in other fungi, but also enables the further biological function development of AD in agriculture and pharmaceutical. KEY POINTS: • The Aspochalasin D (AD) production was improved by optimizing culture conditions. • The deletion of the shunt gene aspoA increased the AD production. • Overexpression of the pathway regulator aspoG further improved the AD production.

Structural analysis of an anthrol reductase inspires enantioselective synthesis of enantiopure hydroxycycloketones and ß-halohydrins.

Asymmetric reduction of prochiral ketones, particularly, reductive desymmetrization of 2,2-disubstituted prochiral 1,3-cyclodiketones to produce enantiopure chiral alcohols is challenging. Herein, an anthrol reductase CbAR with the ability to accommodate diverse bulky substrates, like emodin, for asymmetric reduction is identified. We firstly solve crystal structures of CbAR and CbAR-Emodin complex. It reveals that Tyr210 is critical for emodin recognition and binding, as it forms a hydrogen-bond interaction with His162 and π-π stacking interactions with emodin. This ensures the correct orientation for the stereoselectivity. Then, through structure-guided engineering, variant CbAR-H162F can convert various 2,2-disubstituted 1,3-cyclodiketones and α-haloacetophenones to optically pure (2S, 3S)-ketols and (R)-ß-halohydrins, respectively. More importantly, their stereoselectivity mechanisms are also well explained by the respective crystal structures of CbAR-H162F-substrate complex. Therefore, this study demonstrates that an in-depth understanding of catalytic mechanism is valuable for exploiting the promiscuity of anthrol reductases to prepare diverse enantiopure chiral alcohols.

Highly efficient synthesis of mono-ß-1,6-Glucosylated Rebaudioside A derivative catalyzed by glycosyltransferase YjiC.

Steviol glycosides have attracted great interest because of their high levels of sweetness and safety, and absence of calories. Improvement of their sensory qualities via glycosylation modification by glycosyltransferase is a research hotspot. In this study, YjiC, a uridine diphosphate-dependent glycosyltransferase from Bacillus subtilis 168, was found with the ability to glycosylate rebaudioside A (Reb A) to produce a novel mono ß-1, 6-glycosylated Reb A derivative rebaudioside L2 (Reb L2). It has an improved sweetness compared with Reb A. Next, a cascade reaction was established by combining YjiC with sucrose synthase AtSuSy from Arabidopsis thaliana for scale-up preparation of Reb L2. It shows that Reb L2 (30.94 mg/mL) could be efficiently synthesized with an excellent yield of 91.34% within 12 h. Therefore, this study provides a potential approach for the production and application of new steviol glycoside Reb L2, expanding the scope of steviol glycosides.

Biosynthetic Pathways of Dimeric Natural Products Containing Bisanthraquinone and Related Xanthones.

Many dimeric natural products containing bisanthraquinone and related xanthones with diverse structures and versatile bioactivities have been isolated over the years. However, the complicated biosynthetic pathways of such natural products, which have remained elusive until recently, negatively impact their mass bioproduction and biosynthetic structural modification for drug discovery. In this concept, we summarize the recent progress in gene cluster mining and biosynthetic pathway elucidation of natural products containing bisanthraquinone and related xanthones. These pioneering works may pave the way for further biosynthetic pathway elucidation and structure modification of dimeric natural products through gene and protein engineering.

Efficient synthesis of rebaudioside D2 through UGT94D1-catalyzed regio-selective glycosylation.

Steviol glycosides have been widely applied as new sweeteners in food, beverages, health care, and daily chemical industry owing to the properties of high-intensity sweetness, low calorie, and good physiological characteristics. However, most of steviol glycosides have a bitter taste. Their organoleptic properties can be effectively improved by modifying the linked glycosyl units. In this study, UGT94D1, a uridine diphosphate-dependent glycosyltransferase from Sesamum indicum, was reported to selectively glycosylate rebaudioside A (Reb A) for the synthesis of rebaudioside D2 (Reb D2). Furthermore, a cascade reaction system was constructed to synthesize Reb D2 with 94.66% yield by coupling UGT94D1 with sucrose synthase AtSuSy from Arabidopsis thaliana. Thus, our study not only introduced a practical method for the synthesis of steviol glycosides but also provided the possibility for further exploration of Reb D2.

Highly efficient production of rebaudioside D enabled by structure-guided engineering of bacterial glycosyltransferase YojK.

