In Memoriam: Mosè Rossi (1938-2023).

On August 26, 2023, we bid farewell to Professor Mosè Rossi, a distinguished figure in the field of enzymology and a beloved member of the International Society for Extremophiles since 1993. Born in Castellabate (Salerno) in 1938, Professor Rossi embarked on a remarkable journey in the world of science.

Discovery of a non-canonical prototype long-chain monoacylglycerol lipase through a structure-based endogenous reaction intermediate complex.

The identification and characterization of enzyme function is largely lacking behind the rapidly increasing availability of large numbers of sequences and associated high-resolution structures. This is often hampered by lack of knowledge on in vivo relevant substrates. Here, we present a case study of a high-resolution structure of an unusual orphan lipase in complex with an endogenous C18 monoacylglycerol ester reaction intermediate from the expression host, which is insoluble under aqueous conditions and thus not accessible for studies in solution. The data allowed its functional characterization as a prototypic long-chain monoacylglycerol lipase, which uses a minimal lid domain to position the substrate through a hydrophobic tunnel directly to the enzyme's active site. Knowledge about the molecular details of the substrate binding site allowed us to modulate the enzymatic activity by adjusting protein/substrate interactions, demonstrating the potential of our findings for future biotechnology applications.

Mining thermophiles for biotechnologically relevant enzymes: evaluating the potential of European and Caucasian hot springs.

The development of sustainable and environmentally friendly industrial processes is becoming very crucial and demanding for the rapid implementation of innovative bio-based technologies. Natural extreme environments harbor the potential for discovering and utilizing highly specific and efficient biocatalysts that are adapted to harsh conditions. This review focuses on extremophilic microorganisms and their enzymes (extremozymes) from various hot springs, shallow marine vents, and other geothermal habitats in Europe and the Caucasus region. These hot environments have been partially investigated and analyzed for microbial diversity and enzymology. Hotspots like Iceland, Italy, and the Azores harbor unique microorganisms, including bacteria and archaea. The latest results demonstrate a great potential for the discovery of new microbial species and unique enzymes that can be explored for the development of Circular Bioeconomy.Different screening approaches have been used to discover enzymes that are active at extremes of temperature (up 120 °C), pH (0.1 to 11), high salt concentration (up to 30%) as well as activity in the presence of solvents (up to 99%). The majority of published enzymes were revealed from bacterial or archaeal isolates by traditional activity-based screening techniques. However, the latest developments in molecular biology, bioinformatics, and genomics have revolutionized life science technologies. Post-genomic era has contributed to the discovery of millions of sequences coding for a huge number of biocatalysts. Both strategies, activity- and sequence-based screening approaches, are complementary and contribute to the discovery of unique enzymes that have not been extensively utilized so far.

Structural and biochemical characterisation of the N-carbamoyl-ß-alanine amidohydrolase from Rhizobium radiobacter MDC 8606.

N-carbamoyl-ß-alanine amidohydrolase (CßAA) constitutes one of the most important groups of industrially relevant enzymes used in the production of optically pure amino acids and derivatives. In this study, a CßAA-encoding gene from Rhizobium radiobacter strain MDC 8606 was cloned and overexpressed in Escherichia coli. The purified recombinant enzyme (RrCßAA) showed a specific activity of 14 U·mg-1 using N-carbamoyl-ß-alanine as a substrate with an optimum activity at 55 °C and pH 8.0. In this work, we report also the first prokaryotic CßAA structure at a resolution of 2.0 Å. A discontinuous catalytic domain and a dimerisation domain attached through a flexible hinge region at the domain interface have been revealed. We identify key ligand binding residues, including a conserved glutamic acid (Glu131), histidine (H385) and arginine (Arg291). Our results allowed us to explain the preference of the enzyme for linear carbamoyl substrates, as large and branched carbamoyl substrates cannot fit in the active site of the enzyme. This work envisages the use of RrCßAA from R. radiobacter MDC 8606 for the industrial production of L-α-, L-ß- and L-γ-amino acids. The structural analysis provides new insights on enzyme-substrate interaction, which shed light on engineering of CßAAs for high catalytic activity and broad substrate specificity.

Towards Sustainable Recycling of Epoxy-Based Polymers: Approaches and Challenges of Epoxy Biodegradation.

Epoxy resins are highly valued for their remarkable mechanical and chemical properties and are extensively used in various applications such as coatings, adhesives, and fiber-reinforced composites in lightweight construction. Composites are especially important for the development and implementation of sustainable technologies such as wind power, energy-efficient aircrafts, and electric cars. Despite their advantages, their non-biodegradability raises challenges for the recycling of polymer and composites in particular. Conventional methods employed for epoxy recycling are characterized by their high energy consumption and the utilization of toxic chemicals, rendering them rather unsustainable. Recent progress has been made in the field of plastic biodegradation, which is considered more sustainable than energy-intensive mechanical or thermal recycling methods. However, the current successful approaches in plastic biodegradation are predominantly focused on polyester-based polymers, leaving more recalcitrant plastics underrepresented in this area of research. Epoxy polymers, characterized by their strong cross-linking and predominantly ether-based backbone, exhibit a highly rigid and durable structure, placing them within this category. Therefore, the objective of this review paper is to examine the various approaches that have been employed for the biodegradation of epoxy so far. Additionally, the paper sheds light on the analytical techniques utilized in the development of these recycling methods. Moreover, the review addresses the challenges and opportunities entailed in epoxy recycling through bio-based approaches.

Valorization of whey-based side streams for microbial biomass, molecular hydrogen, and hydrogenase production.

Side streams of the dairy industry are a suitable nutrient source for cultivating microorganisms, producing enzymes, and high-value chemical compounds. The heterotrophic Escherichia coli and chemolithoautotroph Ralstonia eutropha are of major biotechnological interest. R. eutropha is a model organism for producing O2-tolerant [NiFe]-hydrogenases (Hyds) (biocatalysts), and E. coli has found widespread use as an expression platform for producing recombinant proteins, molecular hydrogen (H2), and other valuable products. Aiming at developing suitable cultivation media from side streams of the dairy industry, the pre-treatment (filtration, dilution, and pH adjustment) of cheese (sweet) whey (SW) and curd (acid) whey (AW), with and without the use of ß-glucosidase, has been performed. Growth parameters (oxidation-reduction potential (ORP), pH changes, specific growth rate, biomass formation) of E. coli BW25113 and R. eutropha H16 type strains were monitored during cultivation on filtered and non-filtered SW and AW at 37 °C, pH 7.5 and 30 °C, pH 7.0, respectively. Along with microbial growth, measurements of pH and ORP indicated good fermentative growth. Compared to growth on fructose-nitrogen minimal salt medium (control), a maximum cell yield (OD600 4.0) and H2-oxidizing Hyd activity were achieved in the stationary growth phase for R. eutropha. Hyd-3-dependent H2 production by E. coli utilizing whey as a growth substrate was demonstrated. Moreover, good biomass production and prolonged H2 yields of ~ 5 mmol/L and cumulative H2 ~ 94 mL g/L dry whey (DW) (ß-glucosidase-treated) were observed during the cultivation of the engineered E. coli strain. These results open new avenues for effective whey treatment using thermostable ß-glucosidase and confirm whey as an economically viable commodity for biomass and biocatalyst production. KEY POINTS: • Archaeal thermostable ß-glucosidase isolated from the metagenome of a hydrothermal spring was used for lactose hydrolysis in whey. • Hydrogenase enzyme activity was induced during the growth of Ralstonia eutropha H16 on whey. • Enhanced biomass and H2 production was shown in a genetically modified strain of Escherichia coli.

A host-vector toolbox for improved secretory protein overproduction in Bacillus subtilis.

Target proteins in biotechnological applications are highly diverse. Therefore, versatile flexible expression systems for their functional overproduction are required. In order to find the right heterologous gene expression strategy, suitable host-vector systems, which combine different genetic circuits, are useful. In this study, we designed a novel Bacillus subtilis expression toolbox, which allows the overproduction and secretion of potentially toxic enzymes. This toolbox comprises a set of 60 expression vectors, which combine two promoter variants, four strong secretion signals, a translation-enhancing downstream box, and three plasmid backbones. This B. subtilis toolbox is based on a tailor-made, clean deletion mutant strain, which is protease and sporulation deficient and exhibits reduced autolysis and secondary metabolism. The appropriateness of this alternative expression platform was tested for the overproduction of two difficult-to-produce eukaryotic model proteins. These included the sulfhydryl oxidase Sox from Saccharomyces cerevisiae, which forms reactive hydrogen peroxide and undesired cross-linking of functional proteins, and the human interleukin-1ß, a pro-inflammatory cytokine. For the best performing Sox and interleukin, overproducing and secreting variants of these new B. subtilis toolbox fermentation strategies were developed and tested. This study demonstrates the suitability of the prokaryotic B. subtilis host-vector system for the extracellular production of two eukaryotic proteins with biotechnological relevance. KEY POINTS: • Construction of a versatile Bacillus subtilis gene expression toolbox. • Verification of the toolbox by the secretory overproduction of two difficult-to-express proteins. • Fermentation strategy for an acetoin-controlled overproduction of heterologous proteins.

Microorganisms harbor keys to a circular bioeconomy making them useful tools in fighting plastic pollution and rising CO2 levels.

The major global and man-made challenges of our time are the fossil fuel-driven climate change a global plastic pollution and rapidly emerging plant, human and animal infections. To meet the necessary global changes, a dramatic transformation must take place in science and society. This transformation will involve very intense and forward oriented industrial and basic research strongly focusing on (bio)technology and industrial bioprocesses developments towards engineering a zero-carbon sustainable bioeconomy. Within this transition microorganisms-and especially extremophiles-will play a significant and global role as technology drivers. They harbor the keys and blueprints to a sustainable biotechnology in their genomes. Within this article, we outline urgent and important areas of microbial research and technology advancements and that will ultimately make major contributions during the transition from a linear towards a circular bioeconomy.

A multi-omic screening approach for the discovery of thermoactive glycoside hydrolases.

Next-generation sequencing and computational biology have facilitated the implementation of new combinatorial screening approaches to discover novel enzymes of biotechnological interest. In this study, we describe the successful establishment of a multi-omic approach for the identification of thermostable hydrolase-encoding genes by determination of gene expression levels. We applied this combinatorial approach using an anaerobic enrichment culture from an Azorean hot spring sample grown on green coffee beans as recalcitrant substrate. An in-depth analysis of the microbial community resulted in microorganisms capable of metabolizing the selected substrate, such as the genera Caloramator, Dictyoglomus and Thermoanaerobacter as active and abundant microorganisms. To discover glycoside hydrolases, 90,342 annotated genes were screened for specific reaction types. A total number of 106 genes encoding cellulases (EC 3.2.1.4), beta-glucosidases (EC 3.2.1.21) and endo-1,4-beta-mannosidases (EC 3.2.1.78) were selected. Mapping of RNA-Seq reads to the related metagenome led to expression levels for each gene. Amongst those, 14 genes, encoding glycoside hydrolases, showed highest expression values, and were used for further cloning. Four proteins were biochemically characterized and were identified as thermoactive glycoside hydrolases with a broad substrate range. This work demonstrated that a combinatory omic approach is a suitable strategy identifying unique thermoactive enzymes from environmental samples.

Biomass-degrading glycoside hydrolases of archaeal origin.

During the last decades, the impact of hyperthermophiles and their enzymes has been intensively investigated for implementation in various high-temperature biotechnological processes. Biocatalysts of hyperthermophiles have proven to show extremely high thermo-activities and thermo-stabilities and are identified as suitable candidates for numerous industrial processes with harsh conditions, including the process of an efficient plant biomass pretreatment and conversion. Already-characterized archaea-originated glycoside hydrolases (GHs) have shown highly impressive features and numerous enzyme characterizations indicated that these biocatalysts show maximum activities at a higher temperature range compared to bacterial ones. However, compared to bacterial biomass-degrading enzymes, the number of characterized archaeal ones remains low. To discover new promising archaeal GH candidates, it is necessary to study in detail the microbiology and enzymology of extremely high-temperature habitats, ranging from terrestrial to marine hydrothermal systems. State-of-the art technologies such as sequencing of genomes and metagenomes and automated binning of genomes out of metagenomes, combined with classical microbiological culture-dependent approaches, have been successfully performed to detect novel promising biomass-degrading hyperthermozymes. In this review, we will focus on the detection, characterization and similarities of archaeal GHs and their unique characteristics. The potential of hyperthermozymes and their impact on high-temperature industrial applications have not yet been exhausted.

Digitalization in microbiology - Paving the path to sustainable circular bioeconomy.

The transition to a sustainable bio-based circular economy requires cutting edge technologies that ensure economic growth with environmentally responsible action. This transition will only be feasible when the opportunities of digitalization are also exploited. Digital methods and big data handling have already found their way into life sciences and generally offer huge potential in various research areas. While computational analyses of microbial metagenome data have become state of the art, the true potential of bioinformatics remains mostly untapped so far. In this article we present challenges and opportunities of digitalization including multi-omics approaches in discovering and exploiting the microbial diversity of the planet with the aim to identify robust biocatalysts for application in sustainable bioprocesses as part of the transition from a fossil-based to a bio-based circular economy. This will contribute to solving global challenges, including utilization of natural resources, food supply, health, energy and the environment.

Microbial biofilm formation and degradation of octocrylene, a UV absorber found in sunscreen.

Octocrylene is a widely used synthetic UV absorber of sunscreens and found in several environments. Ecological consequences of the accumulation of UV filters are widely discussed. This is the first report revealing the microbial potential to transform octocrylene. A microbial community comprising four bacterial species was enriched from a landfill site using octocrylene as carbon source. From these microorganisms Mycobacterium agri and Gordonia cholesterolivorans were identified as most potent applying a new "reverse discovery" approach. This relies on the possibility that efficient strains that are already isolated and deposited can be identified through enrichment cultures. These strains formed massive biofilms on the octocrylene droplets. GC-MS analysis after cultivation for 10 days with M. agri revealed a decrease in octocrylene concentration of 19.1%. LC-MS/MS analysis was utilized in the detection and quantification of transformation products of octocrylene. M. agri thus represents an ideal candidate for bioremediation studies with octocrylene and related compounds.

Characterization of an extremely thermo-active archaeal ß-glucosidase and its activity towards glucan and mannan in concert with an endoglucanase.

A metagenome from an enrichment culture of a hydrothermal vent sample taken at Vulcano Island (Italy) was sequenced and an endoglucanase-encoding gene (vul_cel5A) was identified in a previous work. Vul_Cel5A with maximal activity at 115 °C was characterized as the most heat-active endoglucanase to date. Based on metagenome sequences, genomes were binned and bin4 included vul_cel5A as well as a putative GH1 ß-glycosidase-encoding gene (vul_bgl1A) with highest identities to sequences from the archaeal genus Thermococcus. The recombinant ß-glucosidase Vul_Bgl1A produced in E. coli BL21 pQE-80L exhibited highest activity at 105 °C and pH 7.0 (76.12 ± 5.4 U/mg, 100%) using 4NP ß-D-glucopyranoside as substrate and 61% relative activity at 120 °C. Accordingly, Vul_Bgl1A represents one of the most heat-active ß-glucosidases to date. The enzyme has a broad substrate specificity with 155% activity towards 4NP ß-D-mannopyranoside in comparison with 4NP ß-D-glucopyranoside. Moreover, nearly complete hydrolysis of cellobiose was demonstrated. The enzyme exhibited a high glucose tolerance with 26% residual activity in presence of 2 M glucose and was furthermore activated at glucose concentrations of up to 0.5 M. When the endoglucanase Vul_Cel5A and the ß-glucosidase Vul_Bgl1A were applied simultaneously at 99 °C, 158% activity towards barley ß-glucan and 215% towards mannan were achieved compared with the activity of Vul_Cel5A alone (100%). Consequently, a significant increase in glucose formation was observed when both enzymes were incubated with ß-glucan and mannan suggesting a synergistic effect. Hence, the two archaeal extremozymes are ideal candidates for complete glucan and mannan saccharification at temperatures above the boiling point of water.

Characterization of a thermoactive endoglucanase isolated from a biogas plant metagenome.

A metagenomic library from DNA isolated from a biogas plant was constructed and screened for thermoactive endoglucanases to gain insight into the enzymatic diversity involved in plant biomass breakdown at elevated temperatures. Two cellulase-encoding genes were identified and the corresponding proteins showed sequence similarities of 59% for Cel5A to a putative cellulase from Anaerolinea thermolimosa and 99% for Cel5B to a characterized endoglucanase isolated from a biogas plant reactor. The cellulase Cel5A consists of one catalytical domain showing sequence similarities to glycoside hydrolase family 5 and comprises 358 amino acids with a predicted molecular mass of 41.2 kDa. The gene coding for cel5A was successfully cloned and expressed in Escherichia coli C43(DE3). The recombinant protein was purified to homogeneity using affinity chromatography with a specific activity of 182 U/mg, and a yield of 74%. Enzymatic activity was detectable towards cellulose and mannan containing substrates and over a broad temperature range from 40 °C to 70 °C and a pH range from 4.0 to 7.0 with maximal activity at 55 °C and pH 5.0. Cel5A showed high thermostability at 60 °C without loss of activity after 24 h. Due to the enzymatic characteristics, Cel5A is an attractive candidate for the degradation of lignocellulosic material.

