海洋芽孢杆菌B-9987中macrolactin生物合成基因簇分析及反式酰基转移酶高表达

目的 对海洋芽孢杆菌B-9987中macrolactin生物合成基因簇及其关键基因进行分析、鉴定及功能研究。方法 通过生物信息学手段对基因簇的基因组成和功能结构域进行了分析;构建了用于酰基转移酶基因高表达的大肠杆菌—芽孢杆菌穿梭载体,采用电击转化方法导入B-9987之中进行高表达。 结果 macrolactins是由位于同一个操纵子的8个基因bmmA-I所编码的反式酰基转移酶聚酮合酶体系组装而成,反式酰基转移酶(trans-acyltransferase,trans-AT)BmmA的高表达使macrolactin A的产量提高了约0.6倍。结论 macrolactin生物合成基因簇在具有生物防治活性的芽孢杆菌中普遍存在,且高度保守;反式酰基转移酶的高表达能够增加macrolactin A的产量。     

红景天内生真菌Penicillium thomii C18中聚酮类次生代谢产物研究

目的 研究红景天Rhodiola rosea内生真菌 Penicillium thomii C18的次生代谢产物。方法 采用硅胶和Sephadex LH-20 柱色谱及制备型高效液相色谱（HPLC）进行分离纯化,利用核磁共振、质谱、旋光等谱学技术对化合物进行结构鉴定。结果 从Penicillium thomii C18培养物的醋酸乙酯萃取物中共分离得到9个聚酮类化合物,包括1个新的化合物与8个已知化合物,分别鉴定为 (2''S)-5,7-二羟基-2-(2''-羟基-4''-氧代戊基])-色原酮 [(2''S)-5,7-dihydroxy-2-(2''-hydroxy-4''-oxopentyl)-chromone,1]、spirocitromycetin（2）、异决明酮（isotorachrysone,3）、1,6-二羟基-3-甲基-8-甲酯基(口山)酮[1,6-dihydroxy-3-methyl-8-carbomethoxyxanthone,4]、1,5-二羟基-3-甲氧基-7-甲基-蒽-9,10-二酮 (1,5-dihydroxy-3-methoxy-7-methyl-anthracene-9,10-dione,5）、9-O-methylneuchromenin（6）、neuchromenin（7）、星形酸（asterric acid,8）、星形酸甲酯（methyl asterrate,9）。结论 化合物1为新的聚酮类化合物,命名为拖姆酮A,化合物3和5为首次从青霉属中分离得到。     

Synthesis of C11-Desmethoxy Soraphen A1α: A Natural Product Analogue That Inhibits Acetyl-CoA Carboxylase

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Divergent evolution of an atypical S-adenosyl-l-methionine–dependent monooxygenase involved in anthracycline biosynthesis

