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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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Untargeted GC-MS Metabolic Profiling of Anaerobic Gut Fungi Reveals Putative Terpenoids and Strain-Specific Metabolites

Background/Objectives: Anaerobic gut fungi (Neocallimastigomycota) are biotechnologically relevant, lignocellulose-degrading microbes with under-explored biosynthetic potential for secondary metabolites. Untargeted metabolomic profiling with gas chromatography–mass spectrometry (GC-MS) was applied to two gut fungal strains, Anaeromyces robustus and Caecomyces churrovis, to establish a foundational metabolomic dataset to identify metabolites and provide insights into gut fungal metabolic capabilities. Methods: Gut fungi were cultured anaerobically in rumen-fluid-based media with a soluble substrate (cellobiose), and metabolites were extracted using the Metabolite, Protein, and Lipid Extraction (MPLEx) method, enabling metabolomic and proteomic analysis from the same cell samples. Samples were derivatized and analyzed via GC-MS, followed by compound identification by spectral matching to reference databases, molecular networking, and statistical analyses. Results: Distinct metabolites were identified between A. robustus and C. churrovis, including 2,3-dihydroxyisovaleric acid produced by A. robustus and maltotriitol, maltotriose, and melibiose produced by C. churrovis. C. churrovis may polymerize maltotriose to form an extracellular polysaccharide, like pullulan. GC-MS profiling potentially captured sufficiently volatile products of proteomically detected, putative non-ribosomal peptide synthetases and polyketide synthases of A. robustus and C. churrovis. The triterpene squalene and triterpenoid tetrahymanol were putatively identified in A. robustus and C. churrovis. Their conserved, predicted biosynthetic genes—squalene synthase and squalene tetrahymanol cyclase—were identified in A. robustus, C. churrovis, and other anaerobic gut fungal genera. Conclusions: This study provides a foundational, untargeted metabolomic dataset to unmask gut fungal metabolic pathways and biosynthetic potential and to prioritize future efforts for compound isolation and identification.

Biochemistry & Molecular Biology↗

Amylases in the Human Vagina

ABSTRACT Dominance of Lactobacillus species in vaginal communities is a hallmark of healthy conditions in the female genital tract. Key nutrients for lactobacilli include sugars produced when glycogen is degraded by α-amylase in the vagina. While α-amylase activity has been demonstrated in vaginal fluids, it is unclear whether α-amylases are produced solely by the host, bacteria in the vagina, or both. We screened cervicovaginal mucus from 23 reproductive-age women, characterized the species composition of vaginal communities, measured vaginal pH, and determined levels of amylase activity, glycogen, and lactic acid. Based on differences in these measured variables, one sample from each of four individual donors was selected for metagenomic and proteomic analyses. Of eight putative bacterial amylases identified in the assembled bacterial metagenomes, we detected four in vaginal fluids. These amylases were produced by various bacteria in different vaginal communities. Moreover, no two communities were the same in terms of which bacteria were producing amylases. Although we detected bacterial amylases in vaginal fluids, there was no clear association between the bacterial species that was dominant in a community and the level of amylase activity. This association was likely masked by the presence of human α-amylase, which was also detected in vaginal fluids. Finally, the levels of amylase activity and glycogen were only weakly associated. Our findings show, for the first time, that multiple amylases from both bacterial and human origins can be present simultaneously in the vagina. This work also suggests that the link between glycogen, amylase, and Lactobacillus in the vagina is complex. IMPORTANCE In this study, we show that multiple bacteria in the vaginal community produce amylases that hydrolyze glycogen into simpler sugars (i.e., maltose and maltotriose). These sugars serve as “common goods” that sustain bacterial populations in vaginal communities. Given the temporal changes that are observed in the human vaginal microbiome, we expect the kinds of bacterial amylases produced will also vary over time. These differences influence the pool of resources that are broadly shared and shape the species composition of the vaginal bacterial community.

59 BASIC BIOLOGICAL SCIENCES↗

Polypeptide and yeast cell compositions and methods of using the same

Polypeptides comprising maltose/maltotriose transporters are provided. Additionally, polynucleotides, DNA constructs, and vectors encoding a maltose/maltotriose transporter, or yeast cells harboring such polynucleotides are provided. The yeast cell may be a Saccharomyces eubayanus cell modified to increase the expression or transport activity of a maltose/maltotriose transporter at the plasma membrane of the cell. Further, methods are provided for making a fermentation product by culturing any one of the yeast cells described herein with a fermentable substrate. Finally, methods are provided to select for and isolate maltotriose-utilizing strains of Saccharomyces eubayanus.

Hittinger, Chris Todd↗

Polypeptide and yeast cell compositions and methods of using the same

Polypeptides comprising maltose/maltotriose transporters are provided. Additionally, polynucleotides, DNA constructs, and vectors encoding a maltose/maltotriose transporter, or yeast cells harboring such polynucleotides are provided. The yeast cell may be a Saccharomyces eubayanus cell modified to increase the expression or transport activity of a maltose/maltotriose transporter at the plasma membrane of the cell. Further, methods are provided for making a fermentation product by culturing any one of the yeast cells described herein with a fermentable substrate. Finally, methods are provided to select for and isolate maltotriose-utilizing strains of Saccharomyces eubayanus .

Hittinger, Chris Todd↗

Specialization Restricts the Evolutionary Paths Available to Yeast Sugar Transporters

Functional innovation at the protein level is a key source of evolutionary novelties. The constraints on functional innovations are likely to be highly specific in different proteins, which are shaped by their unique histories and the extent of global epistasis that arises from their structures and biochemistries. These contextual nuances in the sequence–function relationship have implications both for a basic understanding of the evolutionary process and for engineering proteins with desirable properties. Here, we have investigated the molecular basis of novel function in a model member of an ancient, conserved, and biotechnologically relevant protein family. These Major Facilitator Superfamily sugar porters are a functionally diverse group of proteins that are thought to be highly plastic and evolvable. By dissecting a recent evolutionary innovation in an α-glucoside transporter from the yeast Saccharomyces eubayanus, we show that the ability to transport a novel substrate requires high-order interactions between many protein regions and numerous specific residues proximal to the transport channel. To reconcile the functional diversity of this family with the constrained evolution of this model protein, we generated new, state-of-the-art genome annotations for 332 Saccharomycotina yeast species spanning ~400 My of evolution. By integrating phylogenetic and phenotypic analyses across these species, we show that the model yeast α-glucoside transporters likely evolved from a multifunctional ancestor and became subfunctionalized. The accumulation of additive and epistatic substitutions likely entrenched this subfunction, which made the simultaneous acquisition of multiple interacting substitutions the only reasonably accessible path to novelty.

59 BASIC BIOLOGICAL SCIENCES↗