Shinta Ijichi

The unique bioactivities of arsenic-containing secondary metabolites have been revealed recently, but studies on arsenic secondary metabolism in microorganisms have been extremely limited. Here, we focused on the organoarsenic metabolite with an unknown chemical structure, named bisenarsan, produced by well-studied model actinomycetes and elucidated its structure by combining feeding of the putative biosynthetic precursor (2-hydroxyethyl)arsonic acid to Streptomyces lividans 1326 and detailed NMR analyses. Bisenarsan is the first characterized actinomycete-derived arsenic secondary metabolite and may function as a prototoxin form of an antibacterial agent or be a detoxification product of inorganic arsenic species. We also verified the previously proposed genes responsible for bisenarsan biosynthesis, especially the (2-hydroxyethyl)arsonic acid moiety. Notably, we suggest that a C-As bond in bisenarsan is formed by the intramolecular rearrangement of a pentavalent arsenic species (arsenoenolpyruvate) by the cofactor-independent phosphoglycerate mutase homologue BsnN, that is entirely distinct from the conventional biological C-As bond formation through As-alkylation of trivalent arsenic species by S-adenosylmethionine-dependent enzymes. Our findings will speed up the development of arsenic natural product biosynthesis.

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) with polar-functionalized fatty acyl groups are newly found lipopeptide-class natural products. We recently employed a combined approach of genome mining and stable isotope labeling and discovered solabiomycins as one of the polar-functionalized fatty-acylated RiPPs (PFARs) from Streptomyces lydicus NBRC13058. The solabiomycins contained a characteristic sulfoxide group in the labionin moiety referred to as the 'solabionin' structure for the RiPP moiety. A previous gene knockout experiment indicated that solS, which encodes a putative flavin adenine dinucleotide (FAD)-nicotinamide adenine dinucleotide (phosphate) (NAD(P))-binding protein, is involved in the sulfoxidation of an alkyl sulfide in the solabionin. In this study, we isolated deoxysolabiomycins A and B from ΔsolS mutant and fully determined the chemical structures using a series of NMR experiments. We also tested the bioactivity of deoxysolabiomycins against Gram-positive bacteria, including Mycolicibacterium smegmatis, and notably found that the sulfoxide is critical for the antibacterial activity. To characterize the catalytic activity of SolS, the recombinant protein was incubated with a putative substrate, deoxysolabiomycins, and the cofactors FAD and NADPH. In vitro reactions demonstrated that SolS catalyzes the sulfoxidation, converting deoxysolabiomycins to solabiomycins.

The thermophilic bacterium Thermosporothrix hazakensis belongs to a class of Ktedonobacteria in the phylum Chloroflexota. Lanthipeptides are a naturally occurring peptide group that contains antibacterial compounds such as nisin. To find a new lanthipeptide that is a possible candidate for an antibacterial reagent, we performed genome-mining of T. hazakensis and heterologous expression experiments. Based on genome-mining, the presence of a total of ten putative biosynthetic gene clusters for class I and class II lanthipeptides was indicated from the genome sequence of T. hazakensis. New lanthipeptides named hazakensins A and B were produced by heterologous expression of a class I lanthipeptide biosynthetic gene cluster in the expression host Escherichia coli. Co-expression of the biosynthetic gene cluster with tRNA-Glu and glutamyl-tRNA synthetase coding genes derived from T. hazakensis increased the production yield of both lanthipeptides by about 4-6 times. The chemical structures of hazakensins A and B including the bridging pattern of lanthionine/methyllanthionine rings were determined by NMR and MS experiments. Since production of hazakensins A and B was not observed in the native strain T. hazakensis, heterologous production was an effective method to obtain the lanthipeptides derived from the biosynthetic gene cluster. This is the first report of heterologous production of class I lanthipeptides originating from the filamentous green non-sulfur bacteria, to the best of our knowledge. The success of heterologous production of hazakensins may lead to the discovery and development of new lanthipeptides derived from the origins of bacteria in the phylum Chloroflexota.

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) with polar-functionalized fatty acyl groups are a rarely found untapped class of natural products. Although polar-functionalized fatty-acylated RiPPs (PFARs) have potential as antimicrobial agents, the repertoire is still limited. Therefore, expanding the chemical space is expected to contribute to the development of pharmaceutical agents. In this study, we performed genome mining and stable isotope-guided comparative metabolomics to discover new PFAR natural products. We focused on the feature that PFARs incorporate l-arginine or l-lysine as the starter unit of the fatty acyl group and fed 13C6,15N4-l-arginine or 13C6,15N2-l-lysine to bacterial cultures. Metabolites were extracted and compared with those extracted from nonlabeled l-arginine or l-lysine fed cultures. We identified putative PFARs and successfully isolated solabiomycin A and B from Streptomyces lydicus NBRC 13 058 and albopeptin B from Streptomyces nigrescens HEK616, which contained a sulfoxide group in the labionin moiety. The gene disruption experiment indicated that solS, which encodes a putative flavin adenine dinucleotide (FAD)-nicotinamide adenine dinucleotide (phosphate) (NAD(P))-binding protein, is involved in the sulfoxidation of aryl sulfides. The solabiomycins showed antibacterial activity against Gram-positive bacteria, including Mycobacterium tuberculosis H37Rv with a minimum 95% inhibitory concentration (MIC95) of 3.125 μg/mL, suggesting their potential as antituberculosis agents.

AIMS: The aim of this study was to utilize a cryptic biosynthetic gene cluster (BGC) of a marine proteobacterium Thalassomonas actiniarum for production of new lanthipeptides by heterologous expression system. METHODS AND RESULTS: Based on genome mining, a new BGC of class I lanthipeptide was found in the genome sequence of a marine proteobacterium T. actiniarum. Molecular cloning was performed to construct an expression vector derived from commercially available plasmid pET-41a(+). Heterologous production of new lanthipeptides named thalassomonasins A and B was performed using the host Escherichia coli BL21(DE3) harbouring the expression vector. The structure of thalassomonasin A was determined by the interpretation of NMR and MS data. As a result, thalassomonasin A was determined to be a lanthipeptide with three units of lanthionine. The bridging pattern of the lanthionine rings in thalassomonasin A was determined by interpretation of NOESY data. The structure of thalassomonasin B was proposed by MS/MS experiment. CONCLUSIONS: We succeeded in heterologous production of new class I lanthipeptides using a BGC of a marine proteobacterium T. actiniarum. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report of heterologous production of lanthipeptides derived from proteobacterial origin. There are many cryptic biosynthetic gene clusters (BCGs) of this class of lanthipeptides in proteobacterial genomes. This study may lead to the production of new lanthipeptides by utilizing the BCGs.