Bacterial secondary metabolic pathways are responsible for the biosynthesis of thousands of bioactive natural products. Many enzymes residing in these pathways have evolved to catalyze unusual chemical transformations, which is facilitated by an evolutionary pressure promoting chemical diversity. Such divergent enzyme evolution has been observed in S-adenosyl-l-methionine (SAM)-dependent methyltransferases involved in the biosynthesis of anthracycline anticancer antibiotics; whereas DnrK from the daunorubicin pathway is a canonical 4-O-methyltransferase, the closely related RdmB (52% sequence identity) from the rhodomycin pathways is an atypical 10-hydroxylase that requires SAM, a thiol reducing agent, and molecular oxygen for activity. Here, we have used extensive chimeragenesis to gain insight into the functional differentiation of RdmB and show that insertion of a single serine residue to DnrK is sufficient for introduction of the monooxygenation activity. The crystal structure of DnrK-Ser in complex with aclacinomycin T and S-adenosyl-l-homocysteine refined to 1.9-Å resolution revealed that the inserted serine S297 resides in an α-helical segment adjacent to the substrate, but in a manner where the side chain points away from the active site. Further experimental work indicated that the shift in activity is mediated by rotation of a preceding phenylalanine F296 toward the active site, which blocks a channel to the surface of the protein that is present in native DnrK. The channel is also closed in RdmB and may be important for monooxygenation in a solvent-free environment. Finally, we postulate that the hydroxylation ability of RdmB originates from a previously undetected 10-decarboxylation activity of DnrK.Anthracyclines are a medically important class of natural products, which belong to the type II family of aromatic polyketides (1–3). These compounds, produced by Streptomyces bacteria, are among the most effective anticancer drugs available and are currently included in 500 clinical trials worldwide (4). Doxorubicin is widely used as a first-choice chemotherapeutic agent for many tumors, including various carcinomas and sarcomas (5), whereas aclacinomycin A has been used in the treatment of hematological malignancies (6). The biological activity of doxorubicin and aclacinomycin is mediated through topoisomerase II (7), while additional mechanisms of these drugs include the recently discovered histone eviction activity (4). Despite the success of anthracyclines in cancer chemotherapy, improved compounds are still urgently needed, because the clinical efficacy of these metabolites has been limited by severe side effects (8, 9).The great diversity of anthracyclines is generated during tailoring steps in their biosynthesis, where glycosylations, hydroxylations, decarboxylations, and methylations modify the common 7,8,9,10-tetrahydro-5,12-naphthacenoquinone aglycone chromophore to obtain the biologically active molecules of the various pathways (1–3). A detailed understanding of these biosynthetic steps is essential for the rational design of improved bioactive metabolites by pathway and protein engineering. In particular, the discovery of thousands of unknown biosynthetic pathways by next-generation sequencing and the emergence of synthetic biology holds great promise for increasing the chemical space of natural products (10). However, identification of the function of gene products residing in these pathways by bioinformatic analysis alone is complicated by the atypical chemistry the corresponding enzymes may catalyze. Unexpected chemical transformations appear to be especially common in the biosynthesis of aromatic polyketides, where biochemical studies have frequently revealed unusual reaction mechanisms (2, 11), which includes instances where the reactivity of the substrates themselves are used in catalysis (12–14). In addition, homologous enzymes have been noted to catalyze diverse chemical transformations (15). Recently, the use of protein engineering for altering the reaction specificities of biosynthetic enzymes has emerged as another developing field for modification of natural products (16, 17).This unusually rapid differentiation of enzyme function may be related to the evolution of secondary metabolism, where traits enabling a swift increase in the diversity of natural products are beneficial (18). To promote chemical diversity rather than formation of a single product, specialized metabolism enzymes appear to have evolved as generalists, which is manifested as promiscuity and slow reaction rates typical to these proteins (19). One intriguing example of such divergent evolution is the enzyme pair DnrK and RdmB from the daunorubicin and rhodomycin pathways, respectively, that are homologous to classical S-adenosyl-l-methionine (SAM)-dependent methyltransferases.Methyltransferase DnrK is a true methyltransferase that catalyzes the 4-O-methylation of carminomycin (1; Fig. 1) in one of the final steps in the biosynthesis of the antitumor drug daunorubicin (2; Fig. 1) in Streptomyces peucetius (20, 21). The enzyme possesses rather relaxed substrate specificity in regard to modifications in the polyaromatic anthracycline ring system, but it is quite specific with respect to the length of the carbohydrate chain at C-7, accepting only monoglycosides (22). In addition, DnrK has even been shown to be able to methylate various flavonoids (23). The crystal structure of the ternary complex of DnrK revealed that the mechanism of methylation is consistent with canonical S_N2-type reactions of SAM-dependent methyltransferases, although proximity and orientation effects rather than acid/base catalysis appear to be more critical in the case of DnrK (22).Open in a separate windowFig. 1.Similar structures, distinct functions. The structures and reactions catalyzed by (A) DnrK and (B) RdmB. In the overall structures, the N-terminal dimerization, the middle, and the catalytic domains are shown in yellow, green, and red, respectively. In the Inset, the catalytic domains are color coded based on dissimilar (black), similar (gray), and identical (white) residues. SAH and the ligands are shown in red, whereas the three regions selected for chimeragenesis (R1, R2, and R3) are shown in orange. The R3 region of RdmB is disordered in the crystal structure and is shown as a dashed orange line in the Inset. In the reaction scheme, “R” represents a thiol reducing agent such as DTT or glutathione.On the other hand, RdmB from the β-rhodomycin pathway in Streptomyces purpurascens is a very unusual enzyme that is evolutionarily closely related to DnrK with a sequence identity of 52% (24). Determination of the crystal structure of RdmB confirmed that the enzyme binds SAM (25) and that the ternary complex is indeed very similar to that of DnrK with a root-mean-square deviation of 1.14 Å for 335 equivalent Cα atoms (26). Despite these features, RdmB completely lacks methyltransferase activity (24, 27, 28); it is an anthracycline 10-hydroxylase, which requires SAM, molecular oxygen, and a thiol reducing agent for activity (26). Contrary to DnrK, RdmB is able to use both monoglycosylated and triglycosylated anthracyclines as substrates.Furthermore, both rhodomycin and daunorubicin gene clusters encode homologous 15-methylesterases RdmC (28) and DnrP (21), respectively. On the rhodomycin pathway, RdmB works strictly in conjunction with RdmC, which first converts ε-rhodomycin (3; Fig. 1) into 15-demethoxy-ε-rhodomycin (4; Fig. 1), whereas RdmB is responsible for conversion of the RdmC reaction product into β-rhodomycin (5; Fig. 1) (28). This is in contrast to the daunorubicin pathway, where the reaction order of the two enzymes DnrP and DnrK is more relaxed, as DnrK is able to methylate at least five biosynthetic intermediates (21).To gain insight into how a methyltransferase has evolved into a monooxygenase, we report here the generation of several chimeric DnrK/RdmB proteins that were designed based on the available crystal structures. These initial experiments guided us in further structure/function studies, where we were able to demonstrate that the insertion of a single amino acid is sufficient for introduction of the 10-hydroxylase activity into the DnrK scaffold, thus providing an especially illustrative example of divergent enzyme evolution. Protein crystallography and further biochemical experiments were then conducted to reveal the molecular basis for the altered enzymatic activity.     

