RESUMEN
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.
Asunto(s)
Ingeniería Metabólica , Mutagénesis , Fenotipo , Técnicas de Cocultivo , Escherichia coli/genética , Escherichia coli/metabolismo , Pruebas GenéticasRESUMEN
Synthetic microbial consortia that can mimic natural systems have the potential to become a powerful biotechnology for various applications. One highly desirable feature of these consortia is that they can be precisely regulated. In this work we designed a programmable, symbiotic circuit that enables continuous tuning of the growth rate and composition of a synthetic consortium. We implemented our general design through the cross-feeding of tryptophan and tyrosine by two E. coli auxotrophs. By regulating the expression of genes related to the export or production of these amino acids, we were able to tune the metabolite exchanges and achieve a wide range of growth rates and strain ratios. In addition, by inverting the relationship of growth/ratio vs. inducer concentrations, we were able to "program" the co-culture for pre-specified attributes with the proper addition of inducing chemicals. This programmable proof-of-concept circuit or its variants can be applied to more complex systems where precise tuning of the consortium would facilitate the optimization of specific objectives, such as increasing the overall efficiency of microbial production of biofuels or pharmaceuticals.
Asunto(s)
Escherichia coli/genética , Consorcios Microbianos , Algoritmos , Proteínas Bacterianas , Biocombustibles , Calibración , Química Farmacéutica/métodos , Técnicas de Cocultivo , Escherichia coli/metabolismo , Ingeniería Genética/métodos , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes , Técnicas Microbiológicas/métodos , Modelos Biológicos , Modelos Estadísticos , Plásmidos/metabolismo , Simbiosis , Triptófano/metabolismo , Tirosina/metabolismoRESUMEN
Microbial interactions in natural microbiota are, in many cases, crucial for the sustenance of the communities, but the precise nature of these interactions remain largely unknown because of the inherent complexity and difficulties in laboratory cultivation. Conventional pure culture-oriented cultivation does not account for these interactions mediated by small molecules, which severely limits its utility in cultivating and studying "unculturable" microorganisms from synergistic communities. In this study, we developed a simple microfluidic device for highly parallel co-cultivation of symbiotic microbial communities and demonstrated its effectiveness in discovering synergistic interactions among microbes. Using aqueous micro-droplets dispersed in a continuous oil phase, the device could readily encapsulate and co-cultivate subsets of a community. A large number of droplets, up to â¼1,400 in a 10 mm × 5 mm chamber, were generated with a frequency of 500 droplets/sec. A synthetic model system consisting of cross-feeding E. coli mutants was used to mimic compositions of symbionts and other microbes in natural microbial communities. Our device was able to detect a pair-wise symbiotic relationship when one partner accounted for as low as 1% of the total population or each symbiont was about 3% of the artificial community.
Asunto(s)
Biota , Técnicas Microbiológicas/métodos , Técnicas de Cocultivo/métodos , Medios de Cultivo/química , Composición de Medicamentos/métodos , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/fisiología , Consorcios Microbianos/fisiología , Interacciones Microbianas/fisiología , Técnicas Analíticas Microfluídicas , Miniaturización , Modelos Biológicos , Organismos Modificados Genéticamente , Simbiosis/fisiologíaRESUMEN
Delta-like protein 1 (Dlk1) is a transmembrane protein characterized by epidermal growth factor (EGF)-like repeats. Dlk1, which is also known as preadipocyte factor 1 (pref-1) because of its ability to inhibit preadipocyte differentiation, regulates the differentiation of several other cell types through unknown mechanisms. To elucidate Dlk1 functions, identification of Dlk1-regulated target genes is critical. The observation that Dlk1 is expressed in many endocrine tissues suggests that Dlk1 may have endocrine-related functions. Because Dlk1 is expressed in GH producing cells, we hypothesize that one function of Dlk1 is to regulate GH expression. We found that GH mRNA, protein, and secretion were significantly decreased in GH3 pituitary cell clones that stably express Dlk1. In contrast, Dlk1 expression was unable to alter prolactin expression. Co-transfection of GH3 cells with a GH promoter-regulated reporter gene showed that Dlk1 repressed GH promoter activity. Deletion and mutation analysis of the GH promoter indicated that Pit-1 binding sites in the GH promoter are required for Dlk1-mediated repression. Furthermore, Dlk1 expression represses Pit-1-mediated transcription when both proteins are co-expressed in MCF-7 cells. Deletion analysis of Dlk1 revealed that the ability of Dlk1 to regulate GH promoter activity is independent of both its EGF-like repeats and its ability to modulate MAP kinase activity. The observation that Dlk1 regulates GH expression identifies the first endocrine function of Dlk1, establishes GH as a Dlk1-regulated target gene, and provides a model system to facilitate studies of Dlk1-mediated signaling.