Draft Genome Sequence of the Lignocellulolytic and Thermophilic Bacterium Thermobacillus xylanilyticus XE.

Thermobacillus xylanilyticus is a thermophilic and hemicellulolytic bacterium able to use several lignocelluloses as its main carbon source. This draft genome sequence gives insight into the genomic potential of this bacterium and provides new resources to understand the enzymatic mechanisms used by the bacterium during lignocellulose degradation and will allow the identification of robust lignocellulolytic enzymes.

ANNOUNCEMENT

Thermophilic microorganisms may provide robust and thermostable enzymes for plant biorefining to produce biofuels and biomolecules (1, 2). Thermobacillus xylanilyticus XE (deposited at the Collection Nationale de Cultures Microbiennes France under no. CNCM I-1017) is a Gram-positive, thermophilic bacterium, originally isolated from farm soil situated underneath a manure heap, that is able to use nonpretreated lignocelluloses as its carbon source (3–5). This bacterium produces several hemicellulases (xylanase, arabinosidase, and feruloyl esterase) that have resistance to high temperatures (60° to 70°C) and alkaline reaction conditions (4–10) and efficiently liberate sugars and phenolic compounds from various lignocelluloses (8, 10–15).

T. xylanilyticus was grown at 50°C on basal medium complemented with 5 g/L xylan (3). The total genome was isolated from early-stationary-phase cell cultures using the Purelink genomic DNA kit (Thermo Fisher). DNA was quantified using a fluorimetry methodology (Qubit v2.0; Thermo Fisher) and by gel electrophoresis analysis. Genomic DNA was fragmented with a Covaris S220 instrument. DNA libraries were prepared with a TruSeq SBS kit v5 (Illumina) protocol and the SPRIworks fragment library system I. The genome was sequenced by using 74-nucleotide paired-end sequencing with Genome Analyzer IIx instruments (Illumina). Raw read quality was assessed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and quality filtering was performed using Sickle (https://github.com/najoshi/sickle). The genome sequence was assembled de novo using Velvet v1.1. 04 (16) after quality analyses and trimming of raw reads. The annotation was performed with the AGMIAL platform (17). Default parameters were used for all software.

A total of 32,786,406 raw reads were obtained, representing an average coverage of 58×; 240 Mb of raw sequences was assembled into 108 scaffolds (146 contigs). The N50 value of the assembly was 83 kb. The largest contig size was 184,473 bp. The size of the genome was 4,109,925 nucleotides with a G+C content of 61.0%. The draft genome encoded 3,956 proteins and 55 tRNAs; 1 copy each of 23S rRNA and 16S rRNA was correctly assembled. The lignocellulolytic potential of T. xylanilyticus was assessed by identifying the relevant genes for lignocellulose utilization (CAZymes) by using a procedure analogous to that used for the analysis of daily releases of GenBank data by the CAZy database (www.cazy.org) (18) and involving both automated and human curation-based BLASTP (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and HMMER v3 (http://hmmer.janelia.org/) results against libraries of modules derived from the database. A total of 94 genes encoding glycoside hydrolases, 16 genes encoding carbohydrate esterases, 45 genes encoding glycosyl transferases, and 2 genes encoding polysaccharide lyases were identified. Some of these genes were organized in operon structures. Except for genes encoding β-glucosidases and one putative gene encoding an exoglucanase, no other genes related to cellulose degradation were identified. Several oxidoreductases have been annotated. This genome sequence will facilitate the identification of new genes implicated in the enzymatic fractionation of lignocellulosic biomass for the production of new enzymes of interest.

Data availability.

T. xylanilyticus raw reads and the draft genome sequence have been deposited at the European Nucleotide Archive (ENA) under the study accession number PRJEB43105. The raw reads are available under run accession number ERR5840465. The annotated genome sequence is available under WGS sequence set CAJRAY010000000. The version described in this paper is the first version.