RMH

Flowers of the hop plant provide both bitterness and “hoppy” flavor to beer. Hops are, however, both a water and energy intensive crop and vary considerably in essential oil content, making it challenging to achieve a consistent hoppy taste in beer. Here, we report that brewer’s yeast can be engineered to biosynthesize aromatic monoterpene molecules that impart hoppy flavor to beer by incorporating recombinant DNA derived from yeast, mint, and basil. Whereas metabolic engineering of biosynthetic pathways is commonly enlisted to maximize product titers, tuning expression of pathway enzymes to affect target production levels of multiple commercially important metabolites without major collateral metabolic changes represents a unique challenge. By applying state-of-the-art engineering techniques and a framework to guide iterative improvement, strains are generated with target performance characteristics. Beers produced using these strains are perceived as hoppier than traditionally hopped beers by a sensory panel in a double-blind tasting.

Increasing availability of new genomes and putative biosynthetic gene clusters (BGCs) has extended the opportunity to access novel chemical diversity for agriculture, medicine, environmental and industrial purposes. However, functional characterization of BGCs through heterologous expression is limited because expression may require complex regulatory mechanisms, specific folding or activation. We developed an integrated workflow for BGC characterization that integrates pathway identification, modular design, DNA synthesis, assembly and characterization. This workflow was applied to characterize multiple phenazine-modifying enzymes. Phenazine pathways are useful for this workflow because all phenazines are derived from a core scaffold for modification by diverse modifying enzymes (PhzM, PhzS, PhzH, and PhzO) that produce characterized compounds. We expressed refactored synthetic modules of previously uncharacterized phenazine BGCs heterologously in Escherichia coli and were able to identify metabolic intermediates they produced, including a previously unidentified metabolite. These results demonstrate how this approach can accelerate functional characterization of BGCs.

The ferredoxin (Fd) protein family is a structurally diverse group of iron–sulfur proteins that function as electron carriers, linking biochemical pathways important for energy transduction, nutrient assimilation, and primary metabolism. While considerable biochemical information about individual Fd protein electron carriers and their reactions has been acquired, we cannot yet anticipate the proportion of electrons shuttled between different Fd-partner proteins within cells using biochemical parameters that govern electron flow, such as holo-Fd concentration, midpoint potential (driving force), molecular interactions (affinity and kinetics), conformational changes (allostery), and off-pathway electron leakage (chemical oxidation). Herein, we describe functional and structural gaps in our Fd knowledge within the context of a sequence similarity network and phylogenetic tree, and we propose a strategy for improving our understanding of Fd sequence–function relationships. We suggest comparing the functions of divergent Fds within cells whose growth, or other measurable output, requires electron transfer between defined electron donor and acceptor proteins. By comparing Fd-mediated electron transfer with biochemical parameters that govern electron flow, we posit that models that anticipate energy flow across Fd interactomes can be built. This approach is expected to transform our ability to anticipate Fd control over electron flow in cellular settings, an obstacle to the construction of synthetic electron transfer pathways and rational optimization of existing energy-conserving pathways.

Engineering and study of protein function by directed evolution has been limited by the technical requirement to use global mutagenesis or introduce DNA libraries. Here, we develop CRISPR-X, a strategy to repurpose the somatic hypermutation machinery for protein engineering in situ. Using catalytically inactive dCas9 to recruit variants of cytidine deaminase (AID) with MS2-modified sgRNAs, we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations and can target multiple genomic locations simultaneously. We mutagenize GFP and select for spectrum-shifted variants, including EGFP. Additionally, we mutate the target of the cancer therapeutic bortezomib, PSMB5, and identify known and novel mutations that confer bortezomib resistance. Finally, using a hyperactive AID variant, we mutagenize loci both upstream and downstream of transcriptional start sites. These experiments illustrate a powerful approach to create complex libraries of genetic variants in native context, which is broadly applicable to investigate and improve protein function.

Geological storage of CO2 is a fast-developing technology that can mitigate rising carbon emissions. However, there are environmental concerns with the long-term storage and implications of a leak from a carbon capture storage (CCS) site. Traditional monitoring lacks clear protocols and relies heavily on physical methods. Here, we discuss the potential of biotechnology, focusing on microbes with a natural ability to utilize and assimilate CO2 through different metabolic pathways. We propose the use of natural microbial communities for CCS monitoring and CO2 utilization, and, with examples, demonstrate how synthetic biology may maximize CO2 uptake within and above storage sites. An integrated physical and biological approach, combined with metagenomics data and biotechnological advances, will enhance CO2 sequestration and prevent large-scale leakages.

