Mutational robustness and the role of buffer genes in evolvability.

Organisms rely on mutations to fuel adaptive evolution. However, many mutations impose a negative effect on fitness. Cells may have therefore evolved mechanisms that affect the phenotypic effects of mutations, thus conferring mutational robustness. Specifically, so-called buffer genes are hypothesized to interact directly or indirectly with genetic variation and reduce its effect on fitness. Environmental or genetic perturbations can change the interaction between buffer genes and genetic variation, thereby unmasking the genetic variation's phenotypic effects and thus providing a source of variation for natural selection to act on. This review provides an overview of our understanding of mutational robustness and buffer genes, with the chaperone gene HSP90 as a key example. It discusses whether buffer genes merely affect standing variation or also interact with de novo mutations, how mutational robustness could influence evolution, and whether mutational robustness might be an evolved trait or rather a mere side-effect of complex genetic interactions.

Rational evolution for altering the ligand preference of estrogen receptor alpha.

Estrogen receptor α is commonly used in synthetic biology to control the activity of genome editing tools. The activating ligands, estrogens, however, interfere with various cellular processes, thereby limiting the applicability of this receptor. Altering its ligand preference to chemicals of choice solves this hurdle but requires adaptation of unspecified ligand-interacting residues. Here, we provide a solution by combining rational protein design with multi-site-directed mutagenesis and directed evolution of stably integrated variants in Saccharomyces cerevisiae. This method yielded an estrogen receptor variant, named TERRA, that lost its estrogen responsiveness and became activated by tamoxifen, an anti-estrogenic drug used for breast cancer treatment. This tamoxifen preference of TERRA was maintained in mammalian cells and mice, even when fused to Cre recombinase, expanding the mammalian synthetic biology toolbox. Not only is our platform transferable to engineer ligand preference of any steroid receptor, it can also profile drug-resistance landscapes for steroid receptor-targeted therapies.

Predicting and improving complex beer flavor through machine learning.

The perception and appreciation of food flavor depends on many interacting chemical compounds and external factors, and therefore proves challenging to understand and predict. Here, we combine extensive chemical and sensory analyses of 250 different beers to train machine learning models that allow predicting flavor and consumer appreciation. For each beer, we measure over 200 chemical properties, perform quantitative descriptive sensory analysis with a trained tasting panel and map data from over 180,000 consumer reviews to train 10 different machine learning models. The best-performing algorithm, Gradient Boosting, yields models that significantly outperform predictions based on conventional statistics and accurately predict complex food features and consumer appreciation from chemical profiles. Model dissection allows identifying specific and unexpected compounds as drivers of beer flavor and appreciation. Adding these compounds results in variants of commercial alcoholic and non-alcoholic beers with improved consumer appreciation. Together, our study reveals how big data and machine learning uncover complex links between food chemistry, flavor and consumer perception, and lays the foundation to develop novel, tailored foods with superior flavors.

Mutagenesis techniques for evolutionary engineering of microbes - exploiting CRISPR-Cas, oligonucleotides, recombinases, and polymerases.

The natural process of evolutionary adaptation is often exploited as a powerful tool to obtain microbes with desirable traits. For industrial microbes, evolutionary engineering is often used to generate variants that show increased yields or resistance to stressful industrial environments, thus obtaining superior microbial cell factories. However, even in large populations, the natural supply of beneficial mutations is typically low, which implies that obtaining improved microbes is often time-consuming and inefficient. To overcome this limitation, different techniques have been developed that boost mutation rates. While some of these methods simply increase the overall mutation rate across a genome, others use recent developments in DNA synthesis, synthetic biology, and CRISPR-Cas techniques to control the type and location of mutations. This review summarizes the most important recent developments and methods in the field of evolutionary engineering in model microorganisms. It discusses how both in vitro and in vivo approaches can increase the genetic diversity of the host, with a special emphasis on in vivo techniques for the optimization of metabolic pathways for precision fermentation.

Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast.