红色糖多孢菌A226-YA突变体构建及产物分析

目的：用红色糖多孢菌（前称糖多孢红霉菌）KR6突变体合成酮内酯类化合物3-脱氧-3-羰基-红霉内酯B，同时，KR6突变体不合成红霉素。方法：以红色糖多孢菌A226基因组DNA为模板，用重叠PCR技术扩增KR6酶域中Tyr2699密码子TAC突变为A1a密码子GCC的1000bpDNA序列，克隆到载体pWHM3上构建同源重组质粒pWHM3-YA。将pWHM3-YA转化到A226中，并整合到红霉素合成基因座，经染色体二次重组后筛选TAC突变为GCC的突变体A226-YA，再进行突变体A226-YA产物分析。结果：通过染色体同源重组，构建了红色糖多孢菌突变菌株A226-YA，Zabspec Fab质谱分析结果显示A226-YA突变体合成了3-脱氧-3-羰基-红霉内酯B，而没有检测到红霉素。结论：突变Tyr2699能有效失活KR6，但3-脱氧-3-羰基-红霉内酯B的产量较低。     

Systems biology of cyanobacterial secondary metabolite production and its role in drug discovery

Background: Cyanobacteria continue to be an important source of compounds that show unprecedented biological activities of pharmaceutical interest. Cyanobacterial metabolites show an interesting and exciting range of biological activities ranging from antimicrobial and immunosuppressant to anticancer and anti-HIV. Objective: This review explores the possibilities of applying systems biology approaches for harnessing these compounds as drug leads, primarily produced through large multimodular non-ribosomal peptide synthetase (NRPS), polyketide synthase (PKS) and mixed NRPS–PKS enzymatic systems. Methods: A brief survey of the strategies for in silico analysis for drug target identification using genomic and high-throughput proteomics data, virtual screening and receptor–ligand docking based approaches are also discussed. Conclusion: We conclude with an outlook on how the field will evolve, especially in partnership with the new engineering-based, more endpoint exploitative paradigm of synthetic biology.     

