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.

Engineered CBEs based on Macaca fascicularis A3A with improved properties for precise genome editing.

Cytidine deaminase defines the properties of cytosine base editors (CBEs) for C-to-T conversion. Replacing the cytidine deaminase rat APOBEC1 (rA1) in CBEs with a human APOBEC3A (hA3A) improves CBE properties. However, the potential CBE application of macaque A3A orthologs remains undetermined. Our current study develops and evaluates engineered CBEs based on Macaca fascicularis A3A (mA3A). Here, we demonstrate that BE4-mA3A and its RNA-editing-derived variants exhibit improved CBE properties, except for DNA off-target activity, compared to BE3-rA1 and BE4-rA1. Unexpectedly, deleting Ser-Val-Arg (SVR) in BE4-mA3A dramatically reduces DNA and RNA off-target activities and improves editing accuracy, with on-target efficiency unaffected. In contrast, a chimeric BE4-hA3A-SVR+ shows editing efficiency increased by about 50%, with other properties unaffected. Our findings demonstrate that mA3A-based CBEs could provide prototype options with advantages over rA1- and hA3A-based CBEs for further optimization, highlighting the importance of the SVR motif in defining CBE intrinsic properties.

High-Level De Novo Production of (2S)-Eriodictyol in Yarrowia Lipolytica by Metabolic Pathway and NADPH Regeneration Engineering.

(2S)-Eriodictyol, a polyphenolic flavonoid, has found widespread applications in health supplements and food additives. However, the limited availability of plant-derived (2S)-eriodictyol cannot meet the market demand. Microbial production of (2S)-eriodictyol faces challenges, including the low catalytic efficiency of flavone 3'-hydroxylase/cytochrome P450 reductase (F3'H/CPR), insufficient precursor supplementation, and inadequate NADPH regeneration. This study systematically engineered Yarrowia lipolytica for high-level (2S)-eriodictyol production. In doing this, the expression of F3'H/CPR was balanced, and the supply of precursors was enhanced by relieving feedback inhibition of the shikimate pathway, promoting fatty acid ß-oxidation, and increasing the copy number of synthetic pathway genes. These strategies, combined with NADPH regeneration, achieved an (2S)-eriodictyol titer of 423.6 mg/L. Finally, in fed-batch fermentation, a remarkable 6.8 g/L (2S)-eriodictyol was obtained, representing the highest de novo microbial titer reported to date and paving the way for industrial production.

Selective synthesis of rebaudioside M2 through structure-guided engineering of glycosyltransferase UGT94D1.

Rebaudioside M2 (Reb M2), a novel steviol glycoside derivative, has limited industrial applications due to its low synthetic yield and selectivity. Herein, we identify UGT94D1 as a selective glycosyltransferase for rebaudioside D (Reb D), leading to the production of a mono ß-1,6-glycosylated derivative, Reb M2. A variant UGT94D1-F119I/D188P was developed through protein engineering. This mutant exhibited a 6.33-fold improvement in catalytic efficiency, and produced Reb M2 with 92% yield. Moreover, molecular dynamics simulations demonstrated that UGT94D1-F119I/D188P exhibited a shorter distance between the nucleophilic oxygen (OH6) of the substrate Reb D and uridine diphosphate glucose, along with an increased Ophosphate-C1-Oacceptor angle, thus improving the catalytic activity of the enzyme. Therefore, this study provides an efficient method for the selective synthesis of Reb M2 and paves the way for its applications in various fields.

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.

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.

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.

Structural Insight into the Catalytic Mechanisms of an L-Sorbosone Dehydrogenase.

L-Sorbosone dehydrogenase (SNDH) is a key enzyme involved in the biosynthesis of 2-keto-L-gulonic acid , which is a direct precursor for the industrial scale production of vitamin C. Elucidating the structure and the catalytic mechanism is essential for improving SNDH performance. By solving the crystal structures of SNDH from Gluconobacter oxydans WSH-004, a reversible disulfide bond between Cys295 and the catalytic Cys296 residues is discovered. It allowed SNDH to switch between oxidation and reduction states, resulting in opening or closing the substrate pocket. Moreover, the Cys296 is found to affect the NADP+ binding pose with SNDH. Combining the in vitro biochemical and site-directed mutagenesis studies, the redox-based dynamic regulation and the catalytic mechanisms of SNDH are proposed. Moreover, the mutants with enhanced activity are obtained by extending substrate channels. This study not only elucidates the physiological control mechanism of the dehydrogenase, but also provides a theoretical basis for engineering similar enzymes.

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.

Characterization of highly gelatinous patatin storage protein from Pichia pastoris.

Patatin is a useful plant protein with excellent gelation properties that could be used as a gelling agent in the food industry. However, the commercial production of patatin is limited because the traditional extraction methods are inefficient and time consuming. Production of patatin with gelation properties by microorganisms is a promising alternative route. In this study, 1424.5 mg/L patatin storage protein with great gelation properties could be obtained in a 5-L bioreactor after optimization of the signal peptide, the promoter, and the fed-batch process when a Pichia pastoris GS115, but not Escherichia coli, expression system was used. Compared with commercial potato-extracted patatins, P. pastoris-derived patatins showed better gelation properties, such as a lower gel-forming concentration and gelation temperature. In addition, the gel strength of P. pastoris-derived patatins was comparable with that of potato-extracted patatins. These results suggested that P. pastoris-derived patatins have the potential to replace current potato-derived ones, which are now widely used in plant-based meat products.

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.

Stimulating fungal cell wall integrity by exogenous ß-glucanase to improve the production of fungal natural products.

The low production of natural products (NPs) is still the critical restrictive factor in exploiting their potential large-scale applications and a barrier to isolating and identifying other meaningful products. Given that the stimulation of cell wall integrity (CWI) has become a novel strategy to modulate the production of microbial natural products, herein, exogenous ß-glucanase treatment was developed as an external cell wall ß-glucan stress to stimulate the fungal CWI, and then to improve the production of fungal NPs. It was found that the production of fungal NPs cercosporin and sophorolipids, biosynthesized by Cercospora sp. and Starmerella bombicola, respectively, was significantly improved by the treatment of ß-glucanase under a controllable dose. Moreover, it demonstrated that ß-glucanase had an ability to stimulate fungal CWI through slight fungal superficial damage, thus facilitating the secretion of NPs. We expected that this easy-operating method to stimulate fungal CWI could be feasible to improve more fungal NPs production. KEY POINTS: • Exogenous ß-glucanase stimulated the fungal cell wall integrity • Changing fungal cell walls modulated natural product production • ß-glucanase with potential universal effects on more fungal natural products.

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.

Insight into the broadened substrate scope of nitrile hydratase by static and dynamic structure analysis.

The narrow substrate scope limits the wide industrial application of enzymes. Here, we successfully broadened the substrate scope of a nitrile hydratase (NHase) through mutation of two tunnel entrance residues based on rational tunnel calculation. Two variants, with increased specific activity, especially toward bulky substrates, were obtained. Crystal structure analysis revealed that the mutations led to the expansion of the tunnel entrance, which might be conducive to substrate entry. More importantly, molecular dynamics simulations illustrated that the mutations introduced anti-correlated movements to the regions around the substrate tunnel and the active site, which would promote substrate access during the dynamic process of catalysis. Additionally, mutations on the corresponding tunnel entrance residues on other NHases also enhanced their activity toward bulky substrates. These results not only revealed that residues located at the enzyme surface were a key factor in enzyme catalytic performance, but also provided dynamic evidence for insight into enzyme substrate scope broadening.

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.