Transient expression of genes coding for the poly-gamma-glutamate (gammaPGA) synthetase system (pgs) was investigated in tobacco plants. Three genes of the pgs, pgsA, pgsB and pgsC, were separately placed under the control of the CaMV 35S promoter and introduced into tobacco leaves via Agrobacterium infection. Synthesized gammaPGA in plant tissues was detected immunologically with mouse anti-gammaPGA antiserum which specifically reacts with gammaPGA on a nitrocellulose membrane. Confirmation of gammaPGA biosynthesis in the transient expression analysis in tobacco tissue indicates that subunits of pgs complex were expressed and reassembled in a functional form. 相似文献
LanV is involved in the biosynthesis of landomycin A. The exact function of this enzyme was elucidated with combinatorial biosynthesis by using Streptomyces fradiae mutants that produce urdamycin A. After expression of lanV in S. fradiae DeltaurdM, which is a mutant that accumulates rabelomycin, urdamycinon B and urdamycin B were found to be produced by the strain. This result indicates that LanV is involved in the 6-ketoreduction of the angucycline core, which preceeds a 5,6-dehydration reaction. 9-C-D-Olivosyltetrangulol was also produced by this strain; this demonstrates that LanV catalyses the aromatization of ring A of the angucycline structure. Coexpression of lanV and lanGT2 in S. fradiae AO, a mutant that lacks all four urdamycin glycosyltransferases, resulted in the production of tetrangulol and the glycoside landomycin H, both of which have an aromatic ring A. As glycosylated angucyclines were not observed after expression of lanGT2 in the absence of lanV, we conclude that LanGT2 needs an aromatized ring A for substrate recognition. 相似文献
The limits and potential of substrate promiscuity of the adenylation domain of tyrocidine synthetase 1 were systematically explored. Substrate acceptance is governed by hydrophobic effects (as shown by the correlation of kcat/KM and side‐chain log P), shape complementarity and steric exclusion. The quantification of these factors provides ground rules for understanding and possibly evolving substrate specificity in this class of enzymes.
Pacidamycins, mureidomycins and napsamycins are structurally related uridyl peptide antibiotics that inhibit translocase I, an as yet clinically unexploited target. This potentially important bioactivity coupled to the biosynthetically intriguing structure of pacidamycin make this natural product a fascinating subject for study. A precursor‐directed biosynthesis approach was employed in order to access new pacidamycin derivatives. Strikingly, the biosynthetic machinery exhibited highly relaxed substrate specificity with the majority of the tryptophan analogues that were administered; this resulted in the production of new pacidamycin derivatives. Remarkably, 2‐methyl‐, 7‐methyl‐, 7‐chloro‐ and 7‐bromotryptophans produced their corresponding pacidamycin analogues in larger amounts than the natural pacidamycin. Low levels or no incorporation was observed for tryptophans substituted at positions 4, 5 and 6. The ability to generate bromo‐ and chloropacidamycins opens up the possibility of further functionalising these compounds through chemical cross‐coupling in order to access a much larger family of derivatives. 相似文献
Tailor made : We report the rational biosynthesis of C15 hydroxylated non‐quinone geldanamycin analogues by site‐directed mutagenesis of the geldanamycin polyketide synthase (PKS), together with a combination of post‐PKS tailoring genes. Rational biosynthetic engineering allowed the generation of geldanamycin derivatives, such as DHQ3 illustrated in the figure, which had superior pharmacological properties in comparison to the parent compound.
Out of the green! Precursor‐directed biosynthesis allowed for the production of new nostocarboline derivatives that display phytotoxic and algicidal properties—in a phototrophic organism. The mechanism of action includes downregulation of photosynthesis, as demonstrated by chlorophyll‐a fluorescence imaging.