Accurate prediction of secondary metabolite gene clusters in filamentous fungi

Biosynthetic pathways of secondary metabolites from fungi are currently subject to an intense effort to elucidate the genetic basis for these compounds due to their large potential within pharmaceutics and synthetic biochemistry. The preferred method is methodical gene deletions to identify supporting enzymes for key synthases one cluster at a time. In this study, we design and apply a DNA expression array for Aspergillus nidulans in combination with legacy data to form a comprehensive gene expression compendium. We apply a guilt-by-association–based analysis to predict the extent of the biosynthetic clusters for the 58 synthases active in our set of experimental conditions. A comparison with legacy data shows the method to be accurate in 13 of 16 known clusters and nearly accurate for the remaining 3 clusters. Furthermore, we apply a data clustering approach, which identifies cross-chemistry between physically separate gene clusters (superclusters), and validate this both with legacy data and experimentally by prediction and verification of a supercluster consisting of the synthase AN1242 and the prenyltransferase AN11080, as well as identification of the product compound nidulanin A. We have used A. nidulans for our method development and validation due to the wealth of available biochemical data, but the method can be applied to any fungus with a sequenced and assembled genome, thus supporting further secondary metabolite pathway elucidation in the fungal kingdom.     

The Molecular Genetics and Regulation of Cyanobacterial Peptide Hepatotoxin Biosynthesis

Over the last 10 years, we have witnessed major advances in our understanding of natural product biosynthesis, including the genetic basis for toxin production by numerous groups of cyanobacteria. Cyanobacteria produce an unparalleled array of bioactive secondary metabolites, including alkaloids, polyketides and non-ribosomal peptides, some of which are potent toxins. This review addresses the molecular genetics underlying the production of hepatotoxins, microcystin and nodularin in fresh and brackish water. These toxins pose a serious threat to human health and their occurrence in water supplies is increasing, because of the prevalence of toxic algal blooms worldwide. Toxin biosynthesis gene-cluster-associated transposition and the natural transformability of certain species suggest a broader distribution of toxic cyanobacterial taxa. The information gained from the discovery of these toxin biosynthetic pathways has enabled the genetic screening of various environments for drinking-water quality management. Understanding the role of cyanotoxins in the producing microorganisms and the environmental regulation of their biosynthesis genes may also suggest the means of controlling toxic-bloom events.     

Expression of biosynthetic gene clusters in heterologous hosts for natural product production and combinatorial biosynthesis

Expression of biosynthetic gene clusters in heterologous hosts for natural product production and combinatorial biosynthesis is playing an increasingly important role in natural product-based drug discovery and development programmes. This review highlights the requirements and challenges associated with this conceptually simple strategy of using surrogate hosts for the production of natural products in good yields and for the generation of novel analogues by combinatorial biosynthesis methods, taking advantage of the recombinant DNA technologies and tools available in the model hosts. Specific topics addressed include: i) the mobilisation of biosynthetic gene clusters using different vector systems; ii) the selection of suitable model heterologous hosts; iii) the requirement of post-translational protein modifications and precursor supply within the model hosts; iv) the influence of promoters and pathway regulators; and v) the choice of suitable fermentation conditions. Lastly, the use of heterologous expression in combinatorial biosynthesis is addressed. Future directions for model heterologous host engineering and the optimisation of natural product biosynthetic gene cluster expression in heterologous hosts are also discussed.