Figure 4. Bacterialization of yeast heme biosynthesis pathway genes at their native loci.
(A) A schematic of the yeast heme pathway shows the beginning of the pathway in mitochondria using succinyl-CoA and glycine as precursors. The subsequent enzymatic reactions are cytosolic up until the penultimate and ultimate reactions which are mitochondrial. (B) Growth kinetics of CRISPR-Cas9 engineered yeast heme pathway genes replaced with the corresponding bacterial genes at their native yeast loci show efficient replaceability in both BY4741 (red solid-line) and BY4742 (blue solid-line) yeast strains. The wild type BY4741 growth curve is shown as a comparison (black dotted-line). Mean and standard deviation plotted with N = 3.
Figure 4—figure supplement 1. Constitutive or native plasmid-based expression of the yeast heme biosynthesis genes generally efficiently complemented growth defects in the corresponding yeast gene deletion strains.
Heterologous expression of yeast genes Sc-HEM1, Sc-HEM2, Sc-HEM3, Sc-HEM4, Sc-HEM12, Sc-HEM13, Sc-HEM14 and Sc-HEM15 under the control of constitutive GPD promoter or native promoter efficiently rescued the growth defect of the corresponding yeast gene deletions respectively except in the case of Sc-HEM4. Sc-HEM4, when expressed constitutively, resulted in toxicity in the presence of the yeast gene at the native locus and did not complement the function in the absence of the yeast gene. This toxicity was relieved when the yeast gene was expressed under the control of the native yeast promoter.
Figure 4—figure supplement 2. Ec-hemA and Ec-hemL carry out the initial reaction in E. coli heme biosynthesis and are both required to complement Sc-HEM1 deletion in yeast, and non-orthologous yeast genes are replaced by E. coli genes that carry out the identical reaction.
(A) Expression of heme pathway genes of E. coli, Ec-hemA or Ec-hemL, individually cannot complement the lethal growth defect of the deletion of Sc-HEM1 gene in yeast. Co-expression of Ec-HemA and Ec-HemL efficiently rescued the growth defect of Sc-hem1 gene deletion in yeast. (B) Growth curves of yeast strains with deletions of Sc-hem4 and Sc-hem14 genes (grey solid-line) show functional replaceability (red solid-line) by the non-orthologous E. coli genes Ec-hemD and Ec-hemG that carry out identical enzymatic reactions to the corresponding yeast genes. The wild type BY4741 growth curve is shown as a comparison (black dotted-line). The empty vector control (grey solid-line) showed no such growth rescue in the presence of G418. (C) Growth curve of engineered yeast strain Sc-hem14Δ::Ec-hemG; Sc-hem15Δ::Ec-hemH in YPD medium harboring E. coli genes at the native yeast loci. The strain displayed a growth defect (red solid-line) compared to the wild type BY4741 strain (black dotted-line). Mean and standard deviation plotted with N = 3.
Figure 4—figure supplement 3. The penultimate and ultimate heme pathway enzymes in yeast are replaceable by their bacterial orthologs, in spite of mis-localizing to the plasma membrane.
EGFP-tagged Ec-HemG and Ec-HemH localize to the plasma membrane in yeast. The EGFP-tagged proteins do not localize to the mitochondria since no clear co-localization is observed with the Mitotracker red stain. EGFP-tagged Ec-HemG and Ec-HemH expression (red solid-line) efficiently rescue the growth defects of the respective yeast gene deletions (Sc-hem14 and Sc-hem15) (pink dotted-line) comparable to the wild type yeast (black dotted-line). Empty vector control is incapable of rescuing the growth defect of the deletion strains (grey dotted-line).
Figure 4—figure supplement 4. Confirmation of CRISPR-Cas9 mediated bacterialized yeast strains.
(A) Schematics of the yeast heme pathway gene loci carrying functionally replaceable E. coli genes while retaining their native promoters and terminators. The arrows indicate the primers used to confirm the replacement (refer to Supplementary file 3). (B) PCR amplification of expected size was obtained for each individual bacterialized yeast strains.