No abstract

Anaerobic bacteria represent an overlooked rich source of biological and chemical diversity. Due to the challenge of cultivation and genetic intractability, assessing the capability of their biosynthetic gene clusters (BGCs) for secondary metabolite production requires an efficient heterologous expression system. However, this kind of host system is still unavailable. Here, we use the facultative anaerobe Streptococcus mutans UA159 as a heterologous host for the expression of BGCs from anaerobic bacteria. A natural competence based large DNA fragment cloning (NabLC) technique was developed, which can move DNA fragments up to 40-kb directly and integrate a 73.7-kb BGC to the genome of S. mutans UA159 via three rounds of NabLC cloning. Using this system, we identify an anti-infiltration compound, mutanocyclin, from undefined BGCs from human oral bacteria. We anticipate this host system will be useful for heterologous expression of BGCs from anaerobic bacteria.

Frogeye leaf spot, caused by Cercospora sojina Hara, is a common disease of soybean in most soybean-growing countries of the world. In this study, we report a high-quality genome sequence of C. sojina by Single Molecule Real-Time sequencing method. The 40.8-Mb genome encodes 11,655 predicated genes, and 8,474 genes are revealed by RNA sequencing. Cercospora sojina genome contains large numbers of gene clusters that are involved in synthesis of secondary metabolites, including mycotoxins and pigments. However, much less carbohydrate-binding module protein encoding genes are identified in C. sojina genome, when compared with other phytopathogenic fungi. Bioinformatics analysis reveals that C. sojina harbours about 752 secreted proteins, and 233 of them are effectors. During early infection, the genes for metabolite biosynthesis and effectors are significantly enriched, suggesting that they may play essential roles in pathogenicity. We further identify 13 effectors that can inhibit BAX-induced cell death. Taken together, our results provide insights into the infection mechanisms of C. sojina on soybean.

Seven-carbon-chain-containing sugars exist in several groups of important bacterial natural products. Septacidin represents a group of l-heptopyranoses containing nucleoside antibiotics with antitumor, antifungal, and pain-relief activities. Hygromycin B, an aminoglycoside anthelmintic agent used in swine and poultry farming, represents a group of d-heptopyranoses-containing antibiotics. To date, very little is known about the biosynthesis of these compounds. Here we sequenced the genome of the septacidin producer and identified the septacidin gene cluster by heterologous expression. After determining the boundaries of the septacidin gene cluster, we studied septacidin biosynthesis by in vivo and in vitro experiments and discovered that SepB, SepL, and SepC can convert d-sedoheptulose-7-phosphate (S-7-P) to ADP-l--β-d--heptose, exemplifying the involvement of ADP-sugar in microbial natural product biosynthesis. Interestingly, septacidin, a secondary metabolite from a gram-positive bacterium, shares the same ADP-heptose biosynthesis pathway with the gram-negative bacterium LPS. In addition, two acyltransferase-encoding genes and, were proposed to be involved in septacidin side-chain formation according to the intermediates accumulated in their mutants. In hygromycin B biosynthesis, an isomerase HygP can recognize S-7-P and convert it to ADP-d--β-d--heptose together with GmhA and HldE, two enzymes from the LPS heptose biosynthetic pathway, suggesting that the d-heptopyranose moiety of hygromycin B is also derived from S-7-P. Unlike the other S-7-P isomerases, HygP catalyzes consecutive isomerizations and controls the stereochemistry of both C2 and C3 positions.

Streptomyces is studied intensively for its outstanding ability to produce bioactive secondary metabolites and for its complicated morphological differentiation process. A classical genetic manipulation system for Streptomyces has been developed and widely used in the community for a long time, using antibiotic resistance markers to select for double-crossover mutants. The screening process is always laborious and time-consuming. However, the lack of a suitable chromogenic reporter for Streptomyces has limited the use of color-based screening system to simplify the selection process for double-crossover mutants. In this study, a blue reporter system for Streptomyces has been established by mining an indigoidine synthetase gene (idgS) from Streptomyces lavendulae CGMCC 4.1386, leading to the development of a time-saving gene inactivation system for Streptomyces by simple blue-white screening. A series of Streptomyces suicide and temperature-sensitive plasmids containing the idgS reporter cassette were constructed and used successfully to inactivate genes in Streptomyces, allowing a simple and efficient screening method to differentiate the colonies for double-crossover (white) and single-crossover (blue) mutants. Inactivation of the putative γ-butyrolactone synthase gene afsA-y via the idgS-based blue-white screening method revealed that the paulomycin production is negatively controlled by afsA-y in Streptomyces sp. YN86.

Streptothricins (STNs) are atypical aminoglycosides containing a rare carbamoylated D-gulosamine (D-GulN) moiety, and the antimicrobial activity of STNs has been exploited for crop protection. Herein, the biosynthetic pathway of the carbamoylated D-GulN moiety was delineated. An N-acetyl-D-galactosamine is first attached to the streptolidine lactam by the glycosyltransferse StnG and then epimerized to N-acetyl-D-gulosamine by the putative epimerase StnJ. After carbamoylation by the carbamoyltransferase StnQ, N-acetyl-D-GulN is deacetylated by StnI to furnish the carbamoylated D-GulN moiety. In vitro studies characterized two novel enzymes: StnG is an unprecedented GT-A fold N-glycosyltransferase that glycosylates the imine nitrogen atom of guanidine, and StnI is the first reported N-acetyl-D-GulN deacetylase.

NW-G01 is a novel cyclic hexapeptide antibiotic produced by Streptomyces alboflavus 313. Its relative structure was established by HR-ESI-MS, IR, 1D and 2D NMR techniques, the absolute structure was determined using a combination of single-crystal X-ray diffraction and Marfey's method finally. The antibiotic consists of L-valine, (3S)-and (3R)-piperazic acids, N-methyl-Dalanine and (2S,3aR,8aS)-6-chloro-3a-hydroxy-1, 2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid.

Alboflavusins (AFNs) are a group of cyclohexapeptides with moderate antibacterial and antitumor activities from Streptomyces alboflavus sp. 313. In vivo and in vitro studies proposed that AFNs are biosynthesized by a nonribosomal peptide synthetase machinery, and the 6-Cl-L-Trp precursor is supplied by a tryptophan halogenase gene located outside the afn gene cluster. Guided by the structure–activity relationship knowledge about the AFN-like cyclohexapeptides, two dimeric AFNs (di-AFNs) with regiospecific biaryl linkages were designed and generated biotechnologically by expressing the P450 gene hmtS or clpS in S. alboflavus wild-type and mutant strains. The di-AFNs displayed much better antibacterial and antitumor activities than their monomers as anticipated, exemplifying a rational strategy to generate natural product congeners with improved bioactivities.