Methyl 3‐Hydroxymyristate,a Diffusible Signal Mediating phc Quorum Sensing in Ralstonia solanacearum

Ralstonia solanacearum, a plant pathogenic bacterium causing “bacterial wilt” on crops, uses a quorum sensing (QS) system consisting of phc regulatory elements to control its virulence. Methyl 3‐hydroxypalmitate (3‐OH PAME) was previously identified as the QS signal in strain AW1. However, 3‐OH PAME has not been reportedly detected from any other strains, and this suggests that they produce another unknown QS signal. Here we identify (R)‐methyl 3‐hydroxymyristate [(R)‐3‐OH MAME] as a new QS signal that regulates the production of virulence factors and secondary metabolites. (R)‐3‐OH MAME was synthesized by the methyltransferase PhcB and sensed by the histidine kinase PhcS. The phylogenetic trees of these proteins from R. solanacearum strains were divided into two groups, according to their QS signal types—(R)‐3‐OH MAME or (R)‐3‐OH PAME. These results demonstrate that (R)‐3‐OH MAME is another crucial QS signal and highlight the unique evolution of QS systems in R. solanacearum.     

Leaf-to-Whole Plant Spread Bioassay for Pepper and Ralstonia solanacearum Interaction Determines Inheritance of Resistance to Bacterial Wilt for Further Breeding

Bacterial wilt (BW) disease from Ralstonia solanacearum is a serious disease and causes severe yield losses in chili peppers worldwide. Resistant cultivar breeding is the most effective in controlling BW. Thus, a simple and reliable evaluation method is required to assess disease severity and to investigate the inheritance of resistance for further breeding programs. Here, we developed a reliable leaf-to-whole plant spread bioassay for evaluating BW disease and then, using this, determined the inheritance of resistance to R. solanacearum in peppers. Capsicum annuum ‘MC4′ displayed a completely resistant response with fewer disease symptoms, a low level of bacterial cell growth, and significant up-regulations of defense genes in infected leaves compared to those in susceptible ‘Subicho’. We also observed the spreading of wilt symptoms from the leaves to the whole susceptible plant, which denotes the normal BW wilt symptoms, similar to the drenching method. Through this, we optimized the evaluation method of the resistance to BW. Additionally, we performed genetic analysis for resistance inheritance. The parents, F₁ and 90 F₂ progenies, were evaluated, and the two major complementary genes involved in the BW resistance trait were confirmed. These could provide an accurate evaluation to improve resistant pepper breeding efficiency against BW.     

真氧产碱杆菌利用短链有机酸合成聚羟基烷酸酯

研究了真氧产碱杆菌利用单一以及混合短链有机酸作为碳源进行聚羟基烷酸酯(PHAs)的生物合成. 不同的碳氮浓度比对产物的形成有较大影响；在同样的碳源浓度下，较低氮源浓度更加有利于产物的合成；当短链有机酸浓度为8 g/L、硫酸铵浓度为0.3 g/L时对PHAs的合成最为有利.在4种短链有机酸分别单独用作碳源时，乳酸的消耗速度最快，其次为乙酸、丁酸以及丙酸；在单酸发酵条件下，丁酸的PHAs产量最高(2.72 g/L). 通过比较摇瓶以及5 L罐水平的混合酸发酵发现，发酵罐中的各项发酵指标均优于摇瓶. 研究还发现，真氧产碱杆菌在利用丙酸为碳源进行产物合成时，在发酵后期还伴随有乙酸产生.     

Functional Dissection of a Multimodular Polypeptide of the Pikromycin Polyketide Synthase into Monomodules by Using a Matched Pair of Heterologous Docking Domains

Working together or apart : Separating multimodular PKS enzymes into their respective monomodules by replacing the natural intraprotein linkers (illustrated in red in the figure) with a matched docking domain pair from a heterologous PKS system, leads to only small losses in overall in vivo polyketide product and increased efficiency at utilizing polyketide pathway intermediates to prime the biosynthetic process.

