Xanthobacter-dominated biofilm as a novel source for high-value rhamnose.

Rhamnose is a high-value carbohydrate used in flavorings, aromatics, and pharmaceuticals. Current demand for rhamnose is filled through plant-based sources; however, microbially originated rhamnolipids have been proposed as an alternative source. A mixed microbial biofilm, cultured from a wastewater sludge, was found to comprise > 8 dry weight% rhamnose when provided volatile fatty acids as carbon source, and 24 dry weight% when given glucose. The latter rhamnose concentration is a fourfold higher production mass than the current plant-based origin and is competitive with yields from pure microbial cultures. The biofilm was characterized based on total carbohydrate production at varying nutrient levels, individual carbohydrate monomer production from varying organic acid substrates, and microbial community composition-based on 16s rRNA. Biofilm carbohydrate production was maximized at a C:N ratio of 28 (mol:mol). The production of rhamnose varied significantly based on carbon substrate; glucose had the greatest yield of rhamnose, followed by propionic acid, lactic acid, acetic acid, valeric acid, and butyric acid. Microbial community analysis indicated an abundance of organisms within the Xanthobacter genus, which is known to produce rhamnose as zeaxanthin rhamnoside. Rhamnose production was heavily correlated with ribose production (R2 = 0.96). Results suggest that mixed microbial biofilms could be a competitive source of monomeric rhamnose that may be produced from mixed organic waste streams of variable composition via volatile fatty acids and glucose.

A Genetic Engineering Toolbox for the Lignocellulolytic Anaerobic Gut Fungus Neocallimastix frontalis.

Anaerobic fungi are powerful platforms for biotechnology that remain unexploited due to a lack of genetic tools. These gut fungi encode the largest number of lignocellulolytic carbohydrate active enzymes (CAZymes) in the fungal kingdom, making them attractive for applications in renewable energy and sustainability. However, efforts to genetically modify anaerobic fungi have remained limited due to inefficient methods for DNA uptake and a lack of characterized genetic parts. We demonstrate that anaerobic fungi are naturally competent for DNA and leverage this to develop a nascent genetic toolbox informed by recently acquired genomes for transient transformation of anaerobic fungi. We validate multiple selectable markers (HygR and Neo), an anaerobic reporter protein (iRFP702), enolase and TEF1A promoters, TEF1A terminator, and a nuclear localization tag for protein compartmentalization. This work establishes novel methods to reliably transform the anaerobic fungus Neocallimastix frontalis, thereby paving the way for strain development and various synthetic biology applications.

Repurposing the Homing Endonuclease I-SceI for Positive Selection and Development of Gene-Editing Technologies.

Prokaryote genomes encode diverse programmable DNA endonucleases with significant potential for biotechnology and gene editing. However, these endonucleases differ significantly in their properties, which must be screened and measured. While positive selection screens based on ccdB and barnase have been developed to evaluate such proteins, their high levels of toxicity make them challenging to use. Here, we develop and validate a more robust positive selection screen based on the homing endonuclease I-SceI. Candidate endonucleases target and cure the I-SceI expression plasmid preventing induction of I-SceI-mediated double strand DNA breaks that lead to cell death in E. coli. We validated this screen to measure the relative activity of SpCas9, xCas9, and eSpCas9 and demonstrated an ability to enrich for more active endonuclease variants from a mixed population. This system may be applied in high throughput to rapidly characterize novel programmable endonucleases and be adapted for directed evolution of endonuclease function.

Anaerobic Fungal Mevalonate Pathway Genomic Biases Lead to Heterologous Toxicity Underpredicted by Codon Adaptation Indices.

Anaerobic fungi are emerging biotechnology platforms with genomes rich in biosynthetic potential. Yet, the heterologous expression of their biosynthetic pathways has had limited success in model hosts like E. coli. We find one reason for this is that the genome composition of anaerobic fungi like P. indianae are extremely AT-biased with a particular preference for rare and semi-rare AT-rich tRNAs in E coli, which are not explicitly predicted by standard codon adaptation indices (CAI). Native P. indianae genes with these extreme biases create drastic growth defects in E. coli (up to 69% reduction in growth), which is not seen in genes from other organisms with similar CAIs. However, codon optimization rescues growth, allowing for gene evaluation. In this manner, we demonstrate that anaerobic fungal homologs such as PI.atoB are more active than S. cerevisiae homologs in a hybrid pathway, increasing the production of mevalonate up to 2.5 g/L (more than two-fold) and reducing waste carbon to acetate by ~90% under the conditions tested. This work demonstrates the bioproduction potential of anaerobic fungal enzyme homologs and how the analysis of codon utilization enables the study of otherwise difficult to express genes that have applications in biocatalysis and natural product discovery.

Hydrolysis of lignocellulose by anaerobic fungi produces free sugars and organic acids for two-stage fine chemical production with Kluyveromyces marxianus.