Plant pathogens can cause serious diseases that impact global agriculture. The plant innate immunity, when fully activated, can halt pathogen growth in plants. Despite extensive studies into the molecular and genetic bases of plant immunity against pathogens, the influence of plant immunity in global pathogen metabolism to restrict pathogen growth is poorly understood. Here, we developed RNA sequencing pipelines for analyzing bacterial transcriptomes in planta and determined high-resolution transcriptome patterns of the foliar bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana with a total of 27 combinations of plant immunity mutants and bacterial strains. Bacterial transcriptomes were analyzed at 6 h post infection to capture early effects of plant immunity on bacterial processes and to avoid secondary effects caused by different bacterial population densities in planta . We identified specific “immune-responsive” bacterial genes and processes, including those that are activated in susceptible plants and suppressed by plant immune activation. Expression patterns of immune-responsive bacterial genes at the early time point were tightly linked to later bacterial growth levels in different host genotypes. Moreover, we found that a bacterial iron acquisition pathway is commonly suppressed by multiple plant immune-signaling pathways. Overexpression of a P. syringae sigma factor gene involved in iron regulation and other processes partially countered bacterial growth restriction during the plant immune response triggered by AvrRpt2. Collectively, this study defines the effects of plant immunity on the transcriptome of a bacterial pathogen and sheds light on the enigmatic mechanisms of bacterial growth inhibition during the plant immune response.

Ammonia oxidation is a fundamental core process in the global biogeochemical nitrogen cycle. Oxidation of ammonia (NH3) to nitrite (NO2−) is the first and rate-limiting step in nitrification and is carried out by distinct groups of microorganisms. Ammonia oxidation is essential for nutrient turnover in most terrestrial, aquatic and engineered ecosystems and plays a major role, both directly and indirectly, in greenhouse gas production and environmental damage. Although ammonia oxidation has been studied for over a century, this research field has been galvanised in the past decade by the surprising discoveries of novel ammonia oxidising microorganisms. This review reflects on the ammonia oxidation research to date and discusses the major gaps remaining in our knowledge of the biology of ammonia oxidation.

Microfluidic platforms have the potential to address four major challenges in studying soil systems, namely (1) their enormous spatio-temporal heterogeneity, (2) the lack of current methods to mimic soil realistically at the appropriate scale, (3) the difficulties in studying interactions among soil microbes and (4) the lack of optical access in real soil systems. We discuss these issues in turn, highlighting important advances driving the transfer of this technology to soil systems, but also identify potential challenges.

Transplanting metabolic reactions from one species into another has many uses as a research tool with applications ranging from optogenetics to crop production. Ferredoxin (Fd), the enzyme that most often supplies electrons to these reactions, is often overlooked when transplanting enzymes from one species to another because most cells already contain endogenous Fd. However, we have shown that the production of chromophores used in Phytochrome B (PhyB) optogenetics is greatly enhanced in mammalian cells by expressing bacterial and plant Fds with ferredoxin-NADP+ reductases (FNR). We delineated the rate limiting factors and found that the main metabolic precursor, heme, was not the primary limiting factor for producing either the cyanobacterial or plant chromophores, phycocyanobilin or phytochromobilin, respectively. In fact, Fd is limiting, followed by Fd+FNR and finally heme. Using these findings, we optimized the PCB production system and combined it with a tissue penetrating red/far-red sensing PhyB optogenetic gene switch in animal cells. We further characterized this system in several mammalian cell lines using red and far-red light. Importantly, we found that the light-switchable gene system remains active for several hours upon illumination, even with a short light pulse, and requires very small amounts of light for maximal activation. Boosting chromophore production by matching metabolic pathways with specific ferredoxin systems will enable the unparalleled use of the many PhyB optogenetic tools and has broader implications for optimizing synthetic metabolic pathways.

epimutagenesis introduces random methylation variation via the introduction of a transgeneepimutagenesis introduces random methylation variation via the introduction of a transgeneDNA methylation in the promoters of plant genes sometimes leads to transcriptional repression, and the loss of DNA methylation in methyltransferase mutants results in altered gene expression and severe developmental defects. However, many cases of naturally occurring DNA methylation variations have been reported, whereby altered expression of differentially methylated genes is responsible for agronomically important traits. The ability to manipulate plant methylomes to generate epigenetically distinct individuals could be invaluable for breeding and research purposes. Here, we describe “epimutagenesis,” a method to rapidly generate DNA methylation variation through random demethylation of the Arabidopsis thaliana genome. This method involves the expression of a human ten–eleven translocation (TET) enzyme, and results in widespread hypomethylation that can be inherited to subsequent generations, mimicking mutants in the maintenance of DNA methyltransferase met1. Application of epimutagenesis to agriculturally significant plants may result in differential expression of alleles normally silenced by DNA methylation, uncovering previously hidden phenotypic variations.

