Lin, X. N. | bioRxiv Science

bioRxiv2020

Temperature regulation as a tool to program synthetic microbial community composition

Engineering of synthetic microbial communities is emerging as a powerful new paradigm for performing various industrially, medically, and environmentally important processes. To reach the fullest potential, however, this approach requires further development in many aspects, a key one being regulating the community composition. Here we leverage well established mechanisms in ecology which govern the relative abundance of multi-species ecosystems and develop a new tool for programming the composition of synthetic microbial communities. Using a simple model system consisting of two microorganisms Escherichia coli and Pseudomonas putida, which occupy different but partially overlapping thermal niches, we demonstrate that temperature regulation can be used to enable coexistence and program the community composition. We first investigate a constant temperature regime and show that different temperatures lead to different community compositions. Next, we invent a new cycling temperature regime and show that it can dynamically tune the microbial community, achieving a wide range of compositions depending on parameters that are readily manipulatable. Our work provides conclusive proof of concept that temperature regulation is a versatile and powerful tool capable of programming compositions of synthetic microbial communities.

systems biology↗

bioRxiv2019

Syntrophic co-culture amplification of production phenotype for high-throughput screening of microbial strain libraries

Microbes can be engineered to synthesize a wide array of bioproducts, yet production phenotype evaluation remains a frequent bottleneck in the design-build-test cycle where strain development requires iterative rounds of library construction and testing. Here, we present Syntrophic Co-culture Amplification of Production phenotype (SnoCAP). Through a metabolic cross-feeding circuit, the production level of a target molecule is translated into highly distinguishable co-culture growth characteristics, which amplifies differences in production into highly distinguishable growth phenotypes. We demonstrate SnoCAP with the screening of Escherichia coli strains for production of two target molecules: 2-ketoisovalerate, a precursor of the drop-in biofuel isobutanol, and L-tryptophan. The dynamic range of the screening can be tuned by employing an inhibitory analog of the target molecule. Screening based on this framework requires compartmentalization of individual producers with the sensor strain. We explore three formats of implementation with increasing throughput capability: confinement in microtiter plates (102-104 assays/experiment), spatial separation on agar plates (104-105 assays/experiment), and encapsulation in microdroplets (105-107 assays/experiment). Using SnoCAP, we identified an efficient isobutanol production strain from a random mutagenesis library, reaching a final titer that is 5-fold higher than that of the parent strain. The framework can also be extended to screening for secondary metabolite production using a push-pull strategy. We expect that SnoCAP can be readily adapted to the screening of various microbial species, to improve production of a wide range of target molecules.

synthetic biology↗