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.

Biodiversity Improves Life Cycle Sustainability Metrics in Algal Biofuel Production.

Algal biofuel has yet to realize its potential as a commercial and sustainable bioenergy source, largely due to the challenge of maximizing and sustaining biomass production with respect to energetic and material inputs in large-scale cultivation. Experimental studies have shown that multispecies algal polycultures can be designed to enhance biomass production, stability, and nutrient recycling compared to monocultures. Yet, it remains unclear whether these impacts of biodiversity make polycultures more sustainable than monocultures. Here, we present results of a comparative life cycle assessment (LCA) for algal biorefineries to compare the sustainability metrics of monocultures and polycultures of six fresh-water algal species. Our results showed that when algae were grown in outdoor experimental ponds, certain bicultures improved the energy return on investment (EROI) and greenhouse gas emissions (GHGs) by 20% and 16%, respectively, compared to the best monoculture. Bicultures outperformed monocultures by performing multiple functions simultaneously (e.g., improved stability, nutrient efficiency, biocrude characteristics), which outweighed the higher productivity attainable by a monoculture. Our results demonstrate that algal polycultures with optimized multifunctionality lead to enhanced life cycle metrics, highlighting the significant potential of ecological engineering for enabling future environmentally sustainable algal biorefineries.

Design and characterization of synthetic fungal-bacterial consortia for direct production of isobutanol from cellulosic biomass.

Synergistic microbial communities are ubiquitous in nature and exhibit appealing features, such as sophisticated metabolic capabilities and robustness. This has inspired fast-growing interest in engineering synthetic microbial consortia for biotechnology development. However, there are relatively few reports of their use in real-world applications, and achieving population stability and regulation has proven to be challenging. In this work, we bridge ecology theory with engineering principles to develop robust synthetic fungal-bacterial consortia for efficient biosynthesis of valuable products from lignocellulosic feedstocks. The required biological functions are divided between two specialists: the fungus Trichoderma reesei, which secretes cellulase enzymes to hydrolyze lignocellulosic biomass into soluble saccharides, and the bacterium Escherichia coli, which metabolizes soluble saccharides into desired products. We developed and experimentally validated a comprehensive mathematical model for T. reesei/E. coli consortia, providing insights on key determinants of the system's performance. To illustrate the bioprocessing potential of this consortium, we demonstrate direct conversion of microcrystalline cellulose and pretreated corn stover to isobutanol. Without costly nutrient supplementation, we achieved titers up to 1.88 g/L and yields up to 62% of theoretical maximum. In addition, we show that cooperator-cheater dynamics within T. reesei/E. coli consortia lead to stable population equilibria and provide a mechanism for tuning composition. Although we offer isobutanol production as a proof-of-concept application, our modular system could be readily adapted for production of many other valuable biochemicals.

The effect of droplet size on syntrophic dynamics in droplet-enabled microbial co-cultivation.

Co-cultivation in microfluidic droplets has emerged as a versatile tool for the study of natural and synthetic microbial communities. In particular, the identification and characterization of syntrophic interactions in these communities is attracting increasing interest due to their critical importance for the functioning of environmental and host-associated communities as well as new biotechnological applications. However, one critical parameter in droplet-enabled co-cultivation that has evaded appropriate evaluation is the droplet size. Given the same number of initial cells, a larger droplet size can increase the length scale secreted metabolites must diffuse as well as dilute the initial concentration of cells and exchanged metabolites, impacting the community dynamics. To evaluate the effect of droplet size on a spectrum of syntrophic interactions, we cultivated a synthetic model system consisting of two E. coli auxotrophs, whose interactions could be modulated through supplementation of related amino acids in the medium. Our results demonstrate that the droplet size impacts substantially numerous aspects of the growth of a cross-feeding bi-culture, particularly the growth capacity, maximum specific growth rate, and lag time, depending on the degree of the interaction. This work heavily suggests that one droplet size does not fit all types of interactions; this parameter should be carefully evaluated and chosen in experimental studies that aim to utilize droplet-enabled co-cultivation to characterize or elucidate microbial interactions.

Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coli.