Bacteria are able to coordinate their movement, growth and biochemical activities through cell-cell communication. While the biophysical mechanism of bacterial chemotaxis has been well understood in individual cells, the role of communication in the chemotaxis of bacterial populations is not clear. Here we report experimental evidence for cell-cell communication that significantly enhances the chemotactic migration of bacterial populations, a finding that we further substantiate using numerical simulations. Using a microfluidic approach, we find that E. coli cells respond to the gradient of chemoattractant not only by biasing their own random-walk swimming pattern through the well-understood intracellular chemotaxis signaling, but also by actively secreting a chemical signal into the extracellular medium, possibly through a hitherto unknown communication signal transduction pathway. This extracellular signaling molecule is a strong chemoattractant that attracts distant cells to the food source. The observed behavior may represent a common evolved solution to accelerate the function of biochemical networks of interacting cells.

Fluorescent proteins are ubiquitous tools that are used to monitor the dynamic functions of natural and synthetic genetic circuits. However, these visual reporters can only be used in transparent settings, a limitation that complicates nondisruptive measurements of gene expression within many matrices, such as soils and sediments. We describe a new ratiometric gas reporting method for nondisruptively monitoring gene expression within hard-to-image environmental matrices. With this approach, C2H4 is continuously synthesized by ethylene forming enzyme to provide information on viable cell number, and CH3Br is conditionally synthesized by placing a methyl halide transferase gene under the control of a conditional promoter. We show that ratiometric gas reporting enables the creation of Escherichia coli biosensors that report on acylhomoserine lactone (AHL) autoinducers used for quorum sensing by Gram-negative bacteria. Using these biosensors, we find that an agricultural soil decreases the bioavailable concentration of a long-chain AHL up to 100-fold. We also demonstrate that these biosensors can be used in soil to nondisruptively monitor AHLs synthesized by Rhizobium leguminosarum and degraded by Bacillus thuringiensis. Finally, we show that this new reporting approach can be used in Shewanella oneidensis, a bacterium that lives in sediments.

Microbes typically live alongside many other species in complex communities. These microbial communities are very important for us because they also live in and on our bodies and can determine our health and well-being. The composition and function of these communities, such as who is part of such a community and who is excluded, are decided by the interactions between the microbes. These microbial interactions can be driven by many different factors such as resource competition or toxin production. Although these factors are all different, the interactions are typically mediated through the environment; the microbes modify the environment, and they and other microbes have to live in this new environment. We show here that by understanding how microbes change and react to the environment, it is possible to understand and even predict their interactions. We believe that this way of thinking about microbial interactions will lead to a better understanding of more complex communities that are so important for our well-being.

Digested genome sequencing, or Digenome-seq, is an in vitro assay that has become increasingly popular since its introduction in 2015. It has a simple two-step protocol: in vitro Cas9 cleavage followed by next-gen sequencing. Two newer methods, CIRCLE-Seq and SITE-Seq, are slightly more complex but are also more sensitive, as they enrich for nuclease-cleaved genomic DNA before sequencing. Researchers are continuing to improve the accuracy and throughput of these methods. “I think in the long term, in vitro is going to be the way to go, but that’s an evolving space right now,” says Joung.

Soil aggregation, a key component of soil structure, has mostly been examined from the perspective of soil management and the mediation of ecosystem processes such as soil carbon storage. However, soil aggregation is also a major factor to consider in terms of the fine-scale organization of the soil microbiome. For example, the physico-chemical conditions inside of aggregates usually differ from the conditions prevalent in the bulk soil and aggregates therefore increase the spatial heterogeneity of the soil. In addition, aggregates can provide a refuge for microbes against predation since their interior is not accessible to many predators. Soil aggregates are thus clearly important for microbial community ecology in soils and for microbially driven biogeochemistry, and soil microbial ecologists are increasingly appreciating these aspects of soil aggregation. Soil aggregates have, however, so far been neglected when it comes to evolutionary considerations and we here propose that the process of soil aggregation should be considered as an important driver of evolution in the soil microbial community.

The arms race between bacteria and phages led to the development of sophisticated antiphage defense systems, including CRISPR-Cas and restriction-modification systems. Evidence suggests that known and unknown defense systems are located in “defense islands” in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal by examining gene families that are clustered next to known defense genes in prokaryotic genomes. Candidate defense systems were systematically engineered and validated in model bacteria for their antiphage activities. We report nine previously unknown antiphage systems and one antiplasmid system that are widespread in microbes and strongly protect against foreign invaders. These include systems that adopted components of the bacterial flagella and condensin complexes. Our data also suggest a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria.