Supplementary materials for Phylogenomics and genetic analysis of solvent-producing Clostridium species

The genus Clostridium is a large and diverse group within the Bacillota (formerly Firmicutes), whose members can encode useful complex traits such as solvent production, gas-fermentation, and lignocellulose breakdown. We describe 270 genome sequences of solventogenic clostridia from a comprehensive industrial strain collection assembled by Professor David Jones that includes 194 C. beijerenckiI, 57 C. saccharobutylicum, 4 C. saccharoperbutylacetonicum, 5 C. butyricum, 7 C. acetobutylicum, and 3 C. tetanomorphum genomes. We report methods, analyses and characterization for phylogeny, key attributes, core biosynthetic genes, secondary metabolites, plasmids, prophage/CRISPR diversity, cellulosomes and quorum sensing for the 6 species. The expanded genomic data described here will facilitate engineering of solvent-producing clostridia as well as non-model microorganisms with innately desirable traits. Sequences could be applied in conventional platform biocatalysts such as yeast or Escherichia coli for enhanced chemical production. Recently, gene sequences from this collection were used to engineer Clostridium autoethanogenum, a gas-fermenting autotrophic acetogen, for continuous acetone or isopropanol production, as well as butanol, butanoic acid, hexanol and hexanoic acid production. 

Methods
A modified phenol-chloroform extraction based on Annan et al. (2019) was used to purify genomic DNA. Cells were harvested by centrifugation at 6,000 x g for 15 min at 4°C, and washed in 50 mL potassium phosphate buffer (10 mM, pH 7.5). The cells were resuspended in STE buffer (50 mM Tris-HCl, pH 8.0; 1 mM EDTA; 200 mM sucrose) with 30 μL lysozyme (100 mg/mL), followed by a combination of SDS, EDTA, Tris, RNaseA/T1, and Proteinase K. Each addition was followed by specific incubation steps at 37°C. The lysate was processed using Phase Lock Gel Heavy tubes and a modified phenol::chloroform procedure that employed MaXtract High Density (Qiagen) phase-lock tubes. DNA was precipitated, then washed with -20°C 70% ethanol, dried, and resuspended in heated (60°C) 1x TE buffer (10 mM Tris, pH 8, and 0.1 mM EDTA). Purified DNA underwent 16S rRNA gene sequencing to confirm strain identity before being shipped to the DOE Joint Genome Institute (JGI) for genome sequencing and annotation. Reference: Annan FJ, et al. Appl Microbiol Biotechnol. 2019 Jun;103(11):4633-4648. Genomes were sequenced at the JGI using Pacific Biosciences (PacBio) technology on either an RSII instrument (P6/C4 chemistry), or Sequel (v2.1 v2 or v3.0 chemistries) or using an Illumina NovaSeq (v1 chemistry) and have been reported previously (https://pubmed.ncbi.nlm.nih.gov/37764097/). All genome sequences were annotated using the JGI IMG pipeline, with the vast majority by version v.5.0.10 and details available for each at the JGI IMG database. Genomes used in this study are curated under the GOLD study ID Gs0118866, with links to raw sequence data available via JGI or via the NCBI SRA database. Contigs were classified into plasmid and chromosomal categories to determine plasmid presence or absence using PlasFlow v1.1.0. For convenience, the 270 sub-projects are deposited under NCBI BioProject PRJNA990349. Strains analyzed in this study are shown Supplementary Table 1, and include relevant culture collection details (Supplementary Tables 2-3). Additional methodological details are provided in accepted Scientific Data manucript SDATA-23-01651.