Enhancer architecture and chromatin accessibility constrain phenotypic space during Drosophila development.

Developmental enhancers bind transcription factors and dictate patterns of gene expression during development. Their molecular evolution can underlie phenotypical evolution, but the contributions of the evolutionary pathways involved remain little understood. Here, using mutation libraries in Drosophila melanogaster embryos, we observed that most point mutations in developmental enhancers led to changes in gene expression levels but rarely resulted in novel expression outside of the native pattern. In contrast, random sequences, often acting as developmental enhancers, drove expression across a range of cell types; random sequences including motifs for transcription factors with pioneer activity acted as enhancers even more frequently. Our findings suggest that the phenotypic landscapes of developmental enhancers are constrained by enhancer architecture and chromatin accessibility. We propose that the evolution of existing enhancers is limited in its capacity to generate novel phenotypes, whereas the activity of de novo elements is a primary source of phenotypic novelty.

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.