Heterologous Expression,Biosynthetic Studies,and Ecological Function of the Selective Gq‐Signaling Inhibitor FR900359

The cyclic depsipeptide FR900359 (FR), isolated from the tropical plant Ardisia crenata, is a strong and selective inhibitor of Gq proteins, making it an indispensable pharmacological tool to study Gq‐related processes, as well as a promising drug candidate. Gq inhibition is a novel mode of action for defense chemicals and crucial for the ecological function of FR, as shown by in vivo experiments in mice, its affinity to insect Gq proteins, and insect toxicity studies. The uncultured endosymbiont of A. crenata was sequenced, revealing the FR nonribosomal peptide synthetase (frs) gene cluster. We here provide a detailed model of FR biosynthesis, supported by in vitro enzymatic and bioinformatic studies, and the novel analogue AC‐1, which demonstrates the flexibility of the FR starter condensation domains. Finally, expression of the frs genes in E. coli led to heterologous FR production in a cultivable, bacterial host for the first time.     

Biosynthesis and Heterologous Production of Vioprolides: Rational Biosynthetic Engineering and Unprecedented 4‐Methylazetidinecarboxylic Acid Formation

Vioprolides are a promising class of anticancer and antifungal lead compounds produced by the myxobacterium Cystobacter violaceus Cb vi35. Previously nothing had been reported about their biosynthesis, including the origin of the unusual 4‐methylazetidinecarboxylic acid (MAZ) moiety. We describe the vioprolide biosynthetic gene cluster and solve the production obstacle by expression in three heterologous hosts. Starting from unstable production in the wild type at the single‐digit mg L^?1 scale, we developed a stable host that eventually allowed for yields of up to half a gram per liter in fermenters. Gene inactivations coupled with isotope feeding studies identified an S‐adenosylmethionine (SAM)‐dependent enzyme and a methyltransferase as being responsible for the generation of the MAZ building block by a proposed mechanism unprecedented in bacteria. Furthermore, nonnatural vioprolide derivatives were generated via rational genetic engineering.     

Reconstitution of Biosynthetic Machinery for the Synthesis of the Highly Elaborated Indole Diterpene Penitrem

Penitrem A is one of the most elaborated members of the fungal indole diterpenes. Two separate penitrem gene clusters were identified using genomic and RNA sequencing data, and 13 out of 17 transformations in the penitrem biosynthesis were elucidated by heterologous reconstitution of the relevant genes. These reactions involve 1) a prenylation‐initiated cationic cyclization to install the bicyclo[3.2.0]heptane skeleton (PtmE), 2) a two‐step P450‐catalyzed oxidative processes forming the unique tricyclic penitrem skeleton (PtmK and PtmU), and 3) five sequential oxidative transformations (PtmKULNJ). Importantly, without conventional gene disruption, reconstitution of the biosynthetic machinery provided sufficient data to determine the pathway. It was thus demonstrated that the Aspergillus oryzae reconstitution system is a powerful method for studying the biosynthesis of complex natural products.     

Unveiling the Biosynthetic Pathway of the Ribosomally Synthesized and Post‐translationally Modified Peptide Ustiloxin B in Filamentous Fungi

The biosynthetic machinery of the first fungal ribosomally synthesized and post‐translationally modified peptide (RiPP) ustiloxin B was elucidated through a series of gene inactivation and heterologous expression studies. The results confirmed an essential requirement for novel oxidases possessing the DUF3328 motif for macrocyclization, and highly unique side‐chain modifications by three oxidases (UstCF1F2) and a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme (UstD). These findings provide new insight into the expression of the RiPP gene clusters found in various fungi.     

Heterologous Reconstitution of Ikarugamycin Biosynthesis in E. coli

Polycyclic tetramate macrolactams (PTMs) are a family of biomedically promising natural products with challenging molecular frameworks. Despite these interesting properties, so far only relatively little is known about the biosynthetic origin of PTMs, in particular concerning the mechanism by which their ring systems are formed. Herein we present the first insights into these processes by using the biosynthesis of ikarugamycin as an example. This has been facilitated by the first heterologous expression of a PTM biosynthetic gene cluster in Escherichia coli. With this approach it will not only become possible to mechanistically investigate already known PTM biosynthetic pathways in more detail in the future, but also to interrogate cryptic PTM biosynthetic pathways chemically and biochemically.     

Use of a Phosphonate Methyltransferase in the Identification of the Fosfazinomycin Biosynthetic Gene Cluster

Natural product discovery has been boosted by genome mining approaches, but compound purification is often still challenging. We report an enzymatic strategy for “stable isotope labeling of phosphonates in extract” (SILPE) that facilitates their purification. We used the phosphonate methyltransferase DhpI involved in dehydrophos biosynthesis to methylate a variety of phosphonate natural products in crude spent medium with a mixture of labeled and unlabeled S‐adenosyl methionine. Mass‐guided fractionation then allowed straightforward purification. We illustrate its utility by purifying a phosphonate that led to the identification of the fosfazinomycin biosynthetic gene cluster. This unusual natural product contains a hydrazide linker between a carboxylic acid and a phosphonic acid. Bioinformatic analysis of the gene cluster provides insights into how such a structure might be assembled.     

Homologous and Heterologous Expression of Delta(12)-Desaturase in Mucor circinelloides Enhanced the Production of Linolenic Acid

Linolenic acid (LA) is gaining more interest within the scientific community. This is because it has a potential medical role in reducing the risk of inflammation, carcinogenesis, atherosclerosis and diabetes and is a valuable nutraceutical for human health. The oleaginous fungus Mucor circinelloides produces a high lipid content (36%), including valuable polyunsaturated fatty acids (PUFAs). However, the critical step in which oleic acid (OA) is converted into LA is not efficient at supplying enough substrates for PUFA synthesis. Hence, we propose a method to increase LA production based on genetic engineering. The overexpression of the Δ12-desaturase gene from M. circinelloides and Mortierella alpina increased the LA content and improved the lipid accumulation (from 14.9% to 21.6% in the Δ12-desaturase gene of the M. circinelloides overexpressing strain (Mc-D12MC) and from 14.9% to 18.7% in the Δ12-desaturase gene of M. alpina overexpressing strain (Mc-D12MA)). Additionally, the up-regulated expression levels of these genes targeted the genes involved in NADPH production, implying that the elevated Δ12-desaturase gene may function as a critical regulator of NADPH and lipid synthesis in M. circinelloides. This study provides the first evidence to support the design of metabolic engineering related to LA and PUFA production in M. circinelloides for potential industrial applications.     

Cloning,Heterologous Expression,and Characterization of the Gene Cluster Required for Gougerotin Biosynthesis

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Highlights? The complete gougerotin biosynthetic gene cluster is cloned ? Heterologous production of gougerotin is accomplished in S. coelicolor M1146 ? Biosynthetic pathway is proposed based on bioinformatics, genetic, and chemical analyses ? Preliminary data support an uncommon enzymatic reaction for peptide bond formation     

Genome Mining,Heterologous Expression,Antibacterial and Antioxidant Activities of Lipoamides and Amicoumacins from Compost-Associated Bacillus subtilis fmb60

Bacillus subtilis fmb60, which has broad-spectrum antimicrobial activities, was isolated from plant straw compost. A hybrid NRPS/PKS cluster was screened from the genome. Sixteen secondary metabolites produced by the gene cluster were isolated and identified using LC-HRMS and NMR. Three lipoamides D–F (1–3) and two amicoumacin derivatives, amicoumacins D, E (4, 5), were identified, and are reported here for the first time. Lipoamides D–F exhibited strong antibacterial activities against harmful foodborne bacteria, with the MIC ranging from 6.25 to 25 µg/mL. Amicoumacin E scavenged 38.8% of ABTS⁺ radicals at 1 mg/mL. Direct cloning and heterologous expression of the NRPS/PKS and ace gene cluster identified its importance for the biosynthesis of amicoumacins. This study demonstrated that there is a high potential for biocontrol utilization of B. subtilis fmb60, and genome mining for clusters of secondary metabolites of B. subtilis fmb60 has revealed a greater biosynthetic potential for the production of novel natural products than previously anticipated.     

