Trent R. Northen

Comprehensive in Vitro Analysis of Acyltransferase Domain Exchanges in Modular Polyketide Synthases and Its Application for Short-Chain Ketone Production

Type I modular polyketide synthases (PKSs) are polymerases that utilize acyl-CoAs as substrates. Each polyketide elongation reaction is catalyzed by a set of protein domains called a module. Each module usually contains an acyltransferase (AT) domain, which determines the specific acyl-CoA incorporated into each condensation reaction. Although a successful exchange of individual AT domains can lead to the biosynthesis of a large variety of novel compounds, hybrid PKS modules often show significantly decreased activities. Using monomodular PKSs as models, we have systematically analyzed the segments of AT domains and associated linkers in AT exchanges in vitro and have identified the boundaries within a module that can be used to exchange AT domains while maintaining protein stability and enzyme activity. Importantly, the optimized domain boundary is highly conserved, which facilitates AT domain replacements in most type I PKS modules. To further demonstrate the utility of the optimized AT domain boundary, we have constructed hybrid PKSs to produce industrially important short-chain ketones. Our in vitro and in vivo analysis demonstrated production of predicted ketones without significant loss of activities of the hybrid enzymes. These results greatly enhance the mechanistic understanding of PKS modules and prove the benefit of using engineered PKSs as a synthetic biology tool for chemical production.

Need for Laboratory Ecosystems To Unravel the Structures and Functions of Soil Microbial Communities Mediated by Chemistry

The chemistry underpinning microbial interactions provides an integrative framework for linking the activities of individual microbes, microbial communities, plants, and their environments. Currently, we know very little about the functions of genes and metabolites within these communities because genome annotations and functions are derived from the minority of microbes that have been propagated in the laboratory. ABSTRACT The chemistry underpinning microbial interactions provides an integrative framework for linking the activities of individual microbes, microbial communities, plants, and their environments. Currently, we know very little about the functions of genes and metabolites within these communities because genome annotations and functions are derived from the minority of microbes that have been propagated in the laboratory. Yet the diversity, complexity, inaccessibility, and irreproducibility of native microbial consortia limit our ability to interpret chemical signaling and map metabolic networks. In this perspective, we contend that standardized laboratory ecosystems are needed to dissect the chemistry of soil microbiomes. We argue that dissemination and application of standardized laboratory ecosystems will be transformative for the field, much like how model organisms have played critical roles in advancing biochemistry and molecular and cellular biology. Community consensus on fabricated ecosystems (“EcoFABs”) along with protocols and data standards will integrate efforts and enable rapid improvements in our understanding of the biochemical ecology of microbial communities.