     

Thioether crosslinkages created by a radical SAM enzyme

Unusually versatile: While the β-carbon thioether linkage in lantibiotics has long been appreciated and is relatively well characterized, a recent publication shows that the unusual sulfur-to-α-carbon thioether crosslinks in subtilosin A are produced by a radical SAM enzyme, AlbA, that contains two [4?Fe-4?S] clusters, thus highlighting the versatility of post-translational modifications in natural product biosynthesis.     

Production of the tubulin destabilizer disorazol in Sorangium cellulosum: biosynthetic machinery and regulatory genes

Myxobacteria show a high potential for the production of natural compounds that exhibit a wide variety of antibiotic, antifungal, and cytotoxic activities. The genus Sorangium is of special biotechnological interest because it produces almost half of the secondary metabolites isolated from these microorganisms. We describe a transposon-mutagenesis approach to identifying the disorazol biosynthetic gene cluster in Sorangium cellulosum So ce12, a producer of multiple natural products. In addition to the highly effective disorazol-type tubulin destabilizers, S. cellulosum So ce12 produces sorangicins, potent eubacterial RNA polymerase inhibitors, bactericidal sorangiolides, and the antifungal chivosazoles. To obtain a transposon library of sufficient size suitable for the identification of the presumed biosynthetic gene clusters, an efficient transformation method was developed. We present here the first electroporation protocol for a strain of the genus Sorangium. The transposon library was screened for disorazol-negative mutants. This approach led to the identification of the corresponding trans-acyltransferase core biosynthetic gene cluster together with a region in the chromosome that is likely to be involved in disorazol biosynthesis. A third region in the genome harbors another gene that is presumed to be involved in the regulation of disorazol production. A detailed analysis of the biosynthetic and regulatory genes is presented in this paper.     

Deciphering Pactamycin Biosynthesis and Engineered Production of New Pactamycin Analogues

Pactamycin is an aminocyclopentitol‐derived natural product that has potent antibacterial and antitumor activities. Sequence analysis of an 86 kb continuous region of the chromosome from Streptomyces pactum ATCC 27456 revealed a gene cluster involved in the biosynthesis of pactamycin. Gene inactivation of the Fe‐S radical SAM oxidoreductase (ptmC) and the glycosyltransferase (ptmJ), individually abrogated pactamycin biosynthesis; this confirmed the involvement of the ptm gene cluster in pactamycin biosynthesis. The polyketide synthase gene (ptmQ) was found to support 6‐methylsalicylic acid (6‐MSA) synthesis in a heterologous host, S. lividans T7. In vivo inactivation of ptmQ in S. pactum impaired pactamycin and pactamycate production but led to production of two new pactamycin analogues, de‐6‐MSA‐pactamycin and de‐6‐MSA‐pactamycate. The new compounds showed equivalent cytotoxic and antibacterial activities with the corresponding parent molecules and shed more light on the structure–activity relationship of pactamycin.     

Strategies for the Discovery of New Natural Products by Genome Mining

New drugs from silent gene clusters : Analysis of genome sequence data has identified numerous “cryptic” gene clusters encoding novel natural product biosynthetic assembly lines; this suggests that many new bioactive metabolites remain to be discovered, even in extensively investigated organisms. Several related and complementary strategies for identifying the products of these clusters have emerged recently and revitalized the search for novel bioactive natural products.

