Hamrick, G. S. | bioRxiv Science

bioRxiv2023

Programming dynamic division of labor using horizontal gene transfer

The metabolic engineering of microbes has broad applications, including in biomanufacturing, bioprocessing, and environmental remediation. The introduction of a complex, multi-step pathway often imposes a substantial metabolic burden on the host cell, restraining the accumulation of productive biomass and limiting pathway efficiency. One strategy to alleviate metabolic burden is division of labor (DOL), in which different subpopulations carry out different parts of the pathway and work together to convert a substrate into a final product. However, the maintenance of different engineered subpopulations is challenging due to competition and convoluted inter-strain population dynamics. Through modeling, we show that dynamic division of labor (DDOL) mediated by horizontal gene transfer (HGT) can overcome these limitations and enable the robust maintenance of burdensome, multi-step pathways. We also use plasmid genomics to uncover evidence that DDOL is a strategy utilized by natural microbial communities. Our work suggests that bioengineers can harness HGT to stabilize synthetic metabolic pathways in microbial communities, enabling the development of robust engineered systems for deployment in a variety of contexts.

synthetic biology↗

bioRxiv2021

Engineered chimeras unveil swappable modular features of fatty acid and polyketide synthase acyl carrier proteins

The strategic redesign of microbial biosynthetic pathways is a compelling route to access molecules of diverse structure and function in a potentially environmentally sustainable fashion. The promise of this approach hinges on an improved understanding of acyl carrier proteins (ACPs), which serve as central hubs in biosynthetic pathways. These small, flexible proteins mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural products. Past combinatorial biosynthesis efforts have failed due to incompatible ACP-enzyme pairings. Herein we report the design of chimeric ACPs with features of the actinorhodin polyketide synthase ACP (ACT) and of the E. coli fatty acid synthase (FAS) ACP (AcpP). We evaluate the ability of the chimeric ACPs to interact with the E. coli FAS ketosynthase FabF, which represents an interaction essential to building the carbon backbone of the synthase molecular output. Given that AcpP interacts with FabF but ACT does not, we sought to exchange modular features of ACT with AcpP to confer functionality with FabF. The interactions of chimeric ACPs with FabF were interrogated using sedimentation velocity experiments, surface plasmon resonance analyses, mechanism-based crosslinking assays, and molecular dynamics simulations. Results suggest that the residues guiding AcpP-FabF compatibility and ACT-FabF incompatibility may reside in the loop I, -helix II region. These findings can inform the development of strategic secondary element swaps that expand the enzyme compatibility of ACPs across systems and therefore represent a critical step towards the strategic engineering of unnatural natural products.

biochemistry↗