RESUMEN
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.
Asunto(s)
Alcanos , Esqueleto , Oxidación-ReducciónRESUMEN
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.
Asunto(s)
Ascomicetos , Perileno , Perileno/análogos & derivados , Fotoquimioterapia , Quinonas , Ascomicetos/metabolismo , Biología Sintética , Perileno/farmacología , Perileno/metabolismoRESUMEN
Fungi, as an important industrial microorganism, play an essential role in the production of natural products (NPs) due to their advantages of utilizing cheap raw materials as substrates and strong protein secretion ability. Although many metabolic engineering strategies have been adopted to enhance the biosynthetic pathway of NPs in fungi, the fungal cell wall as a natural barrier tissue is the final and key step that affects the efficiency of NPs synthesis. To date, many important progresses have been achieved in improving the synthesis of NPs by regulating the cell wall structure of fungi. In this review, we systematically summarize and discuss various strategies for modifying the cell wall structure of fungi to improve the synthesis of NPs. At first, the cell wall structure of different types of fungi is systematically described. Then, strategies to disrupt cell wall integrity (CWI) by regulating the synthesis of cell wall polysaccharides and binding proteins are summarized, which have been applied to improve the synthesis of NPs. In addition, we also summarize the studies on the regulation of CWI-related signaling pathway and the addition of exogenous components for regulating CWI to improve the synthesis of NPs. Finally, we propose the current challenges and essential strategies to usher in an era of more extensive manipulation of fungal CWI to improve the production of fungal NPs.
Asunto(s)
Productos Biológicos , Pared Celular , Hongos , Pared Celular/metabolismo , Productos Biológicos/metabolismo , Hongos/metabolismo , Hongos/genética , Ingeniería Metabólica/métodos , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Vías Biosintéticas , Transducción de SeñalRESUMEN
A wide range of perylenequinones (PQs) with diverse structures and versatile bioactivities have long been isolated, positioning them as highly promising agents for photodynamic therapy (PDT). However, the lack of an efficient and cost-effective method to obtain these compounds and to introduce structural diversity and complexity currently hinders their further research and application. In this concept, we present a comprehensive overview of the advancements in the biosynthetic pathways of natural PQs based on their structural classification, and also summarize recent progress in the biosynthesis of natural PQs and derivatives. These pioneering efforts may pave the way for structure modification and large-scale bioproduction of natural and unnatural PQs through synthetic biology strategies to promote their drug development.
RESUMEN
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.
Asunto(s)
Alcaloides , Biocatálisis , Colchicina , PlantasRESUMEN
(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.
Asunto(s)
Flavanonas , Yarrowia , Yarrowia/genética , Yarrowia/metabolismo , NADP/metabolismo , Ingeniería Metabólica , Redes y Vías MetabólicasRESUMEN
Collagen II (COL2) is the major component of cartilage tissue and is widely applied in pharmaceuticals, food, and cosmetics. In this study, COL fragments were extracted from human COL2 for secretory expression in Pichia pastoris. Three variants were successfully secreted by shake flask cultivation with a yield of 73.3-100.7 mg/L. The three COL2 variants were shown to self-assemble into triple-helix at 4 °C and capable of forming higher order assembly of nanofiber and hydrogel. The bioactivities of the COL2 variants were validated, showing that sample 205 exhibited the best performance for inducing fibroblast differentiation and cell migration. Meanwhile, sample 205 and 209 exhibited higher capacity for inducing in vitro blood clotting than commercial mouse COL1. To overexpress sample 205, the expression cassettes were constructed with different promoters and signal peptides, and the fermentation condition was optimized, obtaining a yield of 172 mg/L for sample 205. Fed-batch fermentation was carried out using a 5 L bioreactor, and the secretory protease Pep4 was knocked out to avoid sample degradation, finally obtaining a yield of 3.04 g/L. Here, a bioactive COL2 fragment was successfully identified and can be overexpressed in P. pastoris; the variant may become a potential biomaterial for skin care.
