Energy Center of Wisconsin

Isobutanol is a fusel alcohol that can be produced microbially for use as a biofuel or upgraded into sustainable aviation fuel (SAF). A key enzyme in the isobutanol biosynthetic pathway is 2-ketoacid decarboxylase (KDC), which irreversibly decarboxylates 2-ketoisovalerate (KIV) to yield isobutyraldehyde. However, many previously characterized KDC enzymes also act promiscuously on other 2-ketoacids, (e.g., pyruvate) to produce a related aldehyde (e.g., acetaldehyde). This unwanted side reaction is especially important when isobutanol is produced in Saccharomyces cerevisiae (S. cerevisiae) because it leads to pyruvate being diverted to ethanol. In order to make S. cerevisiae a strict isobutanologen, a KDC enzyme that is specific for KIV must be deployed. In this study, we used a combination of cell-based and in vitro enzyme assays to investigate KDC substrate specificity, characterizing a large set of homologs for KIV, pyruvate, and phenylpyruvate (PPV) activity. A diverse range of substrate specificities was discovered, and some previously uncharacterized KDCs were revealed to have high KIV activity and low pyruvate activity. Multi-site saturation mutagenesis (SSM) of one of these KDCs identified mutants with increased KIV activity, while maintaining low levels of pyruvate activity. In a KIV bioconversion experiment, bioprospected and engineered KDCs allowed similar KIV consumption to when using the previously characterized Lactococcus lactis KdcA, though with some ethanol also produced. The KDCs identified here show promise for production of isobutanol and other alcohols derived from 2-ketoacids, and the dataset of newly characterized KDCs can inform future efforts to understand and engineer substrate specificity in KDCs.

Candida albicans is a major human fungal pathogen that reproduces by mitotic growth of diploid cells but can also undergo a parasexual cycle. The latter involves an uncoordinated process of ploidy reduction via aberrant mitotic divisions following the fusion of opposite- or same-sex cells. The ability of parasex to recapitulate the genome plasticity of meiosis remains largely unclear. Here we induced parasexual crosses between genetically distinct strains and analysed the genotypic make-up of parasexual progeny following whole-genome sequencing or selective genotyping to determine the genomic landscape generated by the parasexual cycle. Our results show that C. albicans parasex leads to high levels of recombination and chromosome shuffling as observed in a conventional meiosis. Parasexual progeny also showed diversity in the pathogenesis-related phenotypes of filamentation, drug response and in vivo fitness that was associated with variation in inherited, recombinant genotypes. We propose that the parasexual cycle in non-meiotic eukaryotes can enable escape from a purely asexual mode of reproduction and confer the ability to rapidly adapt to new or changing host niches among commensal and pathogenic species.

The production of difructose anhydride (DFA) from fructose under mild conditions, in a green/bio-derived solvent. Crystal structure created with VESTA, J. Appl. Crystallogr. , 2011, 44 , 1272–1276.

Codon usage bias, or the unequal use of synonymous codons, is observed across genes, genomes, and between species. The biased use of synonymous codons has been implicated in many cellular functions, such as translation dynamics and transcript stability, but can also be shaped by neutral forces. The Saccharomycotina, the fungal subphylum containing the yeasts Saccharomyces cerevisiae and Candida albicans , has been a model system for studying codon usage. We characterized codon usage across 1,154 strains from 1,051 species to gain insight into the biases, molecular mechanisms, evolution, and genomic features contributing to codon usage patterns across the subphylum. We found evidence of a general preference for A/T-ending codons and correlations between codon usage bias, GC content, and tRNA-ome size. Codon usage bias is also distinct between the 12 orders within the subphylum to such a degree that yeasts can be classified into orders with an accuracy greater than 90% using a machine learning algorithm trained on codon usage. We also characterized the degree to which codon usage bias is impacted by translational selection. Interestingly, the degree of translational selection was influenced by a combination of genome features and assembly metrics that included the number of coding sequences, BUSCO count, and genome length. Our analysis also revealed an extreme bias in codon usage in the Saccharomycodales associated with a lack of predicted arginine tRNAs. The order contains 24 species, and 23 are computationally predicted to lack tRNAs that decode CGN codons, leaving only the AGN codons to encode arginine. Analysis of Saccharomycodales gene expression, tRNA sequences, and codon evolution suggests that extreme avoidance of the CGN codons is associated with a decline in arginine tRNA function. Codon usage bias within the Saccharomycotina is generally consistent with previous investigations in fungi, which show a role for both genomic features and GC bias in shaping codon usage. However, we find cases of extreme codon usage preference and avoidance along yeast lineages, suggesting additional forces may be shaping the evolution of specific codons.

Abstract Plastic surgery relies heavily on clinical photography to document preoperative and postoperative changes, visualize surgical approaches, and evaluate outcomes. However, the contemporary landscape of plastic surgery photography faces challenges, including a lapse in standards due to the prevalence of smartphones, social media platforms, and security concerns related to data storage and cyberattacks. In this comprehensive review, the authors aim to provide plastic surgeons with practical guidelines for achieving standardized, high-quality clinical photography while navigating the evolving landscape of technology, security, and ethical considerations. We explore the security challenges associated with storing clinical photographs, emphasizing the legal obligations under the Health Insurance Portability and Accountability Act (HIPAA). We also discuss various storage options, including HIPAA-compliant cloud services, electronic medical records, and emerging technologies like blockchain and artificial intelligence. Level of Evidence: 5

Siderophores are crucial for iron-scavenging in microorganisms. While many yeasts can uptake siderophores produced by other organisms, they are typically unable to synthesize siderophores themselves. In contrast, Wickerhamiella/Starmerella (W/S) clade yeasts gained the capacity to make the siderophore enterobactin following the remarkable horizontal acquisition of a bacterial operon enabling enterobactin synthesis. Yet, how these yeasts absorb the iron bound by enterobactin remains unresolved. Here, we demonstrate that Enb1 is the key enterobactin importer in the W/S-clade species Starmerella bombicola. Through phylogenomic analyses, we show that ENB1 is present in all W/S clade yeast species that retained the enterobactin biosynthetic genes. Conversely, it is absent in species that lost the ent genes, except for Starmerella stellata, making this species the only cheater in the W/S clade that can utilize enterobactin without producing it. Through phylogenetic analyses, we infer that ENB1 is a fungal gene that likely existed in the W/S clade prior to the acquisition of the ent genes and subsequently experienced multiple gene losses and duplications. Through phylogenetic topology tests, we show that ENB1 likely underwent horizontal gene transfer from an ancient W/S clade yeast to the order Saccharomycetales, which includes the model yeast Saccharomyces cerevisiae, followed by extensive secondary losses. Taken together, these results suggest that the fungal ENB1 and bacterial ent genes were cooperatively integrated into a functional unit within the W/S clade that enabled adaptation to iron-limited environments. This integrated fungal-bacterial circuit and its dynamic evolution determines the extant distribution of yeast enterobactin producers and cheaters.

Journal Article National Network for Cryo-Electron Tomography Application Portal Get access Matt Larson, Matt Larson UW-Madison Cryo-Electron Research Center, Department of Biochemistry, UW-Madison, Madison, WI, United StatesMidwest Center for Cryo-Electron Tomography, Department of Biochemistry, UW-Madison, Madison, WI, United States Search for other works by this author on: Oxford Academic Google Scholar Keith Thompson, Keith Thompson UW-Madison Cryo-Electron Research Center, Department of Biochemistry, UW-Madison, Madison, WI, United StatesMidwest Center for Cryo-Electron Tomography, Department of Biochemistry, UW-Madison, Madison, WI, United States Search for other works by this author on: Oxford Academic Google Scholar Elizabeth Wright Elizabeth Wright UW-Madison Cryo-Electron Research Center, Department of Biochemistry, UW-Madison, Madison, WI, United StatesMidwest Center for Cryo-Electron Tomography, Department of Biochemistry, UW-Madison, Madison, WI, United StatesDepartment of Biochemistry, UW-Madison, Madison, WI, United StatesMorgridge Institute for Research, Madison, WI, United States Corresponding author: erwright2@wisc.edu Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 28, Issue S1, 1 August 2022, Pages 3216–3217, https://doi.org/10.1017/S1431927622011965 Published: 01 August 2022