Refactoring the Concise Biosynthetic Pathway of Cyanogramide Unveils Spirooxindole Formation Catalyzed by a P450 Enzyme

Cyanogramide ( 1 ) from the marine actinomycete Actinoalloteichus cyanogriseus WH1‐2216‐6 features a unique spirooxindole skeleton and exhibits significant bioactivity to efficiently reverse drug resistance in tumor cells. The biosynthetic gene cluster of 1 in A. cyanogriseus WH1‐2216‐6 was identified and refactored by promoter engineering for heterologous expression in Streptomyces coelicolor YF11, thereby enabling the production of 1 and five new derivatives. Interesting, four of them, including 1 , were identified as enantiomeric mixtures in different ratios. The functions of tailoring enzymes, including two methyltransferases (CyaEF), and three cytochrome P450 monooxygenases (CyaGHI) were confirmed by gene inactivation and feeding experiments, leading to the elucidation of a concise biosynthetic pathway for 1 . Notably, CyaH was biochemically verified to catalyze the formation of the spirooxindole skeleton in 1 through an unusual carbocation‐mediated semipinacol‐type rearrangement reaction.     

放线菌来源生物碱的生物合成机制

生物碱类天然产物通常具有复杂多样的化学结构和广泛的生物活性,因此备受生物学、化学、药学领域研究者的关注。微生物是仅次于植物的生物碱类天然产物重要来源,微生物尤其是放线菌产生的众多次生代谢产物中,也包括很多生物碱。对放线菌来源生物碱的骨架结构和药效基团生物合成研究,不仅能够丰富人们对天然产物结构形成原理的理解,还可以为运用合成生物学技术人工合成此类化合物提供重要的遗传元件。本文从模块化生物合成和非模块化生物合成两种方式,综述放线菌来源生物碱的生物合成基因簇、途径及其酶催化反应过程。     

Elucidation of Biosynthetic Pathways of Natural Products

During the last decade, we have revealed biosynthetic pathways responsible for the formation of important and chemically complex natural products isolated from various organisms through genetic manipulation. Detailed in vivo and in vitro characterizations enabled elucidation of unexpected mechanisms of secondary metabolite biosynthesis. This personal account focuses on our recent efforts in identifying the genes responsible for the biosynthesis of spirotryprostatin, aspoquinolone, Sch 210972, pyranonigrin, fumagillin and pseurotin. We exploit heterologous reconstitution of biosynthetic pathways of interest in our study. In particular, extensive involvement of oxidation reactions is discussed. Heterologous hosts employed here are Saccharomyces cerevisiae, Aspergillus nidulans and A. niger that can also be used to prepare biosynthetic intermediates and product analogs by engineering the biosynthetic pathways using the knowledge obtained by detailed characterizations of the enzymes. (998 char.)     

The Biosynthetic Gene Cluster of Mushroom-Derived Antrocin Encodes Two Dual-Functional Haloacid Dehalogenase-like Terpene Cyclases

(−)-Antrocin ( 1 ), produced by the medicinal mushroom Antrodia cinnamomea, is a potent antiproliferative compound. The biosynthetic gene cluster of 1 was identified, and the pathway was characterized by heterologous expression. We characterized a haloacid dehalogenase-like terpene cyclase AncC that biosynthesizes the drimane-type sesquiterpene (+)-albicanol ( 2 ) from farnesyl pyrophosphate (FPP). Biochemical characterization of AncC, including kinetic studies and mutagenesis, demonstrated the functions of two domains: a terpene cyclase (TC) and a pyrophosphatase (PPase). The TC domain first cyclizes FPP to albicanyl pyrophosphate, and the PPase domain then removes the pyrophosphate to form 2 . Intriguingly, AncA (94 % sequence identity to AncC), in the same gene cluster, converts FPP into (R)-trans-γ-monocyclofarnesol instead of 2 . Notably, Y283/F375 in the TC domain of AncA serve as a gatekeeper in controlling the formation of a cyclofarnesoid rather than a drimane-type scaffold.