Characterization of Miharamycin Biosynthesis Reveals a Hybrid NRPS–PKS to Synthesize High-Carbon Sugar from a Complex Nucleoside

Abstract

Miharamycins are peptidyl nucleoside antibiotics with a unique branched C9 pyranosyl amino acid core and a rare 2-aminopurine moiety. Inactivation of 19 genes in the biosynthetic gene cluster and identification of several unexpected intermediates suggest an alternative biosynthetic pathway, which is further supported by feeding experiments and in vitro characterization of an unusual adenylation domain recognizing a complex nucleoside derivative as the substrate. These results thereby provide an unprecedented biosynthetic route of high-carbon sugar catalyzed by atypical hybrid nonribosomal peptide synthetase–polyketide synthase.

Solving the Structural Puzzles of Amipurimycin and Miharamycins Enabled by Stereodivergent Total Synthesis

“…proposed that the formation of the 9 C sugar core employed a “6C+1C+2C” pattern (Figure 17). [33b] In comparison, our “5C+4C” synthesis is strategically much more efficient, nevertheless, we have to admit that extensive manipulation of protecting groups is required in the chemical synthesis.…”

Identification and Characterization of Enzymes Catalyzing Early Steps in Miharamycin and Amipurimycin Biosynthesis

“…Cytosylglucuronic acid (CGA) synthase, a well-known prokaryotic UDP-glucuronosyltransferase, is specifically used to assemble CGA ( 8 ), which is a key intermediate in the biosynthesis of UDP-glucuronic-acid (UDP-GA, 9 )-derived antibiotics (Figure B). − Both GGA 4 and CGA 8 contain the unique glucuronic acid moiety, which hints that their glycosidic bonds may be formed through a similar mechanism. An MIH biosynthetic gene, mihI , encoding for a hypothetical protein, was previously suggested to participate in the formation of the early metabolite 4 because the production of MIHs and other intermediates was completely abolished in the ΔmihR2/ΔmihI mutant . MihI shows low homology to CGA synthases (18% identity to BlsD, for example), just like its homologous protein ApmI from the APM pathway (Table S1).…”

“…Miharamycins (MIHs, 1a/b ) and amipurimycin (APM, 2 ) represent a group of nucleoside antibiotics featuring a backbone composed of a branched C9 pyranosyl amino acid core and a 2-aminopurine (Figure A). − This group of natural products exhibits potent antifungal activity against rice blast disease and is therefore valued for agriculture in East Asia. , The unique 2-aminopurine and high-carbon sugar of APM and MIHs distinguish them from other nucleoside antibiotics. Recently, we identified the biosynthetic gene clusters (BGCs) of APM ( apm , GenBank accession no: MK257770) and MIHs ( mih , GenBank accession no: MN850873) respectively (Figure B) and proposed an overall biosynthetic pathway for them based mainly on the results of in vivo experiments. , It is suggested that the C9 core of MIHs and APM is constructed by an unusual hybrid nonribosomal peptide synthetase–polyketide synthase (NRPS-PKS) system, which performs a two-carbon extension on a complex nucleoside intermediate ( 3 ) (Figure A) . In addition, other groups also discovered the BGCs of APM and MIHs at almost the same time and made their proposal …”

“…It is suggested that the intermediate 3 with deoxy-guanine and branched C8 glycosyl moieties is derived from guanylglucuronic acid (GGA, 4 ). The pathway from 4 to 3 was summarized owing to the identification of compounds 3 – 7 isolated from the corresponding mutants (Figure A) . However, biochemical evidence from in vitro experiments for these steps is absent; in particular, the formation of GGA ( 4 ) remains enigmatic.…”

“…These results confirmed that MihI/ApmI can assemble the initial intermediate GGA 4 using UDP-GA 9 as the glycosyl donor and guanine 10 as the acceptor. It needs to be pointed out that previous feeding experiments showed that C-1′–C-6′ of APM and MIHs is not derived from intact glucose (Figure S5), − which suggests that the C6 carbohydrate may undergo glycolysis and then reconstitute into UDP-GA.…”