Microbes are increasingly employed as cell factories to produce biomolecules. This often involves the expression of complex heterologous biosynthesis pathways in host strains. Achieving maximal product yields and avoiding build-up of (toxic) intermediates requires balanced expression of every pathway gene. However, despite progress in metabolic modeling, the optimization of gene expression still heavily relies on trial-and-error. Here, we report an approach for in vivo, multiplexed Gene Expression Modification by LoxPsym-Cre Recombination (GEMbLeR). GEMbLeR exploits orthogonal LoxPsym sites to independently shuffle promoter and terminator modules at distinct genomic loci. This approach facilitates creation of large strain libraries, in which expression of every pathway gene ranges over 120-fold and each strain harbors a unique expression profile. When applied to the biosynthetic pathway of astaxanthin, an industrially relevant antioxidant, a single round of GEMbLeR improved pathway flux and doubled production titers. Together, this shows that GEMbLeR allows rapid and efficient gene expression optimization in heterologous biosynthetic pathways, offering possibilities for enhancing the performance of microbial cell factories.

Orthogonal LoxPsym sites allow multiplexed site-specific recombination in prokaryotic and eukaryotic hosts.

Site-specific recombinases such as the Cre-LoxP system are routinely used for genome engineering in both prokaryotes and eukaryotes. Importantly, recombinases complement the CRISPR-Cas toolbox and provide the additional benefit of high-efficiency DNA editing without generating toxic DNA double-strand breaks, allowing multiple recombination events at the same time. However, only a handful of independent, orthogonal recombination systems are available, limiting their use in more complex applications that require multiple specific recombination events, such as metabolic engineering and genetic circuits. To address this shortcoming, we develop 63 symmetrical LoxP variants and test 1192 pairwise combinations to determine their cross-reactivity and specificity upon Cre activation. Ultimately, we establish a set of 16 orthogonal LoxPsym variants and demonstrate their use for multiplexed genome engineering in both prokaryotes (E. coli) and eukaryotes (S. cerevisiae and Z. mays). Together, this work yields a significant expansion of the Cre-LoxP toolbox for genome editing, metabolic engineering and other controlled recombination events, and provides insights into the Cre-LoxP recombination process.

Locus-specific proteome decoding reveals Fpt1 as a chromatin-associated negative regulator of RNA polymerase III assembly.

Transcription of tRNA genes by RNA polymerase III (RNAPIII) is tuned by signaling cascades. The emerging notion of differential tRNA gene regulation implies the existence of additional regulatory mechanisms. However, tRNA gene-specific regulators have not been described. Decoding the local chromatin proteome of a native tRNA gene in yeast revealed reprogramming of the RNAPIII transcription machinery upon nutrient perturbation. Among the dynamic proteins, we identified Fpt1, a protein of unknown function that uniquely occupied RNAPIII-regulated genes. Fpt1 binding at tRNA genes correlated with the efficiency of RNAPIII eviction upon nutrient perturbation and required the transcription factors TFIIIB and TFIIIC but not RNAPIII. In the absence of Fpt1, eviction of RNAPIII was reduced, and the shutdown of ribosome biogenesis genes was impaired upon nutrient perturbation. Our findings provide support for a chromatin-associated mechanism required for RNAPIII eviction from tRNA genes and tuning the physiological response to changing metabolic demands.

Kevin J. Verstrepen.

Interview with Kevin Verstrepen, who combines basic research on microbial genetics and evolution with applied research in the fermentation industry at the VIB - KU Leuven Center for Microbiology.

Full-electric microfluidic platform to capture, analyze and selectively release single cells.

Current single-cell technologies require large and expensive equipment, limiting their use to specialized labs. In this paper, we present for the first time a microfluidic device which demonstrates a combined method for full-electric cell capturing, analyzing, and selectively releasing with single-cell resolution. All functionalities are experimentally demonstrated on Saccharomyces cerevisiae. Our microfluidic platform consists of traps centered around a pair of individually accessible coplanar electrodes, positioned under a microfluidic channel. Using this device, we validate our novel Two-Voltage method for trapping single cells by positive dielectrophoresis (pDEP). Cells are attracted to the trap when a high voltage (VH) is applied. A low voltage (VL) holds the already trapped cell in place without attracting additional cells, allowing full control over the number of trapped cells. After trapping, the cells are analyzed by broadband electrochemical impedance spectroscopy. These measurements allow the detection of single cells and the extraction of cell parameters. Additionally, these measurements show a strong correlation between average phase change and cell size, enabling the use of our system for size measurements in biological applications. Finally, our device allows selectively releasing trapped cells by turning off the pDEP signal in their trap. The experimental results show the techniques potential as a full-electric single-cell analysis tool with potential for miniaturization and automation which opens new avenues towards small-scale, high throughput single-cell analysis and sorting lab-on-CMOS devices.

