A novel D-xylose isomerase from the gut of the wood feeding beetle Odontotaenius disjunctus efficiently expressed in Saccharomyces cerevisiae

Carbohydrate rich substrates such as lignocellulosic hydrolysates remain one of the primary sources of potentially renewable fuel and bulk chemicals. The pentose sugar D-xylose is often present in significant amounts along with hexoses. Saccharomyces cerevisiae can acquire the ability to metabolize D-xylose through expression of heterologous D-xylose isomerase (XI). This enzyme is notoriously difficult to express in S. cerevisiae and only fourteen XIs have been reported to be active so far. We cloned a new D-xylose isomerase derived from microorganisms in the gut of the wood-feeding beetle Odontotaenius disjunctus. Although somewhat homologous to the XI from Piromyces sp. E2, the new gene was identified as bacterial in origin and the host as a Parabacteroides sp. Expression of the new XI in S. cerevisiae resulted in faster aerobic growth than the XI from Piromyces on D-xylose media. The D-xylose isomerization rate conferred by the new XI was also 72% higher, while absolute xylitol production was identical in both strains. Interestingly, increasing concentrations of xylitol (up to 8 g L-1) appeared not to inhibit D-xylose consumption. The newly described XI displayed 2.6 times higher specific activity, 37% lower KM for D-xylose, and exhibited higher activity over a broader temperature range, retaining 51% of maximal activity at 30 °C compared with only 29% activity for the Piromyces XI.This work was supported by the project FatVal PTDC/EAM-AMB/32506/2017 (POCI-01-0145-FEDER-032506), co-funded by the European Regional Development Fund (ERDF), through the Operational Programme for Competitiveness and Internationalization (COMPETE 2020), under Portugal 2020, and by the Fundacao para a Ciencia e a Tecnologia-FCT I.P through national funds. CBMA was supported by the "Contrato-Programa" UIDB/04050/2020 funded by national funds through the FCT I.P. PCS is recipient of a FCT PhD fellowship (SFRH/BD/140039/2018), and was supported by a Fulbright Scholarship Portugal grant from January to May 2020 at Lawrence Berkeley National Laboratory, Berkeley, CA, USA. BJ was awarded a Fulbright grant from The Swedish Fulbright Commission for Visiting Lecturers and Research Scholars between September 2014 and January 2015 visiting Lawrence Berkeley National Laboratory, Berkeley, CA, USA. This work was supported in part by the United States Department of Energy's Genomic Science Program (grant SCW1039). Part of this work was performed at Lawrence Berkeley National Laboratory under US Department of Energy contract number DE-AC02-05CH11231. DNA sequencing was performed at the Vincent J. Coates Genomics Sequencing Laboratory at the University of California Berkeley, supported by NIH S10 Instrumentation grants S10RR029668 and S10RR027303

Niche differentiation is spatially and temporally regulated in the rhizosphere.

The rhizosphere is a hotspot for microbial carbon transformations, and is the entry point for root polysaccharides and polymeric carbohydrates that are important precursors to soil organic matter (SOM). However, the ecological mechanisms that underpin rhizosphere carbohydrate depolymerization are poorly understood. Using Avena fatua, a common annual grass, we analyzed time-resolved metatranscriptomes to compare microbial functions in rhizosphere, detritusphere, and combined rhizosphere-detritusphere habitats. Transcripts were binned using a unique reference database generated from soil isolate genomes, single-cell amplified genomes, metagenomes, and stable isotope probing metagenomes. While soil habitat significantly affected both community composition and overall gene expression, the succession of microbial functions occurred at a faster time scale than compositional changes. Using hierarchical clustering of upregulated decomposition genes, we identified four distinct microbial guilds populated by taxa whose functional succession patterns suggest specialization for substrates provided by fresh growing roots, decaying root detritus, the combination of live and decaying root biomass, or aging root material. Carbohydrate depolymerization genes were consistently upregulated in the rhizosphere, and both taxonomic and functional diversity were highest in the combined rhizosphere-detritusphere, suggesting coexistence of rhizosphere guilds is facilitated by niche differentiation. Metatranscriptome-defined guilds provide a framework to model rhizosphere succession and its consequences for soil carbon cycling