Development of the bioeconomy is driven by our ability to access the energy-rich carbon trapped in recalcitrant plant materials. Current strategies to release this carbon rely on expensive enzyme cocktails and physicochemical pretreatment, producing inhibitory compounds that hinder subsequent microbial bioproduction. Anaerobic fungi are an appealing solution as they hydrolyze crude, untreated biomass at ambient conditions into sugars that can be converted into value-added products by partner organisms. However, some carbon is lost to anaerobic fungal fermentation products. To improve efficiency and recapture this lost carbon, we built a two-stage bioprocessing system pairing the anaerobic fungus Piromyces indianae with the yeast Kluyveromyces marxianus, which grows on a wide range of sugars and fermentation products. In doing so we produce fine and commodity chemicals directly from untreated lignocellulose. P. indianae efficiently hydrolyzed substrates such as corn stover and poplar to generate sugars, fermentation acids, and ethanol, which K. marxianus consumed while producing 2.4 g/L ethyl acetate. An engineered strain of K. marxianus was also able to produce 550 mg/L 2-phenylethanol and 150 mg/L isoamyl alcohol from P. indianae hydrolyzed lignocellulosic biomass. Despite the use of crude untreated plant material, production yields were comparable to optimized rich yeast media due to the use of all available carbon including organic acids, which formed up to 97% of free carbon in the fungal hydrolysate. This work demonstrates that anaerobic fungal pretreatment of lignocellulose can sustain the production of fine chemicals at high efficiency by partnering organisms with broad substrate versatility.

Comparative genomics of the genus Roseburia reveals divergent biosynthetic pathways that may influence colonic competition among species.

Roseburia species are important denizens of the human gut microbiome that ferment complex polysaccharides to butyrate as a terminal fermentation product, which influences human physiology and serves as an energy source for colonocytes. Previous comparative genomics analyses of the genus Roseburia have examined polysaccharide degradation genes. Here, we characterize the core and pangenomes of the genus Roseburia with respect to central carbon and energy metabolism, as well as biosynthesis of amino acids and B vitamins using orthology-based methods, uncovering significant differences among species in their biosynthetic capacities. Variation in gene content among Roseburia species and strains was most significant for cofactor biosynthesis. Unlike all other species of Roseburia that we analysed, Roseburia inulinivorans strains lacked biosynthetic genes for riboflavin or pantothenate but possessed folate biosynthesis genes. Differences in gene content for B vitamin synthesis were matched with differences in putative salvage and synthesis strategies among species. For example, we observed extended biotin salvage capabilities in R. intestinalis strains, which further suggest that B vitamin acquisition strategies may impact fitness in the gut ecosystem. As differences in the functional potential to synthesize components of biomass (e.g. amino acids, vitamins) can drive interspecies interactions, variation in auxotrophies of the Roseburia spp. genomes may influence in vivo gut ecology. This study serves to advance our understanding of the potential metabolic interactions that influence the ecology of Roseburia spp. and, ultimately, may provide a basis for rational strategies to manipulate the abundances of these species.

Hydrolysis of untreated lignocellulosic feedstock is independent of S-lignin composition in newly classified anaerobic fungal isolate, Piromyces sp. UH3-1.

BACKGROUND: Plant biomass is an abundant but underused feedstock for bioenergy production due to its complex and variable composition, which resists breakdown into fermentable sugars. These feedstocks, however, are routinely degraded by many uncommercialized microbes such as anaerobic gut fungi. These gut fungi express a broad range of carbohydrate active enzymes and are native to the digestive tracts of ruminants and hindgut fermenters. In this study, we examine gut fungal performance on these substrates as a function of composition, and the ability of this isolate to degrade inhibitory high syringyl lignin-containing forestry residues. RESULTS: We isolated a novel fungal specimen from a donkey in Independence, Indiana, United States. Phylogenetic analysis of the Internal Transcribed Spacer 1 sequence classified the isolate as a member of the genus Piromyces within the phylum Neocallimastigomycota (Piromyces sp. UH3-1, strain UH3-1). The isolate penetrates the substrate with an extensive rhizomycelial network and secretes many cellulose-binding enzymes, which are active on various components of lignocellulose. These activities enable the fungus to hydrolyze at least 58% of the glucan and 28% of the available xylan in untreated corn stover within 168 h and support growth on crude agricultural residues, food waste, and energy crops. Importantly, UH3-1 hydrolyzes high syringyl lignin-containing poplar that is inhibitory to many fungi with efficiencies equal to that of low syringyl lignin-containing poplar with no reduction in fungal growth. This behavior is correlated with slight remodeling of the fungal secretome whose composition adapts with substrate to express an enzyme cocktail optimized to degrade the available biomass. CONCLUSIONS: Piromyces sp. UH3-1, a newly isolated anaerobic gut fungus, grows on diverse untreated substrates through production of a broad range of carbohydrate active enzymes that are robust to variations in substrate composition. Additionally, UH3-1 and potentially other anaerobic fungi are resistant to inhibitory lignin composition possibly due to changes in enzyme secretion with substrate. Thus, anaerobic fungi are an attractive platform for the production of enzymes that efficiently use mixed feedstocks of variable composition for second generation biofuels. More importantly, our work suggests that the study of anaerobic fungi may reveal naturally evolved strategies to circumvent common hydrolytic inhibitors that hinder biomass usage.

Microbial Ecology along the Gastrointestinal Tract.

The ecosystem of the human gastrointestinal (GI) tract traverses a number of environmental, chemical, and physical conditions because it runs from the oral cavity to the anus. These differences in conditions along with food or other ingested substrates affect the composition and density of the microbiota as well as their functional roles by selecting those that are the most suitable for that environment. Previous studies have mostly focused on Bacteria, with the number of studies conducted on Archaea, Eukarya, and Viruses being limited despite their important roles in this ecosystem. Furthermore, due to the challenges associated with collecting samples directly from the inside of humans, many studies are still exploratory, with a primary focus on the composition of microbiomes. Thus, mechanistic studies to investigate functions are conducted using animal models. However, differences in physiology and microbiomes need to be clarified in order to aid in the translation of animal model findings into the context of humans. This review will highlight Bacteria, Archaea, Fungi, and Viruses, discuss differences along the GI tract of healthy humans, and perform comparisons with three common animal models: rats, mice, and pigs.