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.

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 The integrated strains could, with far lower expression levels than plasmid-based expression, produce high titers (10.

Random Chromosomal Integration and Screening Yields 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 operon encoding nonphosphotransferase sucrose catabolism into the K-12 chromosome.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Network benchmarking: a happy marriage between systems and synthetic biology.

In their new Cell paper, Cantone et al. (2009) present exciting results on constructing and utilizing a small synthetic gene regulatory network in yeast that draws from two rapidly developing fields of systems and synthetic biology.