176 Chapter 6 Lysine 2,3-aminomutase (LAM) and β-lysine N6-acetyl transferase (AT) phylogenetic trees To expand the potential of N(ε)-acetyl-β-L-lysine biosynthesis across bacterial and archaeal representatives, we first obtained a list of genomes from the GTDB (Parks et al., 2020). Using this list, we retrieved the corresponding genomes from the NCBI database. We then searched these genomes for the kamA (KEGG: K01843) and ablB (KEGG: K21935) genes, which are responsible for encoding the relevant amino acid sequences. This approach allowed us to identify and analyze the presence of these genes in the selected bacterial and archaeal genomes. “Ca. Methanoperedens”- related kamA and ablB sequences encoded on Borgs were directly downloaded from the reference paper(Al-Shayeb et al., 2022). The Al Shayeb et al. (2022) metagenome was screened for osmolyte biosynthesis genes of interest via HMMs that were stringently KEGG-curated with the default parameters of kofamKOALA (Aramaki et al., 2020). Sequences were first aligned with MUSCLE v3.8.31(Edgar, 2004), trimmed with (--gappy out) using Trimal v1.4.rev15 (Capella-Gutiérrez et al., 2009) and ran with IQ-TREE v2.0.3 using flags -st AA -m MFP -bb (ultrafast bootstrap) 1000 -nt AUTO (model selected according to the Bayesian information criterion (BIC): LG+R10 both for kamA-encoded LAM and for ablB-encoded AT) (Nguyen et al., 2015). The aligned kamA-encoding LAM sequences were categorized in two groups based on whether LAM was found alone (15/50) or with ablB on the same Borg element (14/50) (Supplementary Table 3). For the ablB-encoding AT tree, we only kept 14 sequences (out of the 37 mined) containing contiguous kamA (encoding LAM) and ablB (encoding AT) genes. In fact, both genes are regulated together (same operon) in order to produce N(ε)-acetyl-β-L-lysine (Supplementary Table 4). For certain “Ca. Methanoperdens” MAGs of interest, NCBI biosample was used to retrieve sampling source information, infer whether they were described to harbor a plasmid(Schoelmerich et al., 2022) or recover metatranscriptomic evidence of osmolyte gene expression following the same metatranscriptomic pipeline as described for this paper. We employed iTOL v5 for the annotation of our tree(Letunic & Bork, 2021). Metatranscriptomics Metatranscriptomic sequencing was performed using a TruSeq stranded mRNA library Kit (Illumina, San Diego, CA, United States) on a NovaSeq 6000 (Illumina)
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