Discrete acyltransferases associated with type I polyketide synthases and methods of use
Genetic and biochemical characterization of the leinamycin biosynthesis gene cluster from Streptomyces atroolivaceus S-140 revealed two PKS genes, lnmI and lnmJ, that encode six PKS modules, none of which contains a cognate AT domain. The AT activity is provided in trans by a discrete protein, LnmG, which loads the malonyl coenzyme A extender unit onto the ACP domains of all six PKS modules. This finding provides a basis for methods of engineering modular polyketide synthases and polyketide synthase/nonribosomal peptide synthetases.
1. A method of loading an extender molecule on an acyl carrier protein (ACP) domain LnmI of SEQ ID NO: 45, comprising the step of contacting the ACP domain of LnmI of SEQ ID NO: 45, with the extender molecule and a recombinantly expressed, discrete acyl transferase (AT) LnmG, having the amino acid sequence of SEQ ID NO: 43, whereby the extender molecule is loaded onto the ACP domain encoded by Lnml of SEQ ID NO. 45 by the catalytic activity of the discrete AT LnmG of SEQ ID NO. 43.
2. A method according to claim 1 wherein the method is carried out in vitro.
3. A method according to claim 1 wherein the method is carried out in a host cell.
4. A method according to claim 3 wherein the host cell includes a vector comprising a nucleic acid encoding the discrete AT LnmG of SEQ ID NO. 43.
5. A method according to claim 4 wherein the host cell includes a different vector comprising a nucleic acid encoding the ACP domain Lnml of SEQ ID NO. 45.
6. A method according to claim 3 wherein the host cell is a bacterium.
7. A method according to claim 1 wherein the extender molecule is malonyl CoA.