Methanol synthesized from captured greenhouse gases is an emerging renewable feedstock with great potential for bioproduction. Recent research has raised the prospect of methanol bioconversion to value-added products using synthetic methylotrophic Escherichia coli, as its metabolism can be rewired to enable growth solely on the reduced one-carbon compound. Here we describe the generation of an E. coli strain that grows on methanol at a doubling time of 4.3 h—comparable to many natural methylotrophs. To establish bioproduction from methanol using this synthetic chassis, we demonstrate biosynthesis from four metabolic nodes from which numerous bioproducts can be derived: lactic acid from pyruvate, polyhydroxybutyrate from acetyl coenzyme A, itaconic acid from the tricarboxylic acid cycle and p-aminobenzoic acid from the chorismate pathway. In a step towards carbon-negative chemicals and valorizing greenhouse gases, our work brings synthetic methylotrophy in E. coli within reach of industrial applications.

Microgravity acts on cellular systems on several levels. Cells of the immune system especially react rapidly to changes in gravity. In this study, we performed a correlative metabolomics analysis on short-term and long-term microgravity effects on primary human macrophages. We could detect an increased amino acid concentration after five minutes of altered gravity, that was inverted after 11 days of microgravity. The amino acids that reacted the most to changes in gravity were tightly clustered. The observed effects indicated protein degradation processes in microgravity. Further, glucogenic and ketogenic amino acids were further degraded to Glucose and Ketoleucine. The latter is robustly accumulated in short-term and long-term microgravity but not in hypergravity. We detected highly dynamic and also robust adaptative metabolic changes in altered gravity. Metabolomic studies could contribute significantly to the understanding of gravity-induced integrative effects in human cells.

The transcriptome of human immune cells rapidly reacts to altered gravity in a highly dynamic way. We could show in previous experiments that transcriptional patterns show profound adaption after seconds to minutes of altered gravity. To gain further insight into these transcriptional alteration and adaption dynamics, we conducted a highly standardized RNA-Seq experiment with human Jurkat T cells exposed to 9xg hypergravity for 3 and 15 min, respectively. We investigated the frequency with which individual exons were used during transcription and discovered that differential exon usage broadly appeared after 3 min and became less pronounced after 15 min. Additionally, we observed a shift in the transcript pool from coding towards non-coding transcripts. Thus, adaption of gravity-sensitive differentially expressed genes followed a dynamic transcriptional rebound effect. The general dynamics were compatible with previous studies on the transcriptional effects of short hypergravity on human immune cells and suggest that initial up-regulatory changes mostly result from increased elongation rates. The shift correlated with a general downregulation of the affected genes. All chromosome bands carried homogenous numbers of gravity-sensitive genes but showed a specific tendency towards up- or downregulation. Altered gravity affected transcriptional regulation throughout the entire genome, whereby the direction of differential expression was strongly dependent on the structural location in the genome. A correlation analysis with potential mediators of the early transcriptional response identified a link between initially upregulated genes with certain transcription factors. Based on these findings, we have been able to further develop our model of the transcriptional response to altered gravity.

Synthetic methylotrophy offers opportunities for sustainable chemical and biofuel production. While recently established methylotrophic E. coli can grow on methanol, undesirable formate accumulation occurs during growth and bioproduction. Here, we show that NAD-dependent methanol dehydrogenase Mdh2 from Cupriavidus necator inherently overoxidizes methanol to formate, a trait we find to be widespread among NAD-dependent Mdh enzymes. In contrast, Mdh/Mdh1 enzymes from Bacillus methanolicus exclusively oxidize methanol to formaldehyde without overoxidation, as we validate in vitro for Mdh Bm MGA3 with and without activator protein Act. Since only formaldehyde is assimilated via the ribulose monophosphate pathway, this explains the physiological role of Mdh/Mdh1 paralogs in natural methylotrophs and highlights the importance of selecting appropriate Mdh variants for synthetic methylotrophy. We demonstrate methanol-dependent growth using non-overoxidizing Mdh Bm MGA3, strongly reducing formate accumulation and carbon loss. Our findings reveal a characteristic of NAD-dependent Mdh enzymes and provide insights for engineering synthetic methylotrophs.

Cellular effects of hypergravity have been described in many studies. We investigated the transcriptional dynamics in Jurkat T cells between 20 s and 60 min of 9 g hypergravity and characterized a highly dynamic biphasic time course of gene expression response with a transition point between rapid adaptation and long-term response at approximately 7 min. Upregulated genes were shifted towards the center of the nuclei, whereby downregulated genes were shifted towards the periphery. Upregulated gene expression was mostly located on chromosomes 16–22. Protein-coding transcripts formed the majority with more than 90% of all differentially expressed genes and followed a continuous trend of downregulation, whereas retained introns demonstrated a biphasic time-course. The gene expression pattern of hypergravity response was not comparable with other stress factors such as oxidative stress, heat shock or inflammation. Furthermore, we tested a routine centrifugation protocol that is widely used to harvest cells for subsequent RNA analysis and detected a huge impact on the transcriptome compared to non-centrifuged samples, which did not return to baseline within 15 min. Thus, we recommend carefully studying the response of any cell types used for any experiments regarding the hypergravity time and levels applied during cell culture procedures and analysis.