Biosynthesis and combinatorial biosynthesis of antifungal nucleoside antibiotics

There is an urgent need for new antifungal agents to treat or combat fungal infection in humans and plants.Antifungal nucleoside antibiotics are an important family of natural products with distinctive structural features.Understanding their biosynthetic machinery is of great importance for the improvement of antibiotics titers.More importantly,it is a requisite for combinatorial biosynthesis to create hybrid nucleoside antibiotics.We herein focus on findings on the natural and designed biosynthesis of this important family of nucleoside antibiotics.

Medium optimization for enhanced production of cytosine-substituted mildiomycin analogue (MIL-C) by Streptoverticillium rimofaciens ZJU 5119

Cytosine-substituted mildiomycin analogue （MIL-C） was produced effectively by supplementing cytosine into the culture of Streptoverticillium rimofaciens. In order to improve the yield of MIL-C, statistically-based experimental designs were applied to optimize the fermentation medium for S. rimofaciens ZJU 5119. Fifteen culture conditions were examined for their significances on MIL-C production using Plackett-Burman design. The Plackett-Burrnan design and one-variable-at-a-time design indicated that glucose and rice meal as the complex carbon sources, and peanut cake meal and NH4NO3 as the complex nitrogen sources were beneficial for MIL-C production in S. rimofaciens ZJU 5119. The results of further central composition design （CCD） showed that the optimal concentration of glucose, rice meal and peanut cake meal were 18.7 g/L, 64.8 g/L and 65.1 g/L, respectively. By using this optimal fermentation medium, the MIL-C concentration was increased up to 1336.5 mg/L, an approximate 3.8-fold improvement over the previous concentration （350.0 mg/L） with un-optimized medium. This work will be very helpful to the large-scale production of MIL-C in the future.

An unusual UMP C-5 methylase in nucleoside antibiotic polyoxin biosynthesis

Polyoxin is a group of structurally-related peptidyl nucleoside antibiotics bearing C-5 modifications on the nucleoside skeleton. Although the structural diversity and bioactivity preference of polyoxin are, to some extent, affected by such modifications, the biosynthetic logic for their occurence remains obscure. Here we report the identification of PolB in polyoxin pathway as an unusual UMP C-5 methylase with thymidylate syn- thase activity which is responsible for the C-5 methyla- tion of the nucleoside skeleton. To probe its molecular mechanism, we determined the crystal structures of PolB alone and in complexes with 5-Br UMP and 5-Br dUMP at 2.15 A, 1.76 A and 2.28 A resolutions, respec- tively. Loop 1 （residues 117-131）, Loop 2 （residues 192- 201） and the substrate recognition peptide （residues 94- 102） of PolB exhibit considerable conformational flexi-bility and adopt distinct structures upon binding to different substrate analogs. Consistent with the structural findings, a PolB homolog that harbors an identical function from Streptomyces viridochromogenes DSM 40736 was identified. The discovery of UMP C5-methy-lase opens the way to rational pathway engineering for polyoxin component optimization, and will also enrich the toolbox for natural nucleotide chemistry.