Functional dissection of a multimodular polypeptide of the pikromycin polyketide synthase into monomodules by using a matched pair of heterologous docking domains.
The pikromyin polyketide synthase (PKS) in Streptomyces venezulae is comprised of a loading module and six extension modules, which generate the corresponding 14-membered macrolactone product. PikAI is a multimodular component of this PKS and houses both the loading domain and the first two extension modules, joined by short intraprotein linkers. We have shown that PikAI can be separated into two proteins at either of these linkers, only when matched pairs of docking domains (DDs) from a heterologous modular phoslactomycin PKS are used in place of the intraprotein linker. In both cases the yields of pikromycin produced by the S. venezuelae mutant were 50% of that of a S. venezuelae strain expressing the native trimodular PikAI. This observation provides the first demonstration that such separations do not dramatically impact the efficiency of the entire in vivo biosynthetic process. Expression of module 2 as a monomodular protein fused to a heterologous N-terminal docking domain was also observed to give almost a tenfold improvement in the in vivo generation of pikromycin from a synthetic diketide intermediate. These results demonstrate the utility of DDs to manipulate biosynthetic processes catalyzed by modular PKSs and the quest to generate novel polyketide products.
Department of Chemistry, Portland State University, 262 Science Building 2, 1719 SW 10th Avenue, Portland, OR 97201, USA.
John
J
Yan
Shuchi
S
Gupta
David H
DH
Sherman
Kevin A
KA
Reynolds
D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D005822:Genetic Vectors; D007783:Lactones / Q000737:chemistry / Q000378:metabolism; D018942:Macrolides / Q000737:chemistry / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D010455:Peptides / Q000378:metabolism*; D048630:Polyketide Synthases / Q000235:genetics / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology
D000900:Anti-Bacterial Agents; D007783:Lactones; D018942:Macrolides; D010455:Peptides; D048630:Polyketide Synthases; C003145:picromycin