Animal biosynthesis of complex polyketides in a photosynthetic partnership

Abstract

Complex polyketides are typically associated with microbial metabolism. Here, we report that animals also make complex, microbe-like polyketides. We show there is a widespread branch of fatty acid synthase- (FAS)-like polyketide synthase (PKS) proteins, which sacoglossan animals use to synthesize complex products. The purified sacogolassan protein EcPKS1 uses only methylmalonyl-CoA as a substrate, otherwise unknown in animal lipid metabolism. Sacoglossans are sea slugs, some of which eat algae, digesting the cells but maintaining functional chloroplasts. Here, we provide evidence that polyketides support this unusual photosynthetic partnership. The FAS-like PKS family represents an uncharacterized branch of polyketide and fatty acid metabolism, encoding a large diversity of biomedically relevant animal enzymes and chemicals awaiting discovery. The biochemical characterization of an intact animal polyketide biosynthetic enzyme opens the door to understanding the immense untapped metabolic potential of metazoans.

Fig. 1. Biogenesis of sacoglossan polypropionates.

a Previous feeding studies demonstrated that kleptoplasts in the sacoglossans fix carbon that is used to make polypropionates. Propionate is also a precursor, presumably via methylmalonate. A further series of hypothetical steps, including synthesis by a PKS, methylation, and photochemical and oxidation reactions would lead to b the known natural polypropionates of diverse structures. These are found in c sacoglossan species investigated in this work, including E. chlorotica and E. timida, but not E. cornigera. Photos of E. chlorotica and E. cornigera by Patrick Krug; E. timida by Heike Wägele; used with permission.

Fig. 2. A class of FAS-like PKS enyzmes.

a Sacoglossan PKSs represent a group separate from the known FAS and PKS proteins from bacteria, fungi and animals. Numbers indicate consensus support in percent. Outgroup DpsA is a type II PKS sequence from bacteria, while the remainder are type I enzymes. b The domain architecture of the sacoglossan PKSs, EcPKS1 and EcPKS2 includes ketosynthase (KS), acyltransferase (AT; known as malonyl/acetyltransferase (MAT) in fatty acid biosynthesis), methyltransferase (MT), dehydratase (DH), enoylreductase (ER), ketoreductase (KR) and acyl carrier protein (ACP). This domain architecture is identical to the sacoglossan FAS, EcFAS except for the absence of a C-terminal thioesterase domain. c EcPKS1 is encoded in the genome of E. chlorotica. d The acyltransferase (AT) domain sequence indicates loading preference for methymalonate while the e enoylreductase (ER) domain lacks key NADPH binding site residues making this function inactive.

Fig. 3. EcPKS1 synthesizes the tridachione precursor.

a Incubation of EcPKS1 with methylmalonyl-CoA and NADPH resulted in the synthesis of polyene 1 and tridachione precursor 2. b UV spectra of the synthesized products showing features of polyene propionates consistent with the structural assignment. c Incubation with other acyl-CoA substrates (malonyl-CoA: m-CoA, propionyl-CoA: p-CoA, methylmalonyl-CoA: mM-CoA) with NADPH did not yield new products. d Using propionyl-CoA as the loading molecule inhibited the reaction, as observed in decreased yields. e Using different loading and extender units other than methylmalonate did not produce any products. Graphs shown in a and c–e are HPLC-DAD experiments, where the x-axis gives the elution time in minutes and the y-axis is absorbance units at λ = 325 nm. The plots shown in b are UV spectra with the y-axis showing absorbance and the x-axis showing wavelength in nanometers. Each of these experiments was performed with three biological replicates, each done in triplicate. Representative data are shown.

Fig. 4. Long-chain polyketides are found only in LTR sacoglossans.

Molecular phylogeny of Sacoglossa with genera collapsed, based on maximum likelihood and Bayesian Inference analyses of four gene regions from 219 species. Circles on supported nodes show posterior probability (above branch) and bootstrap percentage (below); black = 1.0 or 100%; grey = significant (≥0.9 or ≥70%) but not complete support; white = not significant. Arrows indicate ancestral nodes in which chloroplast retention and photosynthetic ability likely evolved. Grey bars join a genus to representative polyketides isolated from member species. At the bottom right, a box containing long-chain polypropionates is attached to the LTR sacoglossans.

Fig. 5. EcPKS1 and relatives are associated with photosynthetic sacoglossans.

a. EcPKS1 is specific to LTR sacoglossans. A neighbor-joining tree based on the acyltransfersase domain of sacoglossan PKS and FAS proteins made using UPGMA. Numbers indicate consensus support (%). The PKS1 clade contains all LTR organisms (orange dots), while FAS and PKS2 includes the non-LTR E. cornigera. b Expression of EcPKS1 during development and c during starvation. APO indicates the aposymbiotic state, at the transition to the benthic state when chloroplasts are acquired. The expression data thus show that EcPKS1 production is highest as chloroplasts are being acquired, and during starvation its expression increases as photosynthesis becomes more important to animal survival. Each time point has a sample size of n = 3 and expressed as mean ± standard error. P values (p < 0.000****; p < 0.001***; p < 0.1**; p < 0.05*) between time points were done using T test method.

Fig. 6. EcPKS1 represents a widespread group of FAS-like PKSs in animals.

A phylogenetic tree inferred using Bayesian method was created using protein sequences of ketosynthase domains representing a broad range of characterized and uncharacterized Type I PKSs and FASs. DpsA, a ketosynthase from a Type II PKS, was used as an outgroup. Numbers indicate posterior probability in percent; only key nodes are shown. Asterisk indicates that the protein and / or gene have been characterized. (Animal FAS is also well characterized.) Purple names are for putatively methylmalonate-selective PKSs. In bold are shown sequences discussed in the main text. Letters indicate multiple proteins encoded in the same sequenced genome. The FAS-like PKS group is described in this work.