Asunto(s)
Colágeno Tipo II , Humanos , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Ratones , Animales , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biosíntesis , Fermentación , Pichia/genética , Pichia/metabolismo , Movimiento Celular/genética , Fibroblastos/metabolismo , Diferenciación Celular , Reactores Biológicos , Saccharomycetales/genética , Saccharomycetales/metabolismo , Nanofibras/químicaRESUMEN
Given the low-calorie, high-sweetness characteristics of steviol glycosides (SGs), developing SGs with improved taste profiles is a key focus. Rebaudioside M8 (Reb M8), a novel non-natural SG derivative obtained through glycosylation at the C-13 position of rebaudioside D (Reb D) using glycosyltransferase UGT94E13, holds promise for further development due to its enhanced sweetness. However, the low catalytic activity of UGT94E13 hampers further research and commercialization. This study aimed to improve the enzymatic activity of UGT94E13 through semirational design, and a variant UGT94E13-F169G/I185G was obtained with the catalytic activity improved by 13.90 times. A cascade reaction involving UGT94E13-F169G/I185G and sucrose synthase AtSuSy was established to recycle uridine diphosphate glucose, resulting in an efficient preparation of Reb M8 with a yield of 98%. Moreover, according to the analysis of the distances between the substrate Reb D and enzymes as well as between Reb D and the glucose donor through molecular dynamics simulations, it is found that the positive effect of shortening the distance on glycosylation reaction activity accounts for the improved catalytic activity of UGT94E13-F169G/I185G. Therefore, this study addresses the bottleneck in the efficient production of Reb M8 and provides a foundation for its widespread application in the food industry.
Asunto(s)
Diterpenos de Tipo Kaurano , Glicosiltransferasas , Diterpenos de Tipo Kaurano/química , Diterpenos de Tipo Kaurano/metabolismo , Glicosiltransferasas/metabolismo , Glicosiltransferasas/química , Glicosiltransferasas/genética , Glicosilación , Edulcorantes/química , Edulcorantes/metabolismo , Stevia/química , Stevia/enzimología , Stevia/metabolismo , Stevia/genética , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Ingeniería de Proteínas , Glucosiltransferasas/química , Glucosiltransferasas/metabolismo , Glucosiltransferasas/genética , GlicósidosRESUMEN
Carbohydrate degradation is crucial for living organisms due to their essential functions in providing energy and composing various metabolic pathways. Nevertheless, in the catalytic cycle of polysaccharide degradation, the details of how the substrates bind and how the products release need more case studies. Here, we choose an inulin fructotransferase (SpIFTase) as a model system, which can degrade inulin into functionally difructose anhydride I. At first, the crystal structures of SpIFTase in the absence of carbohydrates and complex with fructosyl-nystose (GF4), difructose anhydride I, and fructose are obtained, giving the substrate trajectory and product path of SpIFTase, which are further supported by steered molecular dynamics simulations (MDSs) along with mutagenesis. Furthermore, structural topology variations at the active centers of inulin fructotransferases are suggested as the structural base for product release, subsequently proven by substitution mutagenesis and MDSs. Therefore, this study provides a case in point for a deep understanding of the catalytic cycle with substrate trajectory and product path.
Asunto(s)
Hexosiltransferasas , Inulina , Hexosiltransferasas/química , Hexosiltransferasas/metabolismo , Hexosiltransferasas/genética , Inulina/metabolismo , Inulina/química , Especificidad por Sustrato , Simulación de Dinámica Molecular , Dominio Catalítico , Biocatálisis , Catálisis , Fructosa/metabolismo , Fructosa/químicaRESUMEN
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.
Asunto(s)
Citosina , Edición Génica , Proteínas , Ratas , Animales , Humanos , Macaca fascicularis , Citidina Desaminasa/genética , ARN/genética , ADN/genética , Sistemas CRISPR-CasRESUMEN
Introduction: The activation of cerebral endothelial cells (CECs) has recently been reported to be the earliest acute neuroinflammation event in the CNS during sepsis-associated encephalopathy (SAE). Importantly, adenosine-to-inosine (A-to-I) RNA editing mediated by ADARs has been associated with SAE, yet its role in acute neuroinflammation in SAE remains unclear. Methods: Our current study systematically analyzed A-to-I RNA editing in cerebral vessels, cerebral endothelial cells (CECs), and microglia sampled during acute neuroinflammation after treatment in a lipopolysaccharide (LPS)-induced SAE mouse model. Results: Our results showed dynamic A-to-I RNA editing activity changes in cerebral vessels during acute neuroinflammation. Differential A-to-I RNA editing (DRE) associated with acute neuroinflammation were identified in these tissue or cells, especially missense editing events such as S367G in antizyme inhibitor 1 (Azin1) and editing events in lincRNAs such as maternally expressed gene 3 (Meg3), AW112010, and macrophage M2 polarization regulator (Mm2pr). Importantly, geranylgeranyl diphosphate synthase 1 (Ggps1) and another three genes were differentially edited across cerebral vessels, CECs, and microglia. Notably, Spearman correlation analysis also revealed dramatic time-dependent DRE during acute neuroinflammation, especially in GTP cyclohydrolase1 (Gch1) and non-coding RNA activated by DNA damage (Norad), both with the editing level positively correlated with both post-LPS treatment time and edited gene expression in cerebral vessels and CECs. Discussion: The findings in our current study demonstrate substantial A-to-I RNA editing changes during acute neuroinflammation in SAE, underlining its potential role in the disease.
RESUMEN
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.