Rationally Engineering pH Adaptation of Acid‐Induced Arginine Decarboxylase from Escherichia coli to Alkaline Environments to Efficiently Biosynthesize Putrescine

Abstract Acid‐induced arginine decarboxylase AdiA is a typical homo‐oligomeric protein biosynthesizing alkaline nylon monomer putrescine. However, upon loss of the AdiA decamer oligomeric state at neutral and alkaline conditions the activity also diminishes, obstructing the whole‐cell biosynthesis of alkaline putrescine. Here, a structure cohesion strategy is proposed to change the pH adaptation of AdiA to alkaline environments based on the rational engineering of meridional and latitudinal oligomerization interfaces. After integrating substitutions of E467K at the latitudinal interface and H736E at the meridional channel interface, the structural stability of AdiA decamer and its substrate transport efficiency at neutral and alkaline conditions are improved. Finally, E467K_H736E is well adapted to neutral and alkaline environments (pH 7.0–9.0), and its enzymatic activity is 35‐fold higher than that of wild AdiA at pH 8.0. Using E467K_H736E in the putrescine synthesis pathway, the titer of putrescine is up to 128.9 g·L−1 with a conversion of 0.94 mol·mol−1 in whole‐cell catalysis. Additionally, the neutral pH adaptation of lysine decarboxylase, with a decamer structure similar to AdiA, is also improved using this cohesion strategy, providing an option for pH‐adaptation engineering of other oligomeric decarboxylases.

Calibration curves were prepared using different concentrations of putrescine.The quantification method for cadaverine was the same with that of putrescine.

Figure S1 . 3 Figure S2 .
Figure S1.Amino acid residue alignment of AdiAs from different species sources.Key residues at the pentameric ring interface and the channel interface are highlighted in yellow.

Figure S4 .
Figure S4.Establishment of a rapid whole-cell screening method for AdiA variants.a: Whole-cell catalytic system for arginine at pH 5.4 using strains BL21Δ3 and BL21Δ3-AdiA.BL21Δ3 was engineered by deleting genes encoding SpeA, SpeB and AdiA.BL21Δ3-AdiA

Figure S5 .
Figure S5.Primary screening of AdiA variants with an engineered substrate channel based on relative enzymatic activity.Primary screening experiments were performed in 1/15 mol• L -1 sodium potassium phosphate buffer (pH 6.0).

Figure S6 .
Figure S6.Substrate channel analysis.a: CaverDock results for AdiA and AdiA-M2 with arginine.b: Structural alignment of substrate channels of AdiA and AdiA-M2.AdiA was colored in green and purple, and AdiA-M2 was colored in bright blue and pink.c: Effect of the glutamate substitution at residue 736 on the AdiA substrate channel.Residues H736 (or

Figure S8 .
Figure S8.Whole-cell conversion of arginine to synthesize putrescine.a: Putrescine synthesis using strains containing different arginine decarboxylase in shake fermentation with 15 g•L -1 argnine as substrate.b: The pH value during whole-cell catalysis with ΔEGAP-SpeAB-AdiA-M2 in pH 7.0 PBS buffer at 42 °C.

Figure S9 .
Figure S9.Amino acid residue alignment of AdiA and CadA from E. coli BL21.Key acidic amino acid residues are highlighted in yellow.

Figure S10 .
Figure S10.Amino acid residue alignment of CadA from different species sources.Key acidic amino acid residues are highlighted in yellow.

Figure S11 .
Figure S11.Specific activity of CadA and CadA-M2 at alkaline pH.Enzymatic activity was determined in Tris-HCl buffer (pH 7.5, 8.0, 8.5, 9.0 or 9.5) at 37 °C.The error bars indicate the standard deviation of three biological replicates (n=3).

Figure S12 .
Figure S12.Whole-cell catalytic using engineered strains BL21-CadA and BL21-CadA-M2 overexpressing CadA and CadA-M2 in pH 7.0 PBS buffer at 37 °C.The error bars indicate the standard deviation of three biological replicates (n=3).

Table S1
Fit parameters of first-order exponential decay model A=A0e -kt for enzyme activity decay

Table S3 .
Information for primers used in this study PrimerSequence (5' to 3')