     

Discovery of the Tiancilactone Antibiotics by Genome Mining of Atypical Bacterial Type II Diterpene Synthases

Although genome mining has advanced the identification, discovery, and study of microbial natural products, the discovery of bacterial diterpenoids continues to lag behind. Herein, we report the identification of 66 putative producers of novel bacterial diterpenoids, and the discovery of the tiancilactone (TNL) family of antibiotics, by genome mining of type II diterpene synthases that do not possess the canonical DXDD motif. The TNLs, which are broad‐spectrum antibiotics with moderate activities, are produced by both Streptomyces sp. CB03234 and Streptomyces sp. CB03238 and feature a highly functionalized diterpenoid skeleton that is further decorated with chloroanthranilate and γ‐butyrolactone moieties. Genetic manipulation of the tnl gene cluster resulted in TNL congeners, which provided insights into their biosynthesis and structure–activity relationships. This work highlights the biosynthetic potential that bacteria possess to produce diterpenoids and should inspire continued efforts to discover terpenoid natural products from bacteria.     

Discovery and Elucidation of the Biosynthesis of Aspernidgulenes: Novel Polyenes from Aspergillus Nidulans by Using Serial Promoter Replacement

Through serial promoter exchanges, we isolated several novel polyenes, the aspernidgulenes, from Aspergillus nidulans and uncovered their succinct biosynthetic pathway involving only four enzymes. An enoyl reductase (ER)-less highly reducing polyketide synthase (HR-PKS) putatively produces a 5,6-dihydro-α-pyrone polyene, which undergoes bisepoxidation, epoxide ring opening, cyclization, and hydrolytic cleavage by three tailoring enzymes to generate aspernidgulene A1 and A2. Our findings demonstrate the prowess of fungal-tailoring enzymes to transform a polyketide scaffold concisely and efficiently into complex structures. Moreover, comparison with citreoviridin and aurovertin biosynthesis suggests that methylation of the α-pyrone hydroxy group by methyltransferase (CtvB or AurB) is the branching point at which the biosynthesis of these two classes of compounds diverge. Therefore, scanning for the presence or absence of the gatekeeping α-pyrone methyltransferase gene in homologous clusters might be a potential way to classify the product bioinformatically as belonging to methylated α-pyrone polyenes or polyenes containing rings derived from the cyclization of the unmethylated 5,6-dihydro-α-pyrone, such as 2,3-dimethyl-γ-lactone and oxabicyclo[2.2.1]heptane.     

Heterologous Production of Glidobactins/Luminmycins in Escherichia coli Nissle Containing the Glidobactin Biosynthetic Gene Cluster from Burkholderia DSM7029

Natural product peptide‐based proteasome inhibitors show great potential as anticancer drugs. Here we have cloned the biosynthetic gene cluster of a potent proteasome inhibitor—glidobactin from Burkholderia DSM7029—and successfully detected glidobactins/luminmycins in E. coli Nissle. We have also improved the yield of glidobactin A tenfold by promoter change in a heterologous host. In addition, two new biosynthetic intermediates were identified by comparative MS/MS fragmentation analysis. Identification of acyclic luminmycin E implies substrate specificity of the TE domain for cyclization. The establishment of a heterologous expression system for syrbactins provided the basis for the generation of new syrbactins as proteasome inhibitors by molecular engineering, but the TE domain's specificity cannot be ignored.     

Models for the Biodegradation Kinetics of Naphthalene by Ralstonia Eutropha

Biodegradation of naphthalene (naph.) by a pure culture of Ralstonia eutropha was studied. Dissolution of naph. in aqueous solution was provided by using Tween-80 nonionic surfactant. By developing the solubility curve, the molar solubility ratio (MSR) was determined (0.8081). The effects of Tween-80, initial-pH, and naph. concentration on the R. eutropha growth and its naph. degradation capability were investigated. Degradation of naph. at 50 mg/L was complete within 52 h in the presence of 1.6 mM Tween-80 at pH 7. Haldane, Webb, Aiba, and Edwards, as the growth inhibitory kinetic models, were used to mathematically describe the inhibitory characteristic of naphthalene and the results were evaluated using nonlinear regression technique. The values of root mean square error (RMSE) and the correlation coefficient (R²) showed appropriateness of approach taken in the present study (RMSE≈ 3×10^?3, R²≈ 0.99). Importance of the mechanistic view of these types of mathematical models has been emphasized.