Structure-guided engineering of branched-chain α-keto acid decarboxylase for improved 1,2,4-butanetriol production by in vitro synthetic enzymatic biosystem.

Efficient synthetic routes for biomanufacturing chemicals often require the overcoming of pathway bottlenecks by tailoring enzymes to improve the catalytic efficiency or even implement non-native activities. 1,2,4-butanetriol (BTO), a valuable commodity chemical, is currently biosynthesized from D-xylose via a four-enzyme reaction cascade, with the ThDP-dependent α-keto acid decarboxylase (KdcA) identified as the potential bottleneck. Here, to further enhance the catalytic activity of KdcA toward the non-native substrate α-keto-3-deoxy-xylonate (KDX), in silico screening and structure-guided evolution were performed. The best mutants, S286L/G402P and V461K, exhibited a 1.8- and 2.5-fold higher enzymatic activity in the conversion of KDX to 3,4-dihydroxybutanal when compared to KdcA, respectively. MD simulations revealed that the two sets of mutations reshaped the substrate binding pocket, thereby increasing the binding affinity for KDX and promoting interactions between KDX and cofactor ThDP. Then, when the V461K mutant instead of wild type KdcA was integrated into the enzyme cascade, a 1.9-fold increase in BTO titