Polyene polyketide antibiotics are widely used for antifungal therapy and food preservation in food industry. Recently, a few chemical and genetic studies have shown that modifications on exocyclic carboxylic group of amphotericin resulted in a significant decrease of the cytotoxicity, which encouraged us to genetically modify natamycin. However, the lower yields of its derivatives are hampered their full application.

Through inactivation of the P450 monooxygenase gene scnG, two natamycin derivatives, compounds 1 and 2, with improved pharmacological activities were detected in scnG-inactivation mutant. In order to obtain high yields, several site-directed mutations were firstly introduced into the thioesterase (TE) domain, under the guidance of TE homology modeling, and a by-product 3 was and converted into a full-length linear compound 6. Alternatively, when DH-KR12 didomain was replaced by DH-KR11 didomain, the yield of 1 and 2 was improved by 3-fold and without any accumulation of 3 and 6. Subsequent specific accumulation of 1 or 2 was achieved via further overexpression or disruption of scnD.

The specificity of TE domain towards natamycin or decarboxylic derivatives has been further investigated through crystallization of pimTE, molecular docking, site-directed mutagenesis, in vitro biochemical assay, and in vivo site-specific modification of the TE domain. The crystal structure of pimTE with a resolution of 2.07 A was obtained, which shows interesting features on dimerization, substrate binding, and interaction with the hydrophobic tetraene region of natamycin. Mutagenesis based on the molecular docking of 6 with pimTE shed new lights on the yield improvement of decarboxylic polyene antibiotics.

Natural products show diverse structures, which are enhanced by a wide variety of chemical reactions. Cyclization is one of the important reactions that contribute to the structural diversity of natural products. Cyclization reactions have been extensively studied by many researchers and diverse reaction mechanisms have been proposed. However, there are still a number of heterocycles whose biosynthetic mechanisms remained unclear. Benzastatin derivatives are natural products isolated from several Streptomyces species. They have unique heterocyclic structures, such as indoline and tetrahydroquinoline, which are probably derived from geranylated p-aminobenzoic acid derivatives. Although they have some interesting bioactivities including an antiviral activity, their biosynthetic pathways remained unclear. We focused on one of the benzastatin producers, Streptomyces sp. RI18, to elucidate the pathways for the biosynthesis of benzastatin derivatives. The benzastatin biosynthetic gene cluster (bez cluster) was identified in the Streptomyces sp. RI18 genome and the functions of six bez genes (bezA, bezB, bezC, bezE, bezG, and bezJ) were analyzed by gene disruption in a heterologous expression system. Furthermore, the functions of six biosynthetic enzymes (BezA, BezB, BezC, BezE, BezF and BezG) were confirmed by in vitro assay systems. Based on the results of these experiments, we proposed the biosynthetic pathways for benzastatin derivatives. In these pathways, geranylated p-acetoxyaminobenzoic acid derivatives are key intermediates for synthesizing indoline and tetrahydroquinoline structures and BezE (cytochrome P450) catalyzes the heterocyclic ring formation. Detailed reaction mechanisms will be discussed.

FR900452 and maremycinE/F/G feature natural indole diketopiperazine that are linked to oxocyclopentene moiety in distinct patterns. Although isolated from different Streptomyces, the closely related structures implied a common biosynthetic mechanism between FR900452 and maremycins. This work confirmed that Streptomyces sp. B9173, the reported producer of maremycins, could also generate FR900452 and its new analogues. Time course of the metabolic profile also showed that FR900452s and maremycins were synthesized successively. Disruption of mar gene cluster abolished both FR900452s and maremycin G, indicating that the biosynthesis of the skeleton of FR900452 and maremycin G used the same PKS/NRPS genes. Further cloning and heterologous expression of the mar gene cluster in S. lividans TK24 confirmed that production of maremycins and FR900452s shared the same biosynthetic machinery. Inactivation of marP, which was annotated as snoaL-like family protein, abolished the productivity of FR900452s and maremycin A, but remarkably accumulated the spiro maremycin E/F/G. Therefore, MarP, the snoaL-like family protein, played the determinant role in leading the pathway divergence.

Streptothricins (STs) produced by Streptomyces strains are broad-spectrum antibiotics and are characterized by a streptothrisamine core structure with the L-β-lysine (β-Lys) residue and its oligomeric side chains [oligo(β-Lys)]. In addition to the STs, it has been reported that Streptomyces strains produce ST-related compound, BD-12, which possess a glycine-derived side chain rather than the β-Lys residue. The amide bonds connecting the side chains in ST and BD-12 are formed via NRPS¹⁾ or tRNA-dependent²⁾ pathways, respectively. Here, the biosynthetic pathway of SF-2111B, which contains two peptide derived side chains including the unique O-acyl peptide moiety, will be discussed.

References

1) C. Maruyama et. al, A stand-alone adenylation domain forms amide bonds in streptothricin biosynthesis, Nat. Chem. Biol., 8, 791-797 (2012).

2) C. Maruyama et. al, tRNA-dependent aminoacylation of an amino-sugar intermediate in the biosynthesis of a streptothricin-related antibiotic, Appl. Environ. Microbiol., 82, 3640-3648 (2016).

Individual Streptomyces species have the genetic potential to produce a diverse array of natural products of considerable commercial, medical and veterinary importance. However, the genetic information flow of their unique high G+C genomes by transcription and translation processes remains largely unexplored. In order to harness their full biosynthetic potential, it will be important to develop a detailed understanding of the regulatory networks that orchestrate their diverse metabolism. Here, we reveal extensive translational control of the secondary metabolic genes of the model streptomycete, Streptomyces coelicolor A3(2) through the genome-scale integration of transcriptome and translatome data. Our systematic study determined 3,570 transcription start sites and identified a high proportion (~21%) of leaderless mRNAs and 230 small RNAs; this enabled deduction of promoter architecture on a genome-scale. The comprehensive translational landscape was determined by using massively parallel sequencing of ribosome-protected mRNA fragments (Ribo-seq). Interestingly, our study reveals that the translation efficiency of secondary metabolic genes was negatively correlated with transcription and that several key antibiotic regulatory genes were translationally-induced at transition growth phase. These findings could lead to the design of new approaches to antibiotic discovery and development.