Assembling the anaerobic gamma-butyrobetaine to TMA metabolic pathway in Escherichia fergusonii and confirming its role in TMA production from dietary L- carnitine in murine models

Abstract

ABSTRACT Trimethylamine-N-oxide (TMAO) is a major pro-atherogenic and pro-thrombotic metaorganismal molecule produced through the initial conversion of the dietary L -carnitine and other precursors into trimethylamine (TMA). We recently identified a dual-microbe anaerobic pathway for the metabolism of L- carnitine into TMA, in which the widely distributed cai operon in Enterobacteriaceae converts L- carnitine into gamma-butyrobetaine (γBB), followed by the degradation of γBB into TMA by the relatively rare g amma- b utyrobetaine u tilization ( gbu ) gene cluster present in Emergencia timonensis and few other related microbes. Studies of this pathway in animal models, however, have been limited by the lack of single microbes harboring the whole L- carnitine→γBB→TMA transformation pathway. Such recombinant microbes would both serve as a tool to further prove the contribution of this pathway to gut microbial TMA production and for future in vivo studies investigating the diet linkage to cardiovascular disease and the involvement of the TMAO pathway in this linkage. Here, we recapitulate the whole pathway in a single microbe by cloning the E. timonensis gbu gene cluster into Escherichia fergusonii , which naturally harbors the cai operon. We then show that the native E. timonensis GroES/GroEL-like proteins are needed for the proper functioning of the gbu cluster at 37°C. Finally, we demonstrate that inoculating germ-free mice with this recombinant E. fergusonii strain is sufficient to raise serum TMAO to pathophysiological levels upon dietary L- carnitine supplementation. The recombinant E. fergusonii strain developed will be a useful tool to facilitate future studies on the role of anaerobic gut microbial L- carnitine metabolism in cardiovascular and metabolic diseases. IMPORTANCE The key atherosclerotic TMAO originates from the initial gut microbial conversion of L -carnitine and other dietary compounds into TMA. Developing therapeutic strategies to block gut microbial TMA production needs a detailed understanding of the different production mechanisms and their relative contributions. Recently, we identified a two-step anaerobic pathway for TMA production from L -carnitine through initial conversion by some microbes into the intermediate γBB which is then metabolized by other microbes into TMA. Investigational studies of this pathway, however, are limited by the lack of single microbes harboring the whole pathway. Here, we engineered E. fergusonii strain to harbor the whole two-step pathway and optimized the expression through cloning a specific chaperone from the original host. Inoculating germ-free mice with this recombinant E. fergusonii is enough to raise serum TMAO to pathophysiological levels upon L-carnitine feeding. This engineered microbe will facilitate future studies investigating the contribution of this pathway to cardiovascular disease.