A Cas3-base editing tool for targetable in vivo mutagenesis.

The generation of genetic diversity via mutagenesis is routinely used for protein engineering and pathway optimization. Current technologies for random mutagenesis often target either the whole genome or relatively narrow windows. To bridge this gap, we developed CoMuTER (Confined Mutagenesis using a Type I-E CRISPR-Cas system), a tool that allows inducible and targetable, in vivo mutagenesis of genomic loci of up to 55 kilobases. CoMuTER employs the targetable helicase Cas3, signature enzyme of the class 1 type I-E CRISPR-Cas system, fused to a cytidine deaminase to unwind and mutate large stretches of DNA at once, including complete metabolic pathways. The tool increases the number of mutations in the target region 350-fold compared to the rest of the genome, with an average of 0.3 mutations per kilobase. We demonstrate the suitability of CoMuTER for pathway optimization by doubling the production of lycopene in Saccharomyces cerevisiae after a single round of mutagenesis.

Metabolism: How a eukaryote adapted to life without respiration.

A new study finds that Schizosaccharomyces japonicus, a eukaryote that lost the ability to respire, modified its central carbon metabolism to maintain efficient ATP production, cofactor regeneration, and amino-acid production. This remarkable metabolic flexibility opens new avenues towards applications.

Novel Saccharomyces cerevisiae variants slow down the accumulation of staling aldehydes and improve beer shelf-life.

Beer quality generally diminishes over time as staling compounds accumulate through various oxidation reactions. Here, we show that refermentation, a traditional practice where Saccharomyces cerevisiae cells are added to beer prior to bottling, diminishes the accumulation of staling aldehydes. However, commonly used beer yeasts only show a limited lifespan in beer. Using high-throughput screening and breeding, we were able to generate novel S. cerevisiae hybrids that survive for over a year in beer. Extensive chemical and sensory analyses of the two most promising hybrids showed that they slow down the accumulation of staling aldehydes, such as furfural and trans-2-nonenal and significantly increased beer flavor stability for up to 12 months. Moreover, the strains did not change the original flavor of the beer, highlighting their potential to be integrated in existing products. Together, these results demonstrate the ability to breed novel microbes that function as natural and sustainable anti-oxidative food preservatives.

Ethanol induces replication fork stalling and membrane stress in immortalized laryngeal cells.

Although ethanol is a class I carcinogen and is linked to more than 700,000 cancer incidences, a clear understanding of the molecular mechanisms underlying ethanol-related carcinogenesis is still lacking. Further understanding of ethanol-related cell damage can contribute to reducing or treating alcohol-related cancers. Here, we investigated the effects of both short- and long-term exposure of human laryngeal epithelial cells to different ethanol concentrations. RNA sequencing shows that ethanol altered gene expression patterns in a time- and concentration-dependent way, affecting genes involved in ribosome biogenesis, cytoskeleton remodeling, Wnt signaling, and transmembrane ion transport. Additionally, ethanol induced a slower cell proliferation, a delayed cell cycle progression, and replication fork stalling. In addition, ethanol exposure resulted in morphological changes, which could be associated with membrane stress. Taken together, our data yields a comprehensive view of molecular changes associated with ethanol stress in epithelial cells of the upper aerodigestive tract.

Saccharomyces cerevisiae as a Model System for Eukaryotic Cell Biology, from Cell Cycle Control to DNA Damage Response.

The yeast Saccharomyces cerevisiae has been used for bread making and beer brewing for thousands of years. In addition, its ease of manipulation, well-annotated genome, expansive molecular toolbox, and its strong conservation of basic eukaryotic biology also make it a prime model for eukaryotic cell biology and genetics. In this review, we discuss the characteristics that made yeast such an extensively used model organism and specifically focus on the DNA damage response pathway as a prime example of how research in S. cerevisiae helped elucidate a highly conserved biological process. In addition, we also highlight differences in the DNA damage response of S. cerevisiae and humans and discuss the challenges of using S. cerevisiae as a model system.

