## 2 Results
### TCDD Enriched for Mevalonate-Dependent Isoprenoid Biosynthesis
Individual boxplots are numbered with the EC number matching the enzymatic step in pathway schematic. Adjusted \(p\)-values (adj. \(p\)) were determined by MAASLIN2. Abbreviations: UDP-N-acetyl-alpha-D-glucosamine (UDP-GlcNAcNac), UDP-N-acetyllmuramate (UDP-MurNAc), UDP-N-acetyl-alpha-D-muramoyl-L-alanine (UDP-MurNAc-ALA), UDP-N-acetyl-alpha-D-muramoyl-L-L-alanyl-D-glutamate(UDP-MurNAc-Ala-D-Glu), UDP-N-acetylmuramoyl-L-alanyl-gamma-D-glutamyl-meso-2,6-diaminopimelate (UDP-MurNAc-Ala-D-Glu-m-DAP), D-Alanyl-D-alanine (D-Ala-D-Ala), UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-6-carboxy-L-lysisyl-D-alanyl-D-alanine (UDP-MurNAc-Ala-D-Glu-m-DAP-D-Ala-D-Ala), Undecaprenyl-diphospho-N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-2,6-diaminopimeloy-D-alanyl-D-alanine (Und-PP-MurNAc-Ala-D-Glu-m-DAP-D-Ala-D-Ala),Undecaprenyl-diphospho-N-acetylmuramoyl-(N-acetylglucosamine)-L-alanyl-D-glutamyl-meso-2,6-diaminopimeloy-D-alanyl-D-alanine (Und-PP-MurNAc-GlcNAcN-Ala-D-Glu-m-DAP-D-Ala-D-Ala). In the mouse ecum dataset, species contributing to \(\alpha\)-succinylbenzene metaquinone biosynthesis pathway included _Escherichia coli_, several Bacteroides (e.g., _Bacteroides vulgaris_ and _Bacteroides caecimuris_), and Lactobacillus species (e.g., _L. reuteri_) (Figure 7B and Table S6). No one species was annotated to the entire set of enzymes needed for de novo biosynthesis from chorimate, however _B. vulgaris_ was annotated for 6 out of 9 genes in the pathway (Table S6). O-Succinylbenzoate synthase (Figure 6A, EC 4.2.1.113, step 4) was increased by 30 \(\upmu\)g/kg TCDD with _L. reuteri_ being the major contributor to relative abundance (Figure 7, step 4). Figure 7: TCDD-elicited effects on menaquinone biosynthesis. (**A**) Relative abundance of menaquinone biosynthesis EC annotations identified in the metagenomic dataset. Individual stacked bar plots are labeled with the EC number matching the enzymatic step in pathway schematic. Stacked bar plots of annotated EC numbers involved in menaquinone biosynthesis. Values are mean relative abundance (\(n=3\)) classified to the respective species and in ecum samples from male C57 BL/6 mice following oral gavage with sesame oil vehicle or 0.3, 3, or 30 \(\upmu\)g/kg TCDD every 4 days for 28 days. (**B**) Menaquinone biosynthesis EC numbers classified to Lactobacillus species in the ecum metagenomic datasets. Adjusted \(p\)-values (adj. \(p\)) were determined by Maaslin2 R package. Lactobacillus species annotated to menaquinone biosynthesis included _L. reuteri_, _L. murinus_, and _L. johnsonii_. Among annotated menaquinone biosynthesis EC numbers, _L. reuteri_ was among the identified Lactobacillus species that had the highest relative abundance and most menaquinone EC annotations (Figure 7B). _L. reuteri_ also had annotations in samples for EC numbers involved in the final steps of the shikimate pathway responsible for chorismate biosynthesis (Table S7). In the cirrhosis samples, several EC numbers representing the initial menaquinone biosynthesis steps were also increased in compensated and decompensated patients (Figure 8, steps 1,3-5), including o-succinylbenzoate synthase (Figure 8, EC 4.2.1.113, step 4). However, _L. reuteri_ was not among species classified to this EC number. Species classified to all EC numbers comprising the complete pathway included _E. coli_, and Klebsiella species, such as _K. pneumoniae_ and Citrobacter species. _L. reuteri_, were not annotated to any menaquinone biosynthesis genes in healthy or compensated patients, but several EC numbers in the decompensated group (EC 6.2.1.26, 4.1.3.6, and 2.1.1.163), which are involved in later stages of menaquinone biosynthesis (Table S8). Figure 8: Menaquinone biosynthesis genes are increased in cirrhotic patients. Human3 analysis of fecal metagenomic dataset of patients with healthy (H, red, \(n\) = 52), compensated (C, green, \(n\) = 48), or decompensated (D, blue, \(n\) = 44) liver cirrhosis diagnosis for EC numbers in menaquinone biosynthesis.