Roles of acyl carrier proteins in ladderane fatty acid producing-organisms

Ladderanes are highly strained hydrocarbons consisting of two or more linearly concatenated cyclobutane rings. Strikingly, ladderane moieties are part of unique fatty acids and fatty alcohols that are exclusively found in the membrane lipids of anaerobic ammonium-oxidizing (anammox) bacteria. These bacteria express a distinctive gene cluster (cluster I) that has been suggested to be responsible for ladderane fatty acid (FA) biosynthesis in addition to a cluster likely involved in canonical FA biosynthesis (cluster III). In the anammox organism Kuenenia stuttgartiensis, cluster I encodes a unique acyl carrier protein (amxACP), whereas the ACP encoded by cluster III (KsACPII) was suggested to be involved in the production of canonical fatty acids. Here we present targeted isotope labeling studies using ¹³C-malonyl-ACPs to distinguish the roles of these ACPs. While in-vitro¹³C incorporation into ladderane FAs was not observed, we show that KsACPII indeed functions in palmitate biosynthesis in the anammox organism Kuenenia stuttgartiensis. We present an experimental framework for continuing studies into fatty acid biosynthesis in anammox- and similar organisms.