Do microbes have a memory? History-dependent behavior in the adaptation to variable environments.

Microbes are constantly confronted with changes and challenges in their environment. A proper response to these environmental cues is needed for optimal cellular functioning and fitness. Interestingly, past exposure to environmental cues can accelerate or boost the response when this condition returns, even in daughter cells that have not directly encountered the initial cue. Moreover, this behavior is mostly epigenetic and often goes hand in hand with strong heterogeneity in the strength and speed of the response between isogenic cells of the same population, which might function as a bet-hedging strategy. In this review, we discuss examples of history-dependent behavior (HDB) or "memory," with a specific focus on HDB in fluctuating environments. In most examples discussed, the lag time before the response to an environmental change is used as an experimentally measurable proxy for HDB. We highlight different mechanisms already implicated in HDB, and by using HDB in fluctuating carbon conditions as a case study, we showcase how the metabolic state of a cell can be a key determining factor for HDB. Finally, we consider possible evolutionary causes and consequences of such HDB.

Impact of endophytic colonization by entomopathogenic fungi on the behavior and life history of the tobacco peach aphid Myzus persicae var. nicotianae.

Entomopathogenic fungi can adopt an endophytic lifestyle and provide protection against insect herbivores and plant pathogens. So far, most studies have focused on Beauveria bassiana to increase plant resistance against abiotic and biotic stresses, while only little is known for other entomopathogenic fungi. In this study, we investigated whether root inoculation of sweet pepper (Capsicum annuum L.) by the entomopathogenic fungi Akanthomyces muscarius ARSEF 5128 and B. bassiana ARSEF 3097 can improve resistance against the tobacco peach aphid Myzus persicae var. nicotianae. First, dual-choice experiments were performed to test the hypothesis that the fungi deter aphids via modifying plant volatile profiles. Next, we tested the hypothesis that endophytic colonization negatively affects aphid life history traits, such as fecundity, development and mortality rate. Aphids were significantly attracted to the odor of plants inoculated with A. muscarius over non-inoculated plants. Plants inoculated with A. muscarius emitted significantly higher amounts of ß-pinene than non-inoculated plants, and significantly higher amounts of indole than B. bassiana-inoculated and non-inoculated plants. Inoculation with the fungal strains also caused significantly higher emission of terpinolene. Further, both aphid longevity and fecundity were significantly reduced by 18% and 10%, respectively, when feeding on plants inoculated with A. muscarius, although intrinsic rate of population increase did not differ between inoculated and non-inoculated plants. Sweet pepper plants inoculated with B. bassiana ARSEF 3097 did not elicit a significant behavioral response nor affected the investigated life history traits. We conclude that endophytic colonization by entomopathogenic fungi has the potential to alter olfactory behavior and performance of M. persicae var. nicotianae, but effects are small and depend on the fungal strain used.

Synchronized, Spontaneous, and Oscillatory Detachment of Eukaryotic Cells: A New Tool for Cell Characterization and Identification.

Despite the importance of cell characterization and identification for diagnostic and therapeutic applications, developing fast and label-free methods without (bio)-chemical markers or surface-engineered receptors remains challenging. Here, we exploit the natural cellular response to mild thermal stimuli and propose a label- and receptor-free method for fast and facile cell characterization. Cell suspensions in a dedicated sensor are exposed to a temperature gradient, which stimulates synchronized and spontaneous cell-detachment with sharply defined time-patterns, a phenomenon unknown from literature. These patterns depend on metabolic activity (controlled through temperature, nutrients, and drugs) and provide a library of cell-type-specific indicators, allowing to distinguish several yeast strains as well as cancer cells. Under specific conditions, synchronized glycolytic-type oscillations are observed during detachment of mammalian and yeast-cell ensembles, providing additional cell-specific signatures. These findings suggest potential applications for cell viability analysis and for assessing the collective response of cancer cells to drugs.

Permissive aggregative group formation favors coexistence between cooperators and defectors in yeast.