A novel D-xylose isomerase from the gut of the wood feeding patent-leather beetle Odontotaenius disjunctus

D-Xylose Isomerase (XI) is a key enzyme for the metabolism of D-xylose in renewable carbohydrate rich feedstocks such as lignocellulosic hydrolysates. The widely used industrial organism baker’s yeast Saccharomyces cerevisiae can metabolize xylose upon heterologous expression of this enzyme. This enzyme is notoriously difficult to express in S. cerevisiae and only about ten active genes are known from prokaryotic and eukaryotic sources. We cloned a new XI from microorganisms in the gut of the wood feeding beetle Odontotaenius disjunctus. The new enzyme was functionally screened from a pool of enzymes with potential XI activity based on its sequence similarity to XI from Piromyces sp. strain E2. Interestingly, the newly identified enzyme and XI from Piromyces shared the highest sequence identity among the assayed enzymes. Cells carrying the new XI grew in media with D-xylose as the sole carbon source at a superior rate to that of XI from Piromyces, yet at a considerably inferior rate to that of the alternative xylose reductase–xylitol dehydrogenase pathway. Furthermore, optimal conditions of temperature and pH, kinetic parameters, and inhibition kinetics by xylitol were determined for the new enzyme. The physiological characterization of D-xylose fermenting S. cerevisiae expressing the new XI will be further discusse

Functional screening for novel D-xylose isomerases from the gut of a wood feeding beetle reveals efficient expression in Saccharomyces cerevisiae

Renewable sugar rich feedstocks such as lignocellulosic hydrolysates remain one of the primary sources of potentially renewable fuel and bulk chemicals. The pentose sugar D-xylose is often present in significant amounts along with hexoses such as glucose and galactose. The yeast Saccharomyces cerevisiae can acquire the ability to metabolize D-xylose through expression of heterologous D-xylose isomerase (XI). This enzyme is notoriously difficult to express in S. cerevisiae and only fourteen genes have been reported to be active. We cloned a new XI from microorganisms in the gut of the wood feeding beetle Odontotaenius disjunctus. The new enzyme, 8454_2 XI, was functionally screened from a pool of enzymes with potential XI activity based on its sequence similarity to the XI from Piromyces sp. strain E2. A phylogenetic analysis revealed that the enzyme 8454_2 XI shares high identity with XIs from Bacteroidia class of the Bacteroidetes phylum, and all XIs from Bacteroidia screened in yeast so far have exhibited high activity. Cells carrying the new XI in D-xylose containing media as the sole carbon source showed higher growth and D-xylose consumption rates to those of XI of Piromyces. Remarkably, the 8454_2 XI also exhibited 2.6 times higher Vmax and 37 % higher affinity, and retained substantially higher relative activity at 30 ºC. The new XI is a useful addition to the molecular toolbox for genetic modification of S. cerevisiae for the metabolism of second-generation substrates.FatVal POCI-01-0145-FEDER-032506. Ph.D. scholarship SFRH/BD/140039/201

A novel D-xylose isomerase: from the gut of a wood feeding beetle for improved conversion in Saccharomyces cerevisiae

