Figure 3
From: Therapeutic enzyme engineering using a generative neural network
Auxotrophic growth assay for evaluation of hOTC variants. (A) Schematic of the urea cycle pathway in humans (left) and arginine biosynthesis in K12 E.coli (right). The first two enzymatic steps, catalyzed by CPS1 and OTC, in humans occur in the mitochondria. The remaining steps occur in the cytosol. Unlike humans, E. coli possess two genes each in place of CPS1 (carA/carB) and OTC (argF/argI). (B) Phenotype of the ∆argF∆argI double knockout E. coli strain. ∆argF∆argI cannot grow in the absence of arginine (top panel). Growth can be rescued either by arginine supplementation (middle panel) or by transformation of an external plasmid encoding hOTC (bottom panel). Black markings on the plates indicate growth media and plating conditions, see Methods for details. (C) Growth curves of the ∆argF∆argI strain expressing different OTC genes. The auxotroph was transformed with plasmids encoding YFP (negative control, black open circles), hOTC (green), E. coli ArgF (pink) or E. coli ArgI (purple) and growth was assessed in liquid medium lacking arginine. Solid black circles represent the growth rate of the strain when arginine is supplemented in the medium. (D) Growth rescue of the auxotroph by consensus and VAE hOTC variants. OD600 values at 8 h are shown for the auxotroph transformed with hOTC (green) or the variants (blue and orange for consensus and VAE, respectively). The hOTC data were collected multiple times within the same experiment and on separate days to show the spread in values. Only single data points were collected for the consensus and VAE variants; however, as seen by the data most of the variants have OD600 values greater than the highest measurement recorded for hOTC.
