I have a sequence for PKS type III, I was planning to express the gene & perform a functional anti-bacterial assay on it, is this a good idea? More importantly, what is the best characterization method for PKSIII?
Thanks a lot!
Assuming you know what polyketide your synthase makes, the best assay would be to measure the production of the polyketide. This could be done by mass spectrometry, or possibly by HPLC if you have a standard so you know where it elutes and what the absorbance spectrum looks like.
If you have the purified synthase and have reconstituted its reaction in vitro, it will probably have reductase activities, among others. The reductase activities can be observed by the decrease in absorbance at 340 nm when NADPH is oxidized to NADP+.
If you express the polyketide synthase gene in bacteria, if the product is antibacterial, and the bacteria in which it is expressed are susceptible to the action of the product, then they will kill themselves when the product is made. Can you co-express an exporter for the product at the same time, or use a species of bacteria that is otherwise not sensitive to the product?
It depends on the substrates, many plant type III PKS utilize cinnamoyl-CoA or similar starting unit then add on acetate units from malonyl-CoA. The prodcuts should be highly colored compared to the starting materials. Thin layer chromatography is the cheapest way to monitor, utilization of C14 labeled malonyl-CoA will have the best sensitivity. Alternatively, HPLC-MS based detection works. Type III PKS don't have reductases, thus looking at the consumption of NADPH at 340 will not work. There are a few good reviews covering this topic.
Methods in Enzymology Volume 515
Chapter Sixteen – Type III Polyketide Synthases in Microorganisms
Yohei Katsuyama, Yasuo Ohnishi
doi:10.1016/B978-0-12-394290-6.00017-3
http://www.sciencedirect.com/science/article/pii/B9780123942906000173
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