Enrichment of anaerobic heterotrophic thermophiles from four Azorean hot springs revealed different community composition and genera abundances using recalcitrant substrates.

DGGE analysis combined with a metagenomic approach was used to get insights into heterotrophic anoxic enrichment cultures of four hot springs of Vale das Furnas, Portugal, using the recalcitrant substrate spent coffee ground (SCG). Parallel enrichment cultures were performed using the major components of spent coffee ground, namely arabinogalactan, galactomannan, cellulose, and proteins. DGGE revealed that heterotrophic thermophilic bacteria are highly abundant in the hydrothermal springs and significant differences in community composition depending on the substrate were observed. DNA, isolated from enrichment cultures of different locations that were grown on the same substrate were pooled, and the respective metagenomes were analyzed. Results indicated that cultures grown on recalcitrant substrate SCG consists of a totally different thermophilic community, dominated by Dictyoglomus. Enrichments with galactomannan and arabinogalactan were dominated by Thermodesulfovibrio, while cultures with casein and cellulose were dominated by Thermus. This study indicates the high potential of thermophilic bacteria degrading recalcitrant substrate such as SCG and furthermore how the accessibility to complex polymers shapes the bacterial community.

Extremely thermoactive archaeal endoglucanase from a shallow marine hydrothermal vent from Vulcano Island.

Already-characterized microbial cellulases have proven to be highly useful for industrial processes, since they can withstand harsh industrial conditions with characteristics such as high thermo- and acid stability. These properties provide promising features for the process of plant biomass degradation and biofuel generation. Nevertheless, the number of known extremely thermoactive archaeal cellulases is low. Hence, the discovery of archaeal cellulases with different characteristics is crucial for the development of efficient and sustainable biorefinery. In this work, the metagenome of a high-temperature enrichment culture from marine environment of Vulcano Island was screened for the presence of novel endoglucanase-encoding genes of archaeal origin. The ORF vul_cel5A was detected, and the deduced protein was characterized as the most thermoactive endoglucanase described to date. Vul_Cel5A was identified as a thermoactive glycoside hydrolase family 5 endoglucanase, with the highest sequence identity (72-75%) to putative endoglucanases from archaeal genera. Vul_Cel5A showed the highest activity at notable 115 °C towards barley ß-glucan (210.7 U/mg), and lichenan (209.9 U/mg), and further towards carboxymethyl cellulose (38.6 U/mg) and locust bean gum (83.0 U/mg). The endoglucanase exhibited a half-life time of 46 min at 100 °C and did not show any loss of activity after incubation for 48 h at 75 °C. Furthermore, Vul_Cel5A showed high affinity to barley ß-glucan with a Km of 0.52 mg/mL and showed tolerance against various chemical reagents. Due to the outstanding high thermoactivity and thermostability and tolerance to acidic conditions, Vul_Cel5A represents a promising novel archaeal endo-ß-glucanase for application in biorefineries for an efficient biomass pre-treatment.

Characterization of a Theme C Glycoside Hydrolase Family 9 Endo-Beta-Glucanase from a Biogas Reactor Metagenome.

From a biogas reactor metagenome an ORF (bp_cel9A) encoding a bacterial theme C glycoside hydrolase family 9 (GH9) enzyme was recombinantly produced in E. coli BL21 pQE-80L. BP_Cel9A exhibited ≤ 55% identity to annotated sequences. Subsequently, the enzyme was purified to homogeneity by affinity chromatography. The endo-beta-glucanase BP_Cel9A hydrolyzed the beta-1,3-1,4-linked barley beta-glucan with 24 U/mg at 30 °C and pH 6.0. More than 62% of activity was measured between 10 and 40 °C. Lichenan and xyloglucan were hydrolyzed with 67% and 40% of activity, respectively. The activity towards different substrates varied with different temperatures. However, the enzyme activity on CMC was extremely low (> 1%). In contrast to BP_Cel9A, most GH9 glucanases act preferably on crystalline or soluble cellulose with only side activities towards related substrates. The addition of calcium or magnesium enhanced the activity of BP_Cel9A, especially at higher temperatures. EDTA inhibited the enzyme, whereas EGTA had no effect, suggesting that Mg2+ may adopt the function of Ca2+. BP_Cel9A exhibited a unique substrate spectrum when compared to other GH9 enzymes with great potential for mixed-linked glucan or xyloglucan degrading processes at moderate temperatures.

