Fig. 2.

Heterologous expression to screen pathway enzymes. (A) Plasmid design for screening enzymes consisted of a pRSF backbone containing psfA and psfB. A pET15b-based plasmid was used to coexpress the other open reading frames (ORFs) in the BGC. (B) Combinatorial expression screen of ORFs in the BGC showed no new product formation except when psfC was included, which resulted in production of compounds 1 and 2. Additional expression of psfD resulted in the production of the penultimate pathway intermediate, (S)-2-HPP. The chemical shifts of phosphonates in ³¹P NMR spectroscopy are very sensitive to pH near their pK_a values, accounting for the small changes in chemical shifts between experiments.