An unusual feature of the 6-module pikromycin polyketide synthase (PikPKS, PikAI-PikAIV) of S. venezuelae is the ability to generate both 12- and 14-membered ring macrolides. The PikAIV component containing the last extension module and a thioesterase domain is responsible for generating both of these products. In the case of the 12-membered ring macrolide, an acyl-enzyme intermediate on PikAIII is able to efficiently "skip" the last extension step and is cyclized by the TE domain of PikAIV, presumably as a result of a PikAIII-PikAIV interaction. Herein we report that plasmid-based expression (pBK3) of DEBS1, which comprises the loading domain and the first two modules of the Saccharopolyspora erythrea 6-deoxyerythronolide B synthase, in S. venezuelae leads to efficient 15 ± 3 mg/L production of triketide lactone products (TKLs). Comparable levels of TKLs were observed with a plasmid (pBK1) which expressed DEBS1 fused to a TE domain (DEBS1-TE). These results are in stark contrast to previous in vivo and in vitro analyses, where only DEBS1-TE efficiently produces TKLs. Levels of TKLs decreased dramatically with expression of DEBS1 in both pikAIV and pikAIII-pikAIV deletion hosts (0.5 mg/L), but not DEBS1-TE, and could be partially restored by addition of a PikAIV complementation plasmid. These data suggest that PikAIV is able to efficiently catalyze formation of 6-membered lactone ring products from acyl-bound intermediates on DEBS1 in a manner analogous to that observed for 12-membered macrolide products from PikAIII. Significant sequence similarity and length of the C-terminal linker region of PikAIII and DEBS1 suggest that this region may be responsible for the interaction with PikAIV. A replacement of this linker region of DEBS1 with the corresponding region of PikAI led to a 95% decrease in TKL levels in S. venezuelae, consistent with this hypothesis.
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