Transcriptomics elucidates metabolic regulation and functional promoters in the basidiomycete red yeast Xanthophyllomyces dendrorhous CBS 6938

Emma E. Tobin, Joseph H. Collins, Celeste B. Marsan, Gillian T. Nadeau, Kim Mori, Anna Lipzen, Stephen Mondo, Igor V. Grigoriev, Eric M. Young

Genomics has become the primary way to explore microbial diversity, because genetic tools are currently difficult to develop in non-model organisms. Here, we demonstrate that -omics can be leveraged to accelerate genetic tool development for the basidiomycete yeast Xanthophyllomyces dendrorhous CBS 6938, the sole biotechnologically relevant organism in the Tremellomycete family. First, we sequence the genome. Then, we perform transcriptomics under a variety of conditions, focusing on light and oxidative stress. This data not only reveals novel photobiology and metabolic regulation, it also allows derivation of constitutive and regulated gene expression parts. Our analysis of X. dendrorhous photobiology shows for the first time that a complex system of white-collar and cryptochrome homologs mediate response to ultraviolet light (UV). Our analysis of metabolic regulation shows that UV activates DNA repair, aromatic amino acid and carotenoid biosynthesis and represses central carbon metabolism and the fungal-like apoptotic pathway. Thus, X. dendrorhous shows a dynamic response toward biosynthetic pathways for light-absorbing compounds and survival and away from energy production. We then define a modular cloning system, including antibiotic selections, integration sites, and reporter genes, and use the transcriptomics to derive strong constitutive and regulated promoters. Notably, we discover a novel promoter from a hypothetical gene that has 9-fold activation upon UV exposure. Thus, -omics-to-parts workflows can simultaneously provide useful genomic data and advance genetic tools for non-model microbes, particularly those without a closely related model organism. This approach will be broadly useful in current efforts to engineer diverse microbes.

Genome-guided analysis of physiological and morphological traits of the fermentative acetate oxidizer Thermacetogenium phaeum

Dirk Oehler, Anja Poehlein, Andreas Leimbach, Nicolai Müller, Rolf Daniel, Gerhard Gottschalk & Bernhard Schink

Background

Thermacetogenium phaeum is a thermophilic strictly anaerobic bacterium oxidizing acetate to CO2 in syntrophic association with a methanogenic partner. It can also grow in pure culture, e.g., by fermentation of methanol to acetate. The key enzymes of homoacetate fermentation (Wood-Ljungdahl pathway) are used both in acetate oxidation and acetate formation. The obvious reversibility of this pathway in this organism is of specific interest since syntrophic acetate oxidation operates close to the energetic limitations of microbial life.

Results

The genome of Th. phaeum is organized on a single circular chromosome and has a total size of 2,939,057 bp. It comprises 3.215 open reading frames of which 75% could be assigned to a gene function. The G+C content is 53.88 mol%. Many CRISPR sequences were found, indicating heavy phage attack in the past. A complete gene set for a phage was found in the genome, and indications of phage action could also be observed in culture. The genome contained all genes required for CO2 reduction through the Wood-Ljungdahl pathway, including two formyl tetrahydrofolate ligases, three carbon monoxide dehydrogenases, one formate hydrogenlyase complex, three further formate dehydrogenases, and three further hydrogenases. The bacterium contains a menaquinone MQ-7. No indications of cytochromes or Rnf complexes could be found in the genome.

Conclusions

The information obtained from the genome sequence indicates that Th. phaeum differs basically from the three homoacetogenic bacteria sequenced so far, i.e., the sodium ion-dependent Acetobacterium woodii, the ethanol-producing Clostridium ljungdahlii, and the cytochrome-containing Moorella thermoacetica. The specific enzyme outfit of Th. phaeum obviously allows ATP formation both in acetate formation and acetate oxidation.