Owing to zero-calorie, high-intensity sweetness and good taste profile, the plant-derived sweetener rebaudioside D (Reb D) has attracted great interest to replace sugars. However, low content of Reb D in stevia rebaudiana Bertoni as well as low soluble expression and enzymatic activity of plant-derived glycosyltransferase in Reb D preparation restrict its commercial usage. To address these problems, a novel glycosyltransferase YojK from Bacillus subtilis 168 with the ability to glycosylate Reb A to produce Reb D was identified. Then, structure-guided engineering was performed after solving its crystal structure. A variant YojK-I241T/G327N with 7.35-fold increase of the catalytic activity was obtained, which allowed to produce Reb D on a scale preparation with a great yield of 91.29%. Moreover, based on the results from molecular docking and molecular dynamics simulations, the improvement of enzymatic activity of YojK-I241T/G327N was ascribed to the formation of new hydrogen bonds between the enzyme and substrate or uridine diphosphate glucose. Therefore, this study provides an engineered bacterial glycosyltransferase YojK-I241T/G327N with high solubility and catalytic efficiency for potential industrial scale-production of Reb D.

Discovery of the Biosynthetic Pathway of Beticolin 1 Reveals a Novel Non-Heme Iron-Dependent Oxygenase for Anthraquinone Ring Cleavage.

This study used light-mediated comparative transcriptomics to identify the biosynthetic gene cluster of beticolin 1 in Cercospora. It contains an anthraquinone moiety and an unusual halogenated xanthone moiety connected by a bicyclo[3.2.2]nonane. During elucidation of the biosynthetic pathway of beticolin 1, a novel non-heme iron oxygenase BTG13 responsible for anthraquinone ring cleavage was discovered. More importantly, the discovery of non-heme iron oxygenase BTG13 is well supported by experimental evidence: (i) crystal structure and the inductively coupled plasma mass spectrometry revealed that its reactive site is built by an atypical iron ion coordination, where the iron ion is uncommonly coordinated by four histidine residues, an unusual carboxylated-lysine (Kcx377) and water; (ii) Kcx377 is mediated by His58 and Thr299 to modulate the catalytic activity of BTG13. Therefore, we believed this study updates our knowledge of metalloenzymes.

Highly efficient decontamination of tetracycline and pathogen by a natural product-derived Emodin/HAp photocatalyst.

To address the water pollution induced by pharmaceuticals, especially antibiotics, and pathogens, natural product emodin, a traditional Chinese medicine with the characteristic large π-conjugation anthraquinone structure, was used to rationally develop a novel Emodin/HAp photocatalyst by integrating with a thermally stable and recyclable support material hydroxyapatite (HAp) through a simple preparation method. It was found that its photocatalytic activity to generate reactive oxygen species (ROS) was greatly improved due to the migration of photogenerated electrons and holes between emodin and HAp upon visible light irradiation. Thus, this Emodin/HAp photocatalyst not only quickly photodegraded tetracycline with 99.0% removal efficiency but also exhibited complete photodisinfection of pathogenic bacteria Staphylococcus aureus upon visible light irradiation. Therefore, this study offers a new route for the design and preparation of multifunctional photocatalysts using widely available natural products for environmental remediation.

Enhancement of Rebaudioside M Production by Structure-Guided Engineering of Glycosyltransferase UGT76G1.

Owing to zero-calorie and advanced organoleptic properties similar to sucrose, the plant-derived rebaudioside M (Reb M) has been considered as a next generation sweetener. However, a low content of Reb M in Stevia rebaudiana Bertoni and low enzymatic activity of UGT76G1, which is an uridine diphosphate glucose (UDPG)-dependent glycosyltransferase with the ability to glycosylate rebaudioside D (Reb D) to produce Reb M through the formation of ß-1,3 glycosidic bond, restrict its commercial usage. To improve the catalytic activity of UGT76G1, a variant UGT76G1-T284S/M88L/L200A was obtained by structure-guided evolution, whose catalytic activity toward Reb D increased by 2.38 times compared with UGT76G1-T284S. This allowed us to prepare Reb M on a large-scale with a great yield of 90.50%. Moreover, molecular dynamics simulation illustrated that UGT76G1-T284S/M88L/L200A reduced distances from Reb D to catalytic residues and UDPG. Hence, we report an efficient method for the potential scale production of Reb M in this study.

Cercosporin-bioinspired photoinactivation of harmful cyanobacteria under natural sunlight via bifunctional mechanisms.

Harmful cyanobacterial blooms (HCBs), mainly caused by eutrophication, have deleterious impacts on water resources and pose a great threat to human health and natural ecosystems. Thus, an environmentally-friendly method to inhibit HCBs is urgently needed. Learning from nature, herein, natural product cercosporin, produced by the fungi Cercospora to damage plant cells under natural sunlight, was developed as a powerful photosensitive algicidal reagent to inhibit HCBs. Microcystis aeruginosa could be severely inactivated by 20 µM cercosporin in 36 h with 95% inhibition ratio under 23 W compact fluorescent light irradiation. Further mechanism investigation showed that algal cell walls and membranes along with the antioxidant and photosynthetic systems were damaged via two mechanisms, those being, reactive oxygen species generation and cell adsorption. More importantly, the practical applicability of cercosporin was demonstrated by its effectiveness in a 2 L-scale photoinactivation experiment using cyanobacterial blooms from Taihu Lake, China under natural sunlight with a lower dosage of cercosporin (7.5 µM). This study established the bifunctional mechanisms by which cercosporin inactivates HCBs, opening design possibilities for the development of novel photosensitive algicidal reagents to control HCBs.