Abstract Yeasts have broad importance as industrially and clinically relevant microbes and as powerful models for fundamental research, but we are only beginning to understand the roles yeasts play in natural ecosystems. Yeast ecology is often more difficult to study compared to other, more abundant microbes, but growing collections of natural yeast isolates are beginning to shed light on fundamental ecological questions. Here we used environmental sampling and isolation to assemble a dataset of 1,962 isolates collected from throughout the contiguous United States of America (USA) and Alaska, which were then used to uncover geographic patterns, along with substrate and temperature associations among yeast taxa. We found some taxa, including the common yeasts Torulaspora delbrueckii and Saccharomyces paradoxus, to be repeatedly isolated from multiple sampled regions of the US, and we classify these as broadly distributed cosmopolitan yeasts. A number of yeast taxon - substrate associations were identified, some of which were novel and some of which support previously reported associations. Further, we found a strong effect of isolation temperature on the phyla of yeasts recovered, as well as for many species. We speculate that substrate and isolation temperature associations reflect the ecological diversity of and niche partitioning by yeast taxa.Take AwayAnalysis of environmental metadata of nearly 2,000 yeast isolates.Individual yeast taxa associate with specific substrates and plant genera.Optimal yeast isolation temperature differs depending on taxonomic rank.Substrate type and isolation temperatures affect isolated yeast diversity.

Fatty acids play many important roles in cells and also in industrial processes. Furan fatty acids (FuFAs) are present in the lipids of some plant, fish, and microbial species and appear to function as second messengers in pathways that protect cells from membrane-damaging agents. We report here the results of chemical, genetic, and synthetic biology experiments to decipher the biosynthesis of the monomethylated FuFA, methyl 9-(3-methyl-5-pentylfuran-2-yl) nonanoate (9M5-FuFA), and its dimethyl counterpart, methyl 9-(3,4-dimethyl-5-pentylfuran-2-yl) nonanoate (9D5-FuFA), in two α-proteobacteria. Each of the steps in FuFA biosynthesis occurs on pre-existing phospholipid fatty acid chains, and we identified pathway intermediates and the gene products that catalyze 9M5-FuFA and 9D5-FuFA synthesis in Rhodobacter sphaeroides 2.4.1 and Rhodopseudomonas palustris CGA009. One previously unknown pathway intermediate was a methylated diunsaturated fatty acid, (10E,12E)-11-methyloctadeca-10,12-dienoic acid (11Me-10t,12t-18:2), produced from (11E)-methyloctadeca-11-enoic acid (11Me-12t-18:1) by a newly identified fatty acid desaturase, UfaD. We also show that molecular oxygen (O2) is the source of the oxygen atom in the furan ring of 9M5-FuFA, and our findings predict that an O2-derived oxygen atom is incorporated into 9M5-FuFA via a protein, UfaO, that uses the 11Me-10t,12t-18:2 fatty acid phospholipid chain as a substrate. We discovered that R. palustris also contains a SAM-dependent methylase, FufM, that produces 9D5-FuFA from 9M5-FuFA. These results uncover the biochemical sequence of intermediates in a bacterial pathway for 9M5-FuFA and 9D5-FuFA biosynthesis and suggest the existence of homologs of the enzymes identified here that could function in FuFA biosynthesis in other organisms.