BACKGROUND: Isobutanol is a promising next-generation biofuel with demonstrated high yield microbial production, but the toxicity of this molecule reduces fermentation volumetric productivity and final titer. Organic solvent tolerance is a complex, multigenic phenotype that has been recalcitrant to rational engineering approaches. We apply experimental evolution followed by genome resequencing and a gene expression study to elucidate genetic bases of adaptation to exogenous isobutanol stress. RESULTS: The adaptations acquired in our evolved lineages exhibit antagonistic pleiotropy between minimal and rich medium, and appear to be specific to the effects of longer chain alcohols. By examining genotypic adaptation in multiple independent lineages, we find evidence of parallel evolution in marC, hfq, mdh, acrAB, gatYZABCD, and rph genes. Many isobutanol tolerant lineages show reduced RpoS activity, perhaps related to mutations in hfq or acrAB. Consistent with the complex, multigenic nature of solvent tolerance, we observe adaptations in a diversity of cellular processes. Many adaptations appear to involve epistasis between different mutations, implying a rugged fitness landscape for isobutanol tolerance. We observe a trend of evolution targeting post-transcriptional regulation and high centrality nodes of biochemical networks. Collectively, the genotypic adaptations we observe suggest mechanisms of adaptation to isobutanol stress based on remodeling the cell envelope and surprisingly, stress response attenuation. CONCLUSIONS: We have discovered a set of genotypic adaptations that confer increased tolerance to exogenous isobutanol stress. Our results are immediately useful to further efforts to engineer more isobutanol tolerant host strains of E. coli for isobutanol production. We suggest that rpoS and post-transcriptional regulators, such as hfq, RNA helicases, and sRNAs may be interesting mutagenesis targets for future global phenotype engineering.

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

Chromosomal integration of recombinant genes is desirable compared with expression from plasmids due to increased stability, reduced cell-to-cell variability, and elimination of the need for antibiotics for plasmid maintenance. Here, we present a new approach for tuning pathway gene expression levels via random integration and high-throughput screening. We demonstrate multiplexed gene integration and expression-level optimization for isobutanol production in Escherichia coli The integrated strains could, with far lower expression levels than plasmid-based expression, produce high titers (10.0 ± 0.9 g/liter isobutanol in 48 hours) and yields (69% of the theoretical maximum). Close examination of pathway expression in the top-performing, as well as other isolates, reveals the complexity of cellular metabolism and regulation, underscoring the need for precise optimization while integrating pathway genes into the chromosome. We expect this method for pathway integration and optimization can be readily extended to a wide range of pathways and chassis to create robust and efficient production strains.

Random Chromosomal Integration and Screening Yields E. coli K-12 Derivatives Capable of Efficient Sucrose Utilization.

Chromosomal expression of heterologous genes offers stability and maintenance advantages over episomal expression, yet remains difficult to optimize through site-specific integration. The challenge has in large part been due to the variability of chromosomal gene expression, which has only recently been shown to be affected by multiple factors, including the local genomic context. In this work we utilize Tn5 transposase to randomly integrate a three-gene csc operon encoding nonphosphotransferase sucrose catabolism into the E. coli K-12 chromosome. Isolates from the transposon library yielded a range of growth rates on sucrose as the sole carbon source, including some that were comparable to that of E. coli K-12 on glucose (µmax = 0.70 ± 0.03 h-1). Narrowness of the growth rate distributions and faster growth compared to plasmids indicate that efficient csc expression is attainable. Furthermore, enhanced growth rate upon transduction into strains that underwent adaptive laboratory evolution indicate that sucrose catabolism is not limiting to cellular growth. We also show that transduction of a csc fast-growth locus into an isobutanol production strain yields high titer (7.56 ± 0.25 g/L) on sucrose as the sole carbon source. Our results demonstrate that random integration is an effective strategy for optimizing heterologous expression within the context of cellular metabolism for both fast growth and biochemical production phenotypes.

Microdroplet co-cultivation and interaction characterization of human vaginal bacteria.

The human vaginal microbiome (HVM) plays a fundamental role in women's reproductive health. For instance, bacterial vaginosis (BV) is characterized by a depletion of lactobacilli and an overgrowth of strict anaerobes. Women with BV may have an increased risk of acquiring sexually transmitted diseases and adverse pregnancy outcomes. Although the HVM is important, the ecological roles of many vaginal species remain unclear and current approaches for investigating them have severe limitations. We previously developed a new high-throughput technology based on the co-cultivation of bacteria in microdroplets to dissect inter-species interactions in microbial communities. Here, we adapted and extended this technology to investigate the HVM and tested it using pairwise model systems. In one case, Lactobacillus jensenii JV-V16, a lactic acid bacterium, and Gardnerella vaginalis ATCC 49145, a bacterium associated with BV, were cultured in microdroplets as pure cultures and co-cultures. Two assays were developed to analyze their growth in microdroplets. First, qPCR was used to quantify the bacteria in pooled microdroplets. Second, cells in individual microdroplets were plated and enumerated on agar media. The results showed that growth of G. vaginalis was severely inhibited by L. jensenii, which recapitulated previous findings of studies conducted in flask batch cultures. Additionally, we validated the general applicability of our technology pipeline with a second co-culture model system by observing that Enterococcus faecalis, another bacterium from the urogenital tract, was also inhibited by L. jensenii. Our results show that co-cultivation and characterization of bacteria in microdroplets provides an effective way to study inter-species interactions in microbial ecosystems.