Energy-Transfer-Mediated Photocatalysis by a Bioinspired Organic Perylenephotosensitizer HiBRCP.

Energy transfer plays a special role in photocatalysis by utilizing the potential energy of the excited state through indirect excitation, in which a photosensitizer determines the thermodynamic feasibility of the reaction. Bioinspired by the energy-transfer ability of natural product cercosporin, here we developed a green and highly efficient organic photosensitizer HiBRCP (hexaisobutyryl reduced cercosporin) through structural modification of cercosporin. After structural manipulation, its triplet energy was greatly improved, and then, it could markedly promote the efficient geometrical isomerization of alkenes from the E-isomer to the Z-isomer. Moreover, it was also effective for energy-transfer-mediated organometallic catalysis, which allowed realization of the cross-coupling of aryl bromides and carboxylic acids through efficient energy transfer from HiBRCP to nickel complexes. Thus, the study on the relationship between structural manipulation and their photophysical properties provided guidance for further modification of cercosporin, which could be applied to more meaningful and challenging energy-transfer reactions.

A bioinspired cercosporin/polymethylmethacrylate photocatalyst with high efficiency for decontamination of pharmaceuticals and pathogens.

Pharmaceuticals have seriously contaminated aquatic environments and resulted in the formation of drug-tolerant bacteria owing to continuous release and accumulation. Therefore, the development of new methods to simultaneously decompose drugs and disinfect pathogens in an environmental-friendly manner with high efficiency is still in great demand. Bioinspired by the great photosensitivity of natural product cercosporin with the ability to efficiently generate reactive oxygen species (ROS) under natural sunlight and its antibacterial activity, here a novel cercosporin/polymethylmethacrylate (CP/PMMA) photocatalyst was rationally developed by incorporating and restricting cercosporin in a "green" macroporous resin PMMA, which greatly improved the ROS generation efficiency and displayed 97.2-100% photodegradation for broad-spectrum pharmaceuticals, including fluoroquinolones, trimethoprim and chloroquine phosphate, upon 15 W compact fluorescent lamp irradiation. More importantly, this decontamination efficiency was greatly improved, and the decontamination time was substantially shortened in a large-scale assay under natural sunlight. Furthermore, it could inactivate the pathogen Staphylococcus aureus. Overall, this work provides new insight into how a multifunctional photocatalyst could be designed using a natural product and macroporous resins for environmental remediation.

Recent Advances in Rapid Synthesis of Non-proteinogenic Amino Acids from Proteinogenic Amino Acids Derivatives via Direct Photo-Mediated C-H Functionalization.

Non-proteinogenic amino acids have attracted tremendous interest for their essential applications in the realm of biology and chemistry. Recently, rising C-H functionalization has been considered an alternative powerful method for the direct synthesis of non-proteinogenic amino acids. Meanwhile, photochemistry has become popular for its predominant advantages of mild conditions and conservation of energy. Therefore, C-H functionalization and photochemistry have been merged to synthesize diverse non-proteinogenic amino acids in a mild and environmentally friendly way. In this review, the recent developments in the photo-mediated C-H functionalization of proteinogenic amino acids derivatives for the rapid synthesis of versatile non-proteinogenic amino acids are presented. Moreover, postulated mechanisms are also described wherever needed.

Constructing chiral bicyclo[3.2.1]octanes via palladium-catalyzed asymmetric tandem Heck/carbonylation desymmetrization of cyclopentenes.

Transition-metal-catalyzed tandem Heck/carbonylation reaction has emerged as a powerful tool for the synthesis of structurally diverse carbonyl molecules, as well as natural products and pharmaceuticals. However, the asymmetric version was rarely reported, and remains a challenging topic. Herein, we describe a palladium-catalyzed asymmetric tandem Heck/carbonylation desymmetrization of cyclopentenes. Alcohols, phenols and amines are employed as versatile coupling reagents for the construction of multifunctional chiral bicyclo[3.2.1]octanes with one all-carbon quaternary and two tertiary carbon stereogenic centers in high diastereo- and enantioselectivities. This study represents an important progress in both the asymmetric tandem Heck/carbonylation reactions and enantioselective difunctionalization of internal alkenes.

Emodin as a novel organic photocatalyst for selective oxidation of sulfides under mild conditions.

Herein, we have developed naturally-occurring Emodin, which is commercially available at low-cost, as a novel organic photocatalyst for the first time. Emodin was successfully employed in the selective oxidation of sulfides promoted by visible-light, delivering valuable sulfoxides with high efficiency. Mechanistic investigations suggested both single-electron transfer (SET) and energy transfer (EnT) pathways might be involved in the oxidation reaction.