Fig. 6: Directed evolution of MFL for screening MFL random mutants generated by EP-PCR. | Nature Communications

Fig. 6: Directed evolution of MFL for screening MFL random mutants generated by EP-PCR.

From: Biosynthesizing structurally diverse diols via a general route combining oxidative and reductive formations of OH-groups

Fig. 6

a Schematic representation of the screening principle: hydroxylation of leucine by MFL is coupled with oxidation of α-KG to succinate (red arrows), and therefore the growth of a succinate-auxotrophic strain is strictly depended on leucine hydroxylation. b Growth curves of Δ3A strain carrying pET28a or pET-mfl in the M9 medium added with 4 g/L yeast. The initial OD was set as 0.05. c Enzymatic activities of MFL and its two mutants MFLm1 and MFLm2 towards leucine and KIC. d Positions of mutation residues found in MFLm1 and MFLm2. Fe2+ is shown as a pink sphere. Leucine, α-KG, and two mutant residues are shown in sticks. L represents loop, αre presents alpha helix. e Growth curve (blue curves) and IPDO production (pink curves) in strain IP10 and IP11 cultivated in shake flasks. f Determination of valine and leucine concentrations in the fermentation broth of strain IP11 cultivated in shake flasks. Samples were taken every 12 h. For c, e, f the average, and s.d. of three biologically independent experiments are shown. Source data are provided as a Source Data file.

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