Coproduction of Phase-Separated Carotenoids and ß-Farnesene as a Yeast Biomass Valorization Strategy.

Valorization, the process whereby waste materials are converted into more valuable products, is rarely practiced in industrial fermentation. We developed a model valorization system whereby Saccharomyces cerevisiae that had previously been engineered to produce high concentrations (>100 g/L) of extracellular ß-farnesene was further engineered to simultaneously produce intracellular carotenoids, both products being isoprenoids. Thus, a single fermentation generates two valuable products, namely, ß-farnesene in the liquid phase and carotenoids in the solid biomass phase. Initial attempts to produce high levels of canthaxanthin (a ketocarotenoid used extensively in animal feed) in a ß-farnesene production strain negatively impacted both biomass growth and ß-farnesene production. A refined approach used a promoter titration strategy to reduce ß-carotene production to a level that had minimal impact on growth and ß-farnesene production in fed-batch fermentations and then engineered the resulting strain to produce canthaxanthin. Further optimization of canthaxanthin coproduction used a bioprospecting approach to identify ketolase enzymes that maximized conversion of ß-carotene to canthaxanthin. Finally, we demonstrated that ß-carotene is not present in the extracellular ß-farnesene at a significant concentration and that which is present can be removed by a simple distillation, indicating that ß-farnesene (the primary fermentation product) purity is unaffected by coproduction of carotenoids.

Cut the noise or couple up: Coordinating circadian and synthetic clocks.

Circadian clocks are important to much of life on Earth and are of inherent interest to humanity, implicated in fields ranging from agriculture and ecology to developmental biology and medicine. New techniques show that it is not simply the presence of clocks, but coordination between them that is critical for complex physiological processes across the kingdoms of life. Recent years have also seen impressive advances in synthetic biology to the point where parallels can be drawn between synthetic biological and circadian oscillators. This review will emphasize theoretical and experimental studies that have revealed a fascinating dichotomy of coupling and heterogeneity among circadian clocks. We will also consolidate the fields of chronobiology and synthetic biology, discussing key design principles of their respective oscillators.

Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain.

Bacterial cellulose is a strong and ultrapure form of cellulose produced naturally by several species of the Acetobacteraceae Its high strength, purity, and biocompatibility make it of great interest to materials science; however, precise control of its biosynthesis has remained a challenge for biotechnology. Here we isolate a strain of Komagataeibacter rhaeticus (K. rhaeticus iGEM) that can produce cellulose at high yields, grow in low-nitrogen conditions, and is highly resistant to toxic chemicals. We achieved external control over its bacterial cellulose production through development of a modular genetic toolkit that enables rational reprogramming of the cell. To further its use as an organism for biotechnology, we sequenced its genome and demonstrate genetic circuits that enable functionalization and patterning of heterologous gene expression within the cellulose matrix. This work lays the foundations for using genetic engineering to produce cellulose-based materials, with numerous applications in basic science, materials engineering, and biotechnology.

Longitudinal Analysis of DNA Methylation in CD34+ Hematopoietic Progenitors in Myelodysplastic Syndrome.

Myelodysplastic syndrome (MDS) is a disorder of hematopoietic stem cells (HSCs) that is often treated with DNA methyltransferase 1 (DNMT1) inhibitors (5-azacytidine [AZA], 5-aza-2'-deoxycytidine), suggesting a role for DNA methylation in disease progression. How DNMT inhibition retards disease progression and how DNA methylation contributes to MDS remain unclear. We analyzed global DNA methylation in purified CD34+ hematopoietic progenitors from MDS patients undergoing multiple rounds of AZA treatment. Differential methylation between MDS phenotypes was observed primarily at developmental regulators not expressed within the hematopoietic compartment and was distinct from that observed between healthy hematopoietic cell types. After AZA treatment, we observed only limited DNA demethylation at sites that varied between patients. This suggests that a subset of the stem cell population is resistant to AZA and provides a basis for disease relapse. Using gene expression data from patient samples and an in vitro AZA treatment study, we identified differentially methylated genes that can be activated following treatment and that remain silent in the CD34+ stem cell compartment of high-risk MDS patients. Haploinsufficiency in mice of one of these genes (NR4A2) has been shown to lead to excessive HSC proliferation, and our data suggest that suppression of NR4A2 by DNA methylation may be involved in MDS progression.