Carbohydrate rich substrates such as lignocellulosic hydrolysates remain one of the primary sources of potentially renewable fuel and bulk chemicals. The pentose sugar D-xylose is often present in significant amounts along with hexoses. For low value/high volume products, yield is of paramount importance for process economy. Saccharomyces cerevisiae can acquire the ability to metabolize D-xylose through expression of heterologous D-xylose isomerase (XI). This enzyme is notoriously difficult to express in S. cerevisiae and only fourteen genes have been reported to be active so far. We cloned a new D-xylose isomerase derived from microorganisms in the gut of the wood-feeding beetle Odontotaenius disjunctus. Although somewhat homologous to the current gold-standard from Piromyces sp. E2, metagenome scaffold gene neighborhoods and metagenome binning identified the gene as of bacterial in origin and the host as a Parabacteroides sp. Expression of the new XI enzyme in S. cerevisiae resulted in faster aerobic growth on D-xylose than the XI from Piromyces. The D-xylose isomerization rate of the yeast expressing this new XI was also 72 % higher. Interestingly, increasing concentrations of xylitol (up to 8 g/L) appeared not to inhibit xylose consumption in both strains. The newly described XI displayed 2.6 times higher specific activity, 37 % higher affinity for D-xylose, and exhibited higher activity over a broader temperature range, retaining 51 % of maximal activity at 30 ºC compared with only 29% activity for the Piromyces XI. This new enzyme represents a highly valuable addition to the S. cerevisiae molecular toolbox and shows promise for improved industrial conversion of carbohydrates.FatVal PTDC/EAM-AMB/32506/2017. “Contrato-Programa” UIDB/04050/2020. PhD fellowship SFRH/BD/140039/201

Legacy effects of intercropping and nitrogen fertilization on soil N cycling, nitrous oxide emissions, and the soil microbial community in tropical maize production

Maize-forage grasses intercropping systems have been increasingly adopted by farmers because of their capacity to recycle nutrients, provide mulch, and add C to soil. However, grasses have been shown to increase nitrous oxide (N2O) emissions. Some tropical grasses cause biological nitrification inhibition (BNI) which could mitigate N2O emissions in the maize cycle but the reactions of the N cycle and the microbial changes that explain the N2O emissions are little known in such intercropping systems. With this in mind, we explored intercropping of forage grasses (Brachiaria brizantha and Brachiaria humidicola) with distinct BNI and yield potential to increase N cycling in no-till maize production systems compared to monocrop with two N rates (0 and 150 kg ha−1) applied during the maize season. These grasses did not strongly compete with maize during the period of maize cycle and did not have a negative effect on grain yield. We observed a legacy of these grasses on N mineralization and nitrification through the soil microbiome during maize growth. We observed that B. humidicola, genotype with higher BNI potential, increased net N mineralization by 0.4 mg N kg−1 day−1 and potential nitrification rates by 1.86 mg NO3-N kg−1 day−1, while B. brizantha increased the soil moisture, fungi diversity, mycorrhizal fungi, and bacterial nitrifiers, and reduced saprotrophs prior to maize growth. Their legacy on soil moisture and cumulative organic inputs (i.e., grass biomass) was strongly associated with enhanced mineralization and nitrification rates at early maize season. These effects contributed to increase cumulative N2O emission by 12.8 and 4.8 mg N2O-N m−2 for maize growing after B. brizantha and B. humidicola, respectively, regardless of the N fertilization rate. Thus, the nitrification inhibition potential of tropical grasses can be outweighed by their impacts on soil moisture, N recycling, and the soil microbiome that together dictate soil N2O fluxes

Legacy effects of intercropping and nitrogen fertilization on soil N cycling, nitrous oxide emissions, and the soil microbial community in tropical maize production