In Saccharomyces cerevisiae, the FLO1 gene encodes flocculins that lead to formation of multicellular flocs, that offer protection to the constituent cells. Flo1p was found to preferentially bind to fellow cooperators compared to defectors lacking FLO1 expression, enriching cooperators within the flocs. Given this dual function in cooperation and kin recognition, FLO1 has been termed a "green beard gene". Because of the heterophilic nature of the Flo1p bond however, we hypothesize that kin recognition is permissive and depends on the relative stability of the FLO1+/flo1- versus FLO1+/FLO1+ detachment force F. We combine single-cell measurements of adhesion, individual cell-based simulations of cluster formation, and in vitro flocculation to study the impact of relative bond stability on the evolutionary stability of cooperation. We identify a trade-off between both aspects of the green beard mechanism, with reduced relative bond stability leading to increased kin recognition at the expense of cooperative benefits. We show that the fitness of FLO1 cooperators decreases as their frequency in the population increases, arising from the observed permissive character (F+- = 0.5 F++) of the Flo1p bond. Considering the costs associated with FLO1 expression, this asymmetric selection often results in a stable coexistence between cooperators and defectors.

Beer ethanol and iso-α-acid level affect microbial community establishment and beer chemistry throughout wood maturation of beer.

Sour beers produced by barrel-aging of conventionally fermented beers are becoming increasingly popular. However, as the intricate interactions between the wood, the microbes and the beer are still unclear, wood maturation often leads to inconsistent end products with undesired sensory properties. Previous research on industrial barrel-aging of beer suggests that beer parameters like the ethanol content and bitterness play an important role in the microbial community composition and beer chemistry, but their exact impact still remains to be investigated. In this study, an experimentally tractable lab-scale system based on an in-vitro community of four key bacteria (Acetobacter malorum, Gluconobacter oxydans, Lactobacillus brevis and Pediococcus damnosus) and four key yeasts (Brettanomyces bruxellensis, Candida friedrichii, Pichia membranifaciens and Saccharomyces cerevisiae) that are consistently associated with barrel-aging of beer, was used to test the hypotheses that beer ethanol and bitterness impact microbial community composition and beer chemistry. Experiments were performed using different levels of ethanol (5.2 v/v%, 8 v/v% and 11 v/v%) and bitterness (13 ppm, 35 ppm and 170 ppm iso-α-acids), and beers were matured for 60 days. Samples were taken after 0, 10, 20, 30 and 60 days to monitor population densities and beer chemistry. Results revealed that all treatments and the maturation time significantly affected the microbial community composition and beer chemistry. More specifically, the ethanol treatments obstructed growth of L. brevis and G. oxydans and delayed fungal growth. The iso-α-acid treatments hindered growth of L. brevis and stimulated growth of P. membranifaciens, while the other strains remained unaffected. Beer chemistry was found to be affected by higher ethanol levels, which led to an increased extraction of wood-derived compounds. Furthermore, the distinct microbial communities also induced changes in the chemical composition of the beer samples, leading to concentration differences in beer- and wood-derived compounds like 4-ethyl guaiacol, 4-ethyl phenol, cis-oak lactone, vanillin, furfural and 5-hydroxymethyl furfural. Altogether, our results indicate that wood-aging of beer is affected by biotic and abiotic parameters, influencing the quality of the final product. Additionally, this work provides a new, cost-effective approach to study the production of barrel-aged beers based on a simplified microbial community model.

An Integrated Approach Reveals DNA Damage and Proteotoxic Stress as Main Effects of Proton Radiation in S. cerevisiae.

Proton radiotherapy (PRT) has the potential to reduce the normal tissue toxicity associated with conventional photon-based radiotherapy (X-ray therapy, XRT) because the active dose can be more directly targeted to a tumor. Although this dosimetric advantage of PRT is well known, the molecular mechanisms affected by PRT remain largely elusive. Here, we combined the molecular toolbox of the eukaryotic model Saccharomyces cerevisiae with a systems biology approach to investigate the physiological effects of PRT compared to XRT. Our data show that the DNA damage response and protein stress response are the major molecular mechanisms activated after both PRT and XRT. However, RNA-Seq revealed that PRT treatment evoked a stronger activation of genes involved in the response to proteotoxic stress, highlighting the molecular differences between PRT and XRT. Moreover, inhibition of the proteasome resulted in decreased survival in combination with PRT compared to XRT, not only further confirming that protons induced a stronger proteotoxic stress response, but also hinting at the potential of using proteasome inhibitors in combination with proton radiotherapy in clinical settings.