Study On Mildiomycin Biosynthesis

Mildiomycin (MIL) is a peptidyl nucleoside antibiotic with strong activity against powdery mildew disease of plants. In present work, the degenerate primers were designed based on blsM, the gene of CMP hydrolase in the blsticidin S biosynthetic gene cluster, together with its homologues. With the primers, six overlapping cosmids were screened from the genomic library of Streptoverticillum rimofaciens ZJU5119. 14A6, one of them can confer Streptomyces lividans 1326 to produce MIL, implying that it contains all the essential genes for biosynthesis of MIL.The insertion sequence of 14A6 was determined and assembled into 43,561bp on which 41 ORFs were predicted by FramePlot beta4.0. The mutant of milA, a gene encoding CMP hydroxymethylase, could produce only dehydroxymethyl MIL (dHM-MIL), while neither of MIL and dHM-MIL can be biosynthesized when milB, homolog of blsM was disrupted. Recombinant MilA was purified in E. coli and shown to specifically introduce a C-5 hydroxymethyl group on CMP but not able to accept either cytosine or dCMP as substrate. The kinetic parameters of recombinant MilB showed that it hydrolyzed hydroxymethyl-CMP to hydroxymethyl-cytosine (HMC) more efficiently than did CMP to cytosine. With the CMP as substrate, the ratio of free HMC to cytosine generated by MilB and MilA was ca. 9:1 in in vitro assays while the ratio of MIL to dHM-MIL was 3:1 in the extracted broth of Streptoverticillum rimofaciens. The inconsistence was partly compensated by the substrate bias of MilC, which can catalyse the coupling of the cytosine or HMC with UDP-glucuronic acid into cytosylglucuronic acid (CGA) or HMCGA, respectively. The Kcat/Km of MilC to cytosine and HMC were 0.5250±0.0019 and 0.2719±0.0012, respectively.A puzzling and interesting question regarding MIL biosynthesis was the origin of C-6 in carboxyl group. Using the stable isotope labeled L-arginine (U-13C6) to feed Streptoverticillum rimofaciens, the productivity of MIL with a mass of 520, 6 increments than that without isotope incorporation, increased to 11% from 2% after feeding of labeled L-arginine. Q-TOF/MS analyses of the purified mildiomycin indicated that all the fragments containing the guanidino-side chain gave a mass increment of 6, indicating thet all of six carbon atoms of labeled L-arginine were kept after the coupling of L-arginine or its derivative with glucuronic acid moiety, namely, C6 was derived from L-arginine rather than sugar as the precursors of MIL.To propose the complete biosynthetic pathway of MIL, the boundaries were firstly determined via systamtic gene disruption as well as end sequencing of six overlapped cosmids. Two boundaries were located within orf-1-milA and milQ-orf+1. All included genes within the boundary were disrupted individually, and 16 genes were demonstrated to be involved in the biosynthesis of MIL except for milL, of which knock-out caused no effect on production of MIL.The gene of milG encodes a unique Radical SAM family protein and is the only oxidase candidate supposed to be in charge of the oxidation of the hydroxyl group to carbonyl group in C4 of the hydroxymethyl cytosylglucuronic acid. In accordance with this assumption, intermediate of HMCGA was accumulated highly in the milG mutant, strongly supporting the role of MilG as a novel oxidase.in this key step. This was the first report of Radical SAM protein as the oxidase in the biosynthesis of nucleoside antibiotics.Disruption of milE and milQ, two aminoglycoside phosphotransferase genes, abolished the production of MIL; they are presumably involved in the double bond formation between C2 and C3 of the sugar moiety. The gene of milM and milN, encoding an aspartate/tyrosine/aromatic aminotransferase and a dihydrodipicolinate synthetase respectively, were disrupted, and neither resultant mutant could produce MIL. MilM was supposed to convertα-amino group of L-arginine to carbonyl group, and MilN sequentially catalyze the coupling of the carbonyl group in theα-ketonic acid with the carboanion in the decarboxylated hexose. Another aminotransferase gene, namely milD, encoding a degT/dnrJ/eryC1/strS aminotransferase, was considered to transfer amino group to the carbonyl group in the C-4 of sugar residue. MilH, the homologue of BlsK, might be in charge of the attachment of activated L-serine to C4 group of sugar moiety to form amide-like bond in the MIL biosynthesis.The disruption of a LuxR family regulator gene, milO, abolished the production of MIL, suggesting MilO should be a pathway-specific positive regulator. MilJ shows some homology with the Ubiquitone hydroxylase and postulated to be responsible for the hydroxylation ofγ-carbon in arginine residue. MilI was supposed to be involved in the activivation of L-serine. The mutant of an ABC transporter gene, milP, which was considered as the resistance gene, could not produce the MIL; the mutant of milk, a Major Facilitator Superfamily gene, produced less MIL compared to the wild type. MilK was proposed to be involved in the transportation of phosphogluconate.