Maize-forage grasses intercropping systems have been increasingly adopted by farmers because of their capacity to recycle nutrients, provide mulch, and add C to soil. However, grasses have been shown to increase nitrous oxide (N2O) emissions. Some tropical grasses cause biological nitrification inhibition (BNI) which could mitigate N2O emissions in the maize cycle but the reactions of the N cycle and the microbial changes that explain the N2O emissions are little known in such intercropping systems. With this in mind, we explored intercropping of forage grasses (Brachiaria brizantha and Brachiaria humidicola) with distinct BNI and yield potential to increase N cycling in no-till maize production systems compared to monocrop with two N rates (0 and 150 kg ha−1) applied during the maize season. These grasses did not strongly compete with maize during the period of maize cycle and did not have a negative effect on grain yield. We observed a legacy of these grasses on N mineralization and nitrification through the soil microbiome during maize growth. We observed that B. humidicola, genotype with higher BNI potential, increased net N mineralization by 0.4 mg N kg−1 day−1 and potential nitrification rates by 1.86 mg NO3-N kg−1 day−1, while B. brizantha increased the soil moisture, fungi diversity, mycorrhizal fungi, and bacterial nitrifiers, and reduced saprotrophs prior to maize growth. Their legacy on soil moisture and cumulative organic inputs (i.e., grass biomass) was strongly associated with enhanced mineralization and nitrification rates at early maize season. These effects contributed to increase cumulative N2O emission by 12.8 and 4.8 mg N2O-N m−2 for maize growing after B. brizantha and B. humidicola, respectively, regardless of the N fertilization rate. Thus, the nitrification inhibition potential of tropical grasses can be outweighed by their impacts on soil moisture, N recycling, and the soil microbiome that together dictate soil N2O fluxes

Presence and Persistence of Putative Lytic and Temperate Bacteriophages in Vaginal Metagenomes from South African Adolescents

The interaction between gut bacterial and viral microbiota is thought to be important in human health. While fluctuations in female genital tract (FGT) bacterial microbiota similarly determine sexual health, little is known about the presence, persistence, and function of vaginal bacteriophages. We conducted shotgun metagenome sequencing of cervicovaginal samples from South African adolescents collected longitudinally, who received no antibiotics. We annotated viral reads and circular bacteriophages, identified CRISPR loci and putative prophages, and assessed their diversity, persistence, and associations with bacterial microbiota composition. Siphoviridae was the most prevalent bacteriophage family, followed by Myoviridae, Podoviridae, Herelleviridae, and Inoviridae. Full-length siphoviruses targeting bacterial vaginosis (BV)-associated bacteria were identified, suggesting their presence in vivo. CRISPR loci and prophage-like elements were common, and genomic analysis suggested higher diversity among Gardnerella than Lactobacillus prophages. We found that some prophages were highly persistent within participants, and identical prophages were present in cervicovaginal secretions of multiple participants, suggesting that prophages, and thus bacterial strains, are shared between adolescents. The number of CRISPR loci and prophages were associated with vaginal microbiota stability and absence of BV. Our analysis suggests that (pro)phages are common in the FGT and vaginal bacteria and (pro)phages may interact

Airway Microbiota and Pathogen Abundance in Age-Stratified Cystic Fibrosis Patients

Bacterial communities in the airways of cystic fibrosis (CF) patients are, as in other ecological niches, influenced by autogenic and allogenic factors. However, our understanding of microbial colonization in younger versus older CF airways and the association with pulmonary function is rudimentary at best. Using a phylogenetic microarray, we examine the airway microbiota in age stratified CF patients ranging from neonates (9 months) to adults (72 years). From a cohort of clinically stable patients, we demonstrate that older CF patients who exhibit poorer pulmonary function possess more uneven, phylogenetically-clustered airway communities, compared to younger patients. Using longitudinal samples collected form a subset of these patients a pattern of initial bacterial community diversification was observed in younger patients compared with a progressive loss of diversity over time in older patients. We describe in detail the distinct bacterial community profiles associated with young and old CF patients with a particular focus on the differences between respective “early” and “late” colonizing organisms. Finally we assess the influence of Cystic Fibrosis Transmembrane Regulator (CFTR) mutation on bacterial abundance and identify genotype-specific communities involving members of the Pseudomonadaceae, Xanthomonadaceae, Moraxellaceae and Enterobacteriaceae amongst others. Data presented here provides insights into the CF airway microbiota, including initial diversification events in younger patients and establishment of specialized communities of pathogens associated with poor pulmonary function in older patient populations