Analysis of the Mildiomycin Biosynthesis Gene Cluster in Streptoverticillum remofaciens ZJU5119 and Characterization of MilC, a Hydroxymethyl cytosyl‐glucuronic Acid Synthase

Abstract

Mildiomycin (MIL) is a peptidyl‐nucleoside antibiotic produced by Streptoverticillum remofaciens ZJU5119 that exhibits strong inhibitory activity against powdery mildew. The entire MIL biosynthesis gene cluster was cloned and expressed in Streptomyces lividans 1326. Systematic gene disruptions narrowed down the cluster to 16 functional ORFs and identified the boundaries of the gene cluster. A putative cytosylglucuronic acid (CGA) synthase gene, milC, was disrupted in Sv. remofaciens and heterologously expressed in E. coli. An in vitro assay revealed that purified MilC could utilize either cytosine or hydroxymethylcytosine as substrate to yield CGA or hydroxymethyl‐CGA (HM‐CGA), respectively. MilG is believed to be a key enzyme in the MIL biosynthesis pathway and contains the CXXXCXXC motif characteristic of members of the radical S‐adenosyl methionine (SAM) superfamily. Disruption of milG leads to accumulation of HM‐CGA. Labeling experiments with 13C6‐L‐arginine indicated that decarboxylation at C5 of the pyranoside ring was coupled with the attachment of 5‐guanidino‐2,4‐dihydroxyvalerate side chain through CC bond formation. In contrast, exogenous 13C6‐labeled 4‐hydroxy‐L‐arginine was not incorporated into the MIL structure. Comparative analysis of the 16 MIL ORFs with counterparts involved in the biosynthesis of the structurally similar compound blasticidin S, along with the results above, provide insight into the complete MIL biosynthetic pathway.

Biosynthesis of the β-Methylarginine Residue of Peptidyl Nucleoside Arginomycin in Streptomyces arginensis NRRL 15941

“…Arginomycin, blasticidin S, and mildiomycin share the core moiety cytosylglucuronic acid (CGA), which is catalyzed by the CGA synthase (BlsD/MilC) via coupling of the UDP-glucuronic acid and free cytosine or its analog (see Fig. S1A in the supplemental material) (9,10). Degenerate primers SCR-For and -Rev (Table S1) were designed based on the multiple DNA sequence alignments with milC, blsD, and other homologous genes from the database (Fig.…”

Streptomyces lividans Blasticidin S Deaminase and Its Application in Engineering a Blasticidin S-Producing Strain for Ease of Genetic Manipulation

“…4C) of mildiomycin to deaminohydroxymildiomycin by SLBSD. Consistent with this assumption, mildiomycin was successfully produced in S. lividans (29), whereas all of the blasticidin S was converted into deaminohydroxyblasticidin S. As for cytosylglucuronic acid, another substrate for SLBSD, this compound was nearly completely transformed into deaminohydroxycytosylglucuronic acid. The detection of cytosylglucuronic acid in the fermentation broth of the earlier heterologous expression experiments (15) might be the result of the high yield of this compound.…”

“…To narrow down the genes involved in blasticidin S assembly, blsF a gene homologous to milL in the mildiomycin gene cluster, which is predicted to encode a protein of unknown function (29), was disrupted. Disruption of blsF (Fig.…”

“…In our earlier work, the disruption of milG, a homologue to blsE in the mildiomycin biosynthetic pathway, led to the accumulation of hydroxymethyl cytosylglucuronic acid (29). To analyze the function of blsE in blasticidin S formation, the gene was inactivated, and the resulting mutant, S. lividans WJ5, was confirmed by PCR (Fig.…”

“…This engineered blasticidin S-producing strain provided the necessary means to investigate the blasticidin S biosynthesis pathway in vivo. The disruption of blsF, a homologue of milL that had been confirmed as a gene unnecessary for mildiomycin biosynthesis (29), raised the yields of blasticidin S and other intermediates instead of abolishing their production. This result suggested that blsF might have a negative effect on blasticidin S biosynthesis.…”