Natural variation meets synthetic biology: Promiscuous trichome-expressed acyltransferases from Nicotiana.

Acylsugars are defensive, trichome-synthesized sugar esters produced in plants across the Solanaceae (nightshade) family. Although assembled from simple metabolites and synthesized by a relatively short core biosynthetic pathway, tremendous within- and across-species acylsugar structural variation is documented across the family. To advance our understanding of the diversity and the synthesis of acylsugars within the Nicotiana genus, trichome extracts were profiled across the genus coupled with transcriptomics-guided enzyme discovery and in vivo and in vitro analysis. Differences in the types of sugar cores, numbers of acylations, and acyl chain structures contributed to over 300 unique annotated acylsugars throughout Nicotiana. Placement of acyl chain length into a phylogenetic context revealed that an unsaturated acyl chain type was detected in a few closely related species. A comparative transcriptomics approach identified trichome-enriched Nicotiana acuminata acylsugar biosynthetic candidate enzymes. More than 25 acylsugar variants could be produced in a single enzyme assay with four N. acuminata acylsugar acyltransferases (NacASAT1-4) together with structurally diverse acyl-CoAs and sucrose. Liquid chromatography coupled with mass spectrometry screening of in vitro products revealed the ability of these enzymes to make acylsugars not present in Nicotiana plant extracts. In vitro acylsugar production also provided insights into acyltransferase acyl donor promiscuity and acyl acceptor specificity as well as regiospecificity of some ASATs. This study suggests that promiscuous Nicotiana acyltransferases can be used as synthetic biology tools to produce novel and potentially useful metabolites.

Evolution of a plant gene cluster in Solanaceae and emergence of metabolic diversity.

Plants produce phylogenetically and spatially restricted, as well as structurally diverse specialized metabolites via multistep metabolic pathways. Hallmarks of specialized metabolic evolution include enzymatic promiscuity and recruitment of primary metabolic enzymes and examples of genomic clustering of pathway genes. Solanaceae glandular trichomes produce defensive acylsugars, with sidechains that vary in length across the family. We describe a tomato gene cluster on chromosome 7 involved in medium chain acylsugar accumulation due to trichome specific acyl-CoA synthetase and enoyl-CoA hydratase genes. This cluster co-localizes with a tomato steroidal alkaloid gene cluster and is syntenic to a chromosome 12 region containing another acylsugar pathway gene. We reconstructed the evolutionary events leading to this gene cluster and found that its phylogenetic distribution correlates with medium chain acylsugar accumulation across the Solanaceae. This work reveals insights into the dynamics behind gene cluster evolution and cell-type specific metabolite diversity.

Plants produce a vast variety of different molecules known as secondary or specialized metabolites to attract pollinating insects, such as bees, or protect themselves against herbivores and pests. The secondary metabolites are made from simple building blocks that are readily available in plants, including amino acids, fatty acids and sugars. Different species of plant, and even different parts of the same plant, produce their own sets of secondary metabolites. For example, the hairs on the surface of tomatoes and other members of the nightshade family of plants make metabolites known as acylsugars. These chemicals deter herbivores and pests from damaging the plants. To make acylsugars, the plants attach long chains known as fatty acyl groups to molecules of sugar, such as sucrose. Some members of the nightshade family produce acylsugars with longer chains than others. In particular, acylsugars with long chains are only found in tomatoes and other closely-related species. It remained unclear how the nightshade family evolved to produce acylsugars with chains of different lengths. To address this question, Fan et al. used genetic and biochemical approaches to study tomato plants and other members of the nightshade family. The experiments identified two genes known as AACS and AECH in tomatoes that produce acylsugars with long chains. These two genes originated from the genes of older enzymes that metabolize fatty acids ­ the building blocks of fats ­ in plant cells. Unlike the older genes, AACS and AECH were only active at the tips of the hairs on the plant's surface. Fan et al. then investigated the evolutionary relationship between 11 members of the nightshade family and two other plant species. This revealed that AACS and AECH emerged in the nightshade family around the same time that longer chains of acylsugars started appearing. These findings provide insights into how plants evolved to be able to produce a variety of secondary metabolites that may protect them from a broader range of pests. The gene cluster identified in this work could be used to engineer other species of crop plants to start producing acylsugars as natural pesticides.