Towards a sustainable biobased industry - Highlighting the impact of extremophiles.

The transition of the oil-based economy towards a sustainable economy completely relying on biomass as renewable feedstock requires the concerted action of academia, industry, politics and civil society. An interdisciplinary approach of various fields such as microbiology, molecular biology, chemistry, genetics, chemical engineering and agriculture in addition to cross-sectional technologies such as economy, logistics and digitalization is necessary to meet the future global challenges. The genomic era has contributed significantly to the exploitation of naturés biodiversity also from extreme habitats. By applying modern technologies it is now feasible to deliver robust enzymes (extremozymes) and robust microbial systems that are active at temperatures up to 120°C, at pH 0 and 12 and at 1000bar. In the post-genomic era, different sophisticated "omics" analyses will allow the identification of countless novel enzymes regardless of the lack of cultivability of most microorganisms. Furthermore, elaborate protein-engineering methods are clearing the way towards tailor-made robust biocatalysts. Applying environmentally friendly and efficient biological processes, terrestrial and marine biomass can be converted to high value products e.g. chemicals, building blocks, biomaterials, pharmaceuticals, food, feed and biofuels. Thus, further application of extremophiles has the potential to improve sustainability of existing biotechnological processes towards a greener biobased industry.

Diversity of bacteria and archaea from two shallow marine hydrothermal vents from Vulcano Island.

To obtain new insights into community compositions of hyperthermophilic microorganisms, defined as having optimal growth temperatures of 80 °C and above, sediment and water samples were taken from two shallow marine hydrothermal vents (I and II) with temperatures of 100 °C at Vulcano Island, Italy. A combinatorial approach of denaturant gradient gel electrophoresis (DGGE) and metagenomic sequencing was used for microbial community analyses of the samples. In addition, enrichment cultures, growing anaerobically on selected polysaccharides such as starch and cellulose, were also analyzed by the combinatorial approach. Our results showed a high abundance of hyperthermophilic archaea, especially in sample II, and a comparable diverse archaeal community composition in both samples. In particular, the strains of the hyperthermophilic anaerobic genera Staphylothermus and Thermococcus, and strains of the aerobic hyperthermophilic genus Aeropyrum, were abundant. Regarding the bacterial community, ε-Proteobacteria, especially the genera Sulfurimonas and Sulfurovum, were highly abundant. The microbial diversity of the enrichment cultures changed significantly by showing a high dominance of archaea, particularly the genera Thermococcus and Palaeococcus, depending on the carbon source and the selected temperature.

Complete genome sequence of Thermus brockianus GE-1 reveals key enzymes of xylan/xylose metabolism.

Thermus brockianus strain GE-1 is a thermophilic, Gram-negative, rod-shaped and non-motile bacterium that was isolated from the Geysir geothermal area, Iceland. Like other thermophiles, Thermus species are often used as model organisms to understand the mechanism of action of extremozymes, especially focusing on their heat-activity and thermostability. Genome-specific features of T. brockianus GE-1 and their properties further help to explain processes of the adaption of extremophiles at elevated temperatures. Here we analyze the first whole genome sequence of T. brockianus strain GE-1. Insights of the genome sequence and the methodologies that were applied during de novo assembly and annotation are given in detail. The finished genome shows a phred quality value of QV50. The complete genome size is 2.38 Mb, comprising the chromosome (2,035,182 bp), the megaplasmid pTB1 (342,792 bp) and the smaller plasmid pTB2 (10,299 bp). Gene prediction revealed 2,511 genes in total, including 2,458 protein-encoding genes, 53 RNA and 66 pseudo genes. A unique genomic region on megaplasmid pTB1 was identified encoding key enzymes for xylan depolymerization and xylose metabolism. This is in agreement with the growth experiments in which xylan is utilized as sole source of carbon. Accordingly, we identified sequences encoding the xylanase Xyn10, an endoglucanase, the membrane ABC sugar transporter XylH, the xylose-binding protein XylF, the xylose isomerase XylA catalyzing the first step of xylose metabolism and the xylulokinase XylB, responsible for the second step of xylose metabolism. Our data indicate that an ancestor of T. brockianus obtained the ability to use xylose as alternative carbon source by horizontal gene transfer.