Nonactin Biosynthesis: Unexpected Patterns of Label Incorporation from 4,6-Dioxoheptanoate Show Evidence of a Degradation Pathway for Levulinate through Propionate in Streptomyces griseus

Abstract

The polyketide nonactin, a polyketide possessing antitumor and antibacterial activity, is produced by an unusual biosynthesis pathway in Streptomyces griseus that uses both enantiomers of the nonactin precursor, nonactic acid. Despite many studies with labeled precursors, much of the biosynthesis pathway remains unconfirmed, particularly the identity of the last achiral intermediate in the pathway, which is believed to be 4,6-diketoheptanoyl-CoA. We set out to confirm the latter hypothesis with feeding studies employing [4,5-(13)C(2)]-, [5,6-(13)C(2)]-, and [6,7-(13)C(2)]-4,6-diketoheptanoate thioester derivatives. In each case the isotopic label was incorporated efficiently into nonactin; however, at positions inconsistent with the currently accepted biosynthesis pathway. To resolve the discrepancy, we conducted additional feeding studies with a [3,4-(13)C(2)]levulinate thioester derivative and again observed efficient label incorporation. The latter result was intriguing, as levulinate is not an obvious precursor to nonactin. Levulinate, however, is known to be efficiently degraded into propionate even though the pathway for the conversion is not known. On the basis of both our levulinate and diketoheptanoate isotope incorporation data we can now postulate a pathway from levulinate to propionate that can also account for the conversion of 4,6-diketoheptanoate into levulinate in S. griseus.

Insights into the Pamamycin Biosynthesis

“…The resulting compounds are then rotated by PamB acyltransferase as proposed by Rong and co-workers. [20] The resulting 4-oxoadipyl-CoA and 5-methyl-4-oxoadipyl-CoA are key intermediates in the biosynthetic pathway for 1 and are used as extenders for a Claisen condensation facilitating the incorporation of succinate. PamD catalyzes the first extension of a short-chain acyl starter unit with one of these compounds followed by the PamE-catalyzed addition of malonyl-CoA.…”

“…The first reaction in the pathway is the PamA‐catalyzed condensation of succinyl‐CoA with malonyl‐ or methylmalonyl‐CoA. The resulting compounds are then rotated by PamB acyltransferase as proposed by Rong and co‐workers 20. The resulting 4‐oxoadipyl‐CoA and 5‐methyl‐4‐oxoadipyl‐CoA are key intermediates in the biosynthetic pathway for 1 and are used as extenders for a Claisen condensation facilitating the incorporation of succinate.…”

Recent advances in the biosynthesis of unusual polyketide synthase substrates

“…Subsequent rotation of the 3-oxoadipyl-CoA derivatives by the highly atypical AT domain PamB yields 28 and its methylated derivatives, which are the unusual extender units incorporated into the pamamycins. 44,45 …”

Biosynthesis of oxygen and nitrogen-containing heterocycles in polyketides

“… a) Nonactin ( 70 ) is formed from heterodimers of (−)(+)-dimeric nonactic acid and (+)(−)-dimeric nonactic acid. b) The product of the nonS gene catalyses the cyclisation of (6 R ,8 R , E )-6,8-dihydroxy-2-methylnon-2-enoic acid thioester ( 71a and 71 b ) to (+)-nonactic acid thioester ( 69b / 72 ) [ 53 , 56 , 58 ]. …”