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

Abstract

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 (10 2 -10 4 assays/experiment), spatial separation on agar plates (10 4 -10 5 assays/experiment), and encapsulation in microdroplets (10 5 -10 7 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. We expect the cross-feeding system to have two major advantages for high-throughput screening over traditional auxotrophic biosensor implementation: i) amplification of production improvement through the co-culture's exponential growth, and ii) a wider and more tunable dynamic range. The former lies in the co-culture's amplification of differences between library members (Section 3, Calculation). Since strain development generally proceeds through incremental improvements, it is critical to be able to discern modest increases in production.Amplification of differences through the cross-feeding metabolic circuit makes relatively small improvements conspicuous. The second advantage is achieved because, when neither of the cross-fed molecules is exogenously supplied, the production rates of the cross-fed molecules generally limit total co-culture growth, keeping the target molecule within the dynamic range of the sensor strain. This is in contrast to direct application of the sensor to the supernatant of a secretor strain or in co-culture with a non-auxotrophic secretor strain when the level of target molecule will often exceed sensor dynamic range. 6 Materials and MethodsKey elements are described here. Additional details are provided in the Supplementary Information. Strains and plasmidsStrains and plasmids used are lis...

Optimized gene expression from bacterial chromosome by high-throughput integration and screening

“…The library itself is also auxotrophic for a second, orthogonal molecule (here, lysine) supplied by the sensor strain. This cross-feeding configuration enables conversion of the production phenotype into a fluorescent output, amplified by coculture growth (36). To screen for overproducers of 2-KIV, an intermediate in the isobutanol pathway (Fig.…”

“…This strain can only grow when it is provided an exogenous source of 2-KIV and/or branched-chain amino acids. We demonstrated in our previous work that this sensor can be used to identify strains that will overproduce isobutanol once the genes responsible for converting 2-KIV to isobutanol (kivD and adhA) are added to them (36).…”

“…Increasing the expression level of a gene can improve production level by increasing flux through the pathway, but avoiding too high of an expression level can also provide benefits such as reducing the buildup of toxic intermediates and avoiding unnecessary metabolic burden. (B) Libraries with pathway genes integrated into various genome locations can be screened for production phenotype using syntrophic coculture amplification of production (SnoCAP) (36). A cross-feeding coculture is configured consisting of a secretor strain producing a target molecule and a sensor strain that cannot synthesize the target molecule autonomously.…”

“…In this method, we use the Tn5 transposase to randomly integrate pathway genes in the E. coli genome in a multiplexed fashion. We subsequently screen the libraries using syntrophic coculture amplification of production (SnoCAP), which converts the production phenotype into a readily screenable growth phenotype, as described in our previous work (36). We demonstrate the approach using a pathway for the production of isobutanol, a promising drop-in biofuel (37).…”

In vivo continuous evolution of metabolic pathways for chemical production

“…Recently, another form of selection based on syntrophic interaction called syntrophic co-culture amplification of production phenotype (SnoCAP) was successfully demonstrated. It amplifies distinguishability of production level into growth phenotype through metabolic cross-feeding circuit [96]. In this system, target molecule auxotroph sensor strain and target molecule secreting secretor strain which is an auxotroph to an orthogonal molecule secreted by sensor strain, are required.…”