In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus

Abstract

A stable-isotope assay was used to analyze the effectiveness of various perdeuterated short-chain acyl coenzyme A (acyl-CoA) compounds as starter units for straight-and branched-chain fatty acid biosynthesis in cell extracts of Streptomyces collinus. In these extracts perdeuterated isobutyryl-CoA was converted to isopalmitate (a branched-chain fatty acid), while butyryl-CoA was converted to palmitate (a straight-chain fatty acid). These observations are consistent with previous in vivo analyses of fatty acid biosynthesis in S. collinus, which suggested that butyryl-CoA and isobutyryl-CoA function as starter units for palmitate and isopalmitate biosynthesis, respectively. Additionally, in vitro analysis demonstrated that acetyl-CoA can function as a starter unit for palmitate biosynthesis. Palmitate biosynthesis and isopalmitate biosynthesis in these cell extracts were both effectively inhibited by thiolactomycin, a known type II fatty acid synthase inhibitor. In vivo experiments demonstrated that concentrations of thiolactomycin ranging from 0.1 to 0.2 mg/ml produced both a dramatic decrease in the cellular levels of branched-chain fatty acids and a surprising three-to fivefold increase in the cellular levels of the straight-chain fatty acids palmitate and myristate. Additional in vivo incorporation studies with perdeuterated butyrate suggested that, in accord with the in vitro studies, the biosynthesis of the palmitate from butyryl-CoA decreases in the presence of thiolactomycin. In contrast, in vivo incorporation studies with perdeuterated acetate demonstrated that the biosynthesis of palmitate from acetylCoA increases in the presence of thiolactomycin. These observations clearly demonstrate that isobutyryl-CoA is a starter unit for isopalmitate biosynthesis and that either acetyl-CoA or butyryl-CoA can be a starter unit for palmitate biosynthesis in S. collinus. However, the pathway for palmitate biosynthesis from acetyl-CoA is less sensitive to thiolactomycin, and it is suggested that the basis for this difference is in the initiation step.

Engineered Fatty Acid Biosynthesis in Streptomyces by Altered Catalytic Function of β-Ketoacyl-Acyl Carrier Protein Synthase III

“…These observations support the notion that the increase in SCFA biosynthesis is the result of an increase in the acetyl-ACP pool. Significant increases in SCFA biosynthesis (with concomitant decreases in the levels of BCFA) have previously been observed for addition of nonlethal doses of type II FAS inhibitor thiolactomycin to cultures of S. glaucescens and Streptomyces collinus (7,28). In this case an increase, rather than a decrease, in the intact utilization of a perdeuterated acetyl-CoA starter unit was observed (28).…”

“…5) and the corresponding ␤-ketoacyl-ACP product, as well as the loss of deuterium in the subsequent dehydration step (19). Thus any contributions to the labeled palmitate with a molecular ion of 273 by multiple incorporations of deuterated malonyl-ACP are low and can be readily corrected for (28).…”

“…In the fatty acid analyses the hydrolysis and subsequent methylation of this acyl thioester generate methyl palmitate with a molecular ion of 270 (29). Labeled palmitate with a molecular ion of 273 elutes at a slightly earlier retention time in the gas chromatography-mass spectrometry analyses, and this is indicative of the intact use of perdeuterated acetylCoA as a starter unit (28). It is well established that significantly lower levels of deuterium are incorporated into palmitate via the malonyl-CoA extender units (formed by carboxylation of the perdeuterated acetyl-CoA) (28).…”

“…Labeled palmitate with a molecular ion of 273 elutes at a slightly earlier retention time in the gas chromatography-mass spectrometry analyses, and this is indicative of the intact use of perdeuterated acetylCoA as a starter unit (28). It is well established that significantly lower levels of deuterium are incorporated into palmitate via the malonyl-CoA extender units (formed by carboxylation of the perdeuterated acetyl-CoA) (28). This observation has been attributed to a combination of "washout" of the deuterium label (exchange of deuterium with hydrogen) from the malonyl-CoA and/or malonyl-ACP substrate (Fig.…”

“…The fatty acid quantities were estimated by integrating the area of each major fatty acid peak and expressed as percentages of the total fatty acid pool. The determination of the percent labeling of the palmitate pool from an intact D 3 -acetate was based on the relative intensities of molecular ions (m/z) at 273 (methyl palmitate with three deuterium atoms) and 270 (unlabeled methyl palmitate) after correction for the natural abundance of 13 C and incorporation of deuterated malonate (28).…”