(A) hphd-1 and alh-8 (blue) are tightly connected to acdh-1, ech-6 and hach-1 (green) in a C. elegans intestinal functional network and are candidates to catalyze the fourth and fifth reactions of the propionate shunt, respectively. (B) Structure of CRISPR/Cas9-generated alh-8 mutant. Diagram of the mutation generated by CRISPR/Cas9-mediated genome editing using an sgRNA (red sequence) targeting alh-8. The alh-8(ww48) mutation consists of a 23 bp insertion and 399 bp deletion, and removes a part of the 5’UTR, the start codon, the first and second exons, and part of the third exon. Also shown is the Δhphd-1(ok3590) mutation. (C) Propionate toxicity dose response showing that Δhphd-1 and Δalh-8 mutants phenocopy acdh-1, ech-6 and hach-1 perturbation. (D) Δhphd-1 and Δalh-8 mutants exhibit partial lethality on low-B12 conditions. Like the Δacdh-1 mutant phenotype, Δhphd-1 and Δalh-8 mutant phenotypes were rescued by 64nM B12 supplementation or by Comamonas aquatica DA1877 (Coma.). The partial lethal phenotype of the Δhphd-1;Δpcca-1 double mutant was not rescued by B12. (E) Combined deletion of hphd-1 and pcca-1 renders the animals more sensitive to propionate than mutation in either gene alone. Note that Δhphd-1 may not be a null allele. (F) The C. elegans propionate breakdown shunt pathway comprises five genes: acdh-1, ech-6, hach-1, hphd-1 and alh-8.