High-Resolution Mapping of the Escherichia coli Chromosome Reveals Positions of High and Low Transcription.

Recent studies on targeted gene integrations in bacteria have demonstrated that chromosomal location can substantially affect a gene's expression level. However, these studies have only provided information on a small number of sites. To measure position effects on transcriptional propensity at high resolution across the genome, we built and analyzed a library of over 144,000 genome-integrated, standardized reporters in a single mixed population of Escherichia coli. We observed more than 20-fold variations in transcriptional propensity across the genome when the length of the chromosome was binned into broad 4 kbp regions; greater variability was observed over smaller regions. Our data reveal peaks of high transcriptional propensity centered on ribosomal RNA operons and core metabolic genes, while prophages and mobile genetic elements were enriched in less transcribable regions. In total, our work supports the hypothesis that E. coli has evolved gene-independent mechanisms for regulating expression from specific regions of its genome.

Dissecting the Ecology of Microbes Using a Systems Toolbox.

Study of simplified microbial consortia shows that nitrogen overflow by yeast is a mechanism that supports the stable co-existence of yeasts and lactic acid bacteria.

Bead mediated separation of microparticles in droplets.

Exchange of components such as particles and cells in droplets is important and highly desired in droplet microfluidic assays, and many current technologies use electrical or magnetic fields to accomplish this process. Bead-based microfluidic techniques offer an alternative approach that uses the bead's solid surface to immobilize targets like particles or biological material. In this paper, we demonstrate a bead-based technique for exchanging droplet content by separating fluorescent microparticles in a microfluidic device. The device uses posts to filter surface-functionalized beads from a droplet and re-capture the filtered beads in a new droplet. With post spacing of 7 µm, beads above 10 µm had 100% capture efficiency. We demonstrate the efficacy of this system using targeted particles that bind onto the functionalized beads and are, therefore, transferred from one solution to another in the device. Binding capacity tests performed in the bulk phase showed an average binding capacity of 5 particles to each bead. The microfluidic device successfully separated the targeted particles from the non-targeted particles with up to 98% purity and 100% yield.

Demonstration of transgressive overyielding of algal mixed cultures in microdroplets.

Algae are ubiquitous in natural ecosystems and have been studied extensively for biofuel production due to their unique metabolic capabilities. Most studies to date have approached biofuel optimization through synthetic biology and process engineering with few industrial scale projects considering algal community interactions. Such interactions can potentially lead to increased productivity and reduced community invasability, both important characteristics for scalable algal biofuel production. It is estimated that over a million species of algae exist such that elucidating the interactions that might be beneficial for biofuel production remains extremely resource and time intensive. Here we describe a strategy for rapid, high-throughput screening of algal community combinations using a microfluidic platform to generate millions of parallel, nanoliter-scale algal mixed cultures for estimation of biomass accumulation. Model communities were first studied in a bench scale flask experiment and then examined using microfluidic droplets. These experiments showed consistent results for both positively interacting algal bicultures that increase biomass when together, and negatively interacting bicultures that decrease biomass. Specifically, these included enhanced performance of two bicultures, Ankistrodesmus falcatus and Chlorella sorokiniana, Chlorella sorokiniana and Selenastrum minutum, and reduced performance of a biculture consisting of Selenastrum capricornutum and Scenedesmus ecornis. While the ultimate techno-economic feasibility of algal bioproducts hinges on an amalgamation of scientific fields, rapid screening of algal communities will prove imperative for efficiently discovering community interactions.

Isofunctional enzymes PAD1 and UbiX catalyze formation of a novel cofactor required by ferulic acid decarboxylase and 4-hydroxy-3-polyprenylbenzoic acid decarboxylase.

The decarboxylation of antimicrobial aromatic acids such as phenylacrylic acid (cinnamic acid) and ferulic acid by yeast requires two enzymes described as phenylacrylic acid decarboxylase (PAD1) and ferulic acid decarboxylase (FDC). These enzymes are of interest for various biotechnological applications, such as the production of chemical feedstocks from lignin under mild conditions. However, the specific role of each protein in catalyzing the decarboxylation reaction remains unknown. To examine this, we have overexpressed and purified both PAD1 and FDC from E. coli. We demonstrate that PAD1 is a flavin mononucleotide (FMN)-containing protein. However, it does not function as a decarboxylase. Rather, PAD1 catalyzes the formation of a novel, diffusible cofactor required by FDC for decarboxylase activity. Coexpression of FDC and PAD1 results in the production of FDC with high levels cofactor bound. Holo-FDC catalyzes the decarboxylation of phenylacrylic acid, coumaric acid and ferulic acid with apparent kcat ranging from 1.4-4.6 s(-1). The UV-visible and mass spectra of the cofactor indicate that it appears to be a novel, modified form of reduced FMN; however, its instability precluded determination of its structure. The E. coli enzymes UbiX and UbiD are related by sequence to PAD1 and FDC respectively and are involved in the decarboxylation of 4-hydroxy-3-octaprenylbenzoic acid, an intermediate in ubiquinone biosynthesis. We found that endogenous UbiX can also activate FDC. This implies that the same cofactor is required for decarboxylation of 4-hydroxy-3-polyprenylbenzoic acid by UbiD and suggests a wider role for this cofactor in metabolism.

Improving fatty acid availability for bio-hydrocarbon production in Escherichia coli by metabolic engineering.

Previous studies have demonstrated the feasibility of producing fatty-acid-derived hydrocarbons in Escherichia coli. However, product titers and yields remain low. In this work, we demonstrate new methods for improving fatty acid production by modifying central carbon metabolism and storing fatty acids in triacylglycerol. Based on suggestions from a computational model, we deleted seven genes involved in aerobic respiration, mixed-acid fermentation, and glyoxylate bypass (in the order of cyoA, nuoA, ndh, adhE, dld, pta, and iclR) to modify the central carbon metabolic/regulatory networks. These gene deletions led to increased total fatty acids, which were the highest in the mutants containing five or six gene knockouts. Additionally, when two key enzymes in the fatty acid biosynthesis pathway were over-expressed, we observed further increase in strain △cyoA△adhE△nuoA△ndh△pta△dld, leading to 202 mg/g dry cell weight of total fatty acids, ~250% of that in the wild-type strain. Meanwhile, we successfully introduced a triacylglycerol biosynthesis pathway into E. coli through heterologous expression of wax ester synthase/acyl-coenzyme:diacylglycerol acyltransferase (WS/DGAT) enzymes. The added pathway improved both the amount and fuel quality of the fatty acids. These new metabolic engineering strategies are providing promising directions for future investigation.

Microbial utilization of aqueous co-products from hydrothermal liquefaction of microalgae Nannochloropsis oculata.

Hydrothermal liquefaction of algae biomass is a promising technology for the production of sustainable biofuels, but the non-oil, aqueous co-product of the process has only been examined to a limited extent. The aqueous phase from liquefaction of the alga Nannochloropsis oculata (AqAl) was used to make growth media for model heterotrophic microorganisms Escherichia coli, Pseudomonas putida, and Saccharomyces cerevisiae. Growth rates, yields, and carbon/nitrogen/phosphorus uptake were measured. E. coli and P. putida could grow using AqAl as the sole C, N, and P source in media containing 10 vol.%-40 vol.% AqAl with the best growth occurring at 20 vol.%. S. cerevisiae could grow under these conditions only if the media were supplemented with glucose. The results indicate that in a biorefinery utilizing algae liquefaction, the aqueous co-product may be recycled via microbial cultures with significantly less dilution than previously published methods.

A programmable Escherichia coli consortium via tunable symbiosis.

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.

Microdroplet-enabled highly parallel co-cultivation of microbial communities.

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 ∼1,400 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.

Multisite phosphorylation provides an effective and flexible mechanism for switch-like protein degradation.

Phosphorylation-triggered degradation is a common strategy for elimination of regulatory proteins in many important cell signaling processes. Interesting examples include cyclin-dependent kinase inhibitors such as p27 in human and Sic1 in yeast, which play crucial roles during the G1/S transition in the cell cycle. In this work, we have modeled and analyzed the dynamics of multisite-phosphorylation-triggered protein degradation systematically. Inspired by experimental observations on the Sic1 protein and a previous intriguing theoretical conjecture, we develop a model to examine in detail the degradation dynamics of a protein featuring multiple phosphorylation sites and a threshold site number for elimination in response to a kinase signal. Our model explains the role of multiple phosphorylation sites, compared to a single site, in the regulation of protein degradation. A single-site protein cannot convert a graded input of kinase increase to much sharper output, whereas multisite phosphorylation is capable of generating a highly switch-like temporal profile of the substrate protein with two characteristics: a temporal threshold and rapid decrease beyond the threshold. We introduce a measure termed temporal response coefficient to quantify the extent to which a response in the time domain is switch-like and further investigate how this property is determined by various factors including the kinase input, the total number of sites, the threshold site number for elimination, the order of phosphorylation, the kinetic parameters, and site preference. Some interesting and experimentally verifiable predictions include that the non-degradable fraction of the substrate protein exhibits a more switch-like temporal profile; a sequential system is more switch-like, while a random system has the advantage of increased robustness; all the parameters, including the total number of sites, the threshold site number for elimination and the kinetic parameters synergistically determine the exact extent to which the degradation profile is switch-like. Our results suggest design principles for protein degradation switches which might be a widespread mechanism for precise regulation of cellular processes such as cell cycle progression.