## 2 Results
### TCDD Enriched for Mevalonate-Dependent Isoprenoid Biosynthesis  
To investigate other metabolic pathways imparting competitive advantages to TCDD-elicited gut environmental stresses, functional gene annotations associated with _L. reuteri_, the highest enriched species, were assessed. Among enriched uniref90 annotations in the cecum metagenomic dataset was the aromatic amino acid aminotransferase (UniRef90_A0A2S1 ENB9) also classified to _L. reuteri_ (Table S2). Aromatic amino acid aminotransferase produces a tryptophan metabolite, indole-3-aldehyde, a known AhR ligand reported to induce IL-22 in vivo [26]. Among 39 enzyme commission (EC) annotations that were enriched and associated with _L. reuteri_ were several annotated to the isoprenoid biosynthesis pathway (Figure 3, Figures S1 and S3). Bacteria biosynthesize the isoprenoid, isoptenenyl diphosphate (IPP), either through the mevalonate-dependent pathway, which is also found in mammals, or the methyleryth-thirtl phosphate (MEP)-pathway. Both _L. reuteri_ and _Lactobacillus jolnsonii_ were the major contributors to mevalonate-dependent IPP biosynthesis pathway enrichment with almost all genes in the pathway increased by TCDD; four out of six of the genes significantly increased by TCDD (Figure 3 and Figure S1). Gene enrichment in the alternative MEP-pathway were unchanged by TCDD. For _L. murinus_, only two EC annotations (EC 2.7.1.148, 4-diphosphocytidyl-2-C-methyl-D-erythitol (CDP-ME) kinase, and EC 5.3.3.2, isoptenenyl diphosphate Delta-isomerase) were identified in the MEP pathway also found in _L. reuteri_ (Figure S2). HUMAnN 3.0 analysis of a published metagenomics dataset of fecal samples from human cirrhotic patients ([[https://www.ncbi.nlm.nih.gov/bioproject/PRJEB6337/](https://www.ncbi.nlm.nih.gov/bioproject/PRJEB6337/)]([https://www.ncbi.nlm.nih.gov/bioproject/PRJEB6337/](https://www.ncbi.nlm.nih.gov/bioproject/PRJEB6337/)), accessed on 25 March 2021) [39] revealed strikingly similar results to our caecum samples from TCDD treated mice. Specifically, increased gene abundance associated with the mevalonate-dependent pathways was also evident in patients with compensated and decompensated liver disease (Figure 4). Compensated cirrhosis is defined as no decrease in liver function while decompensated cirrhosis exhibits decreased liver function. Among decompensated patients with cirrhosis, the mevalonate dependent IPP pathway was increased in 7 out of 8 EC numbers required for de novo IPP biosynthesis (Figure 4). Taxa annotated to genes in the pathway exhibited a wide variety in genera for each EC number in human samples compared to murine cecum samples from this study (Figure S3). Taxonomy classified to a majority of the mevalonate-dependent genes were from the Lactobacillusaceae family, including Enterococcus, Lactobacillus, Streptococcus genera (Figure S3 and Table S5). Lactobacillus and Streptococcus species, including _L. reuteri_ and _Streptococcus anginosus_, a known pathogen in liver abscesses [40], were among species classified to the pathway (Tables S4 and S5). Figure 3: TCDD enriched genes from the mevalonate-dependent isoprenoid biosynthesis pathway. Relative abundance of genes involved in isoprenoid biosynthesis and grouped by enzyme commission (EC) numbers for the mevalonate dependent and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways in cecum samples from male C57 BL6 mice following oral gavage with sesame oil vehicle or 0.3, 3, or 30 \(\upmu\)g/kg TCDD every 4 days for 28 days (\(n=3\)). Individual box plots are also numbered with the EC number matching the enzymatic step in pathway schematic. Adjusted \(p\)-values (adj. \(p\)) were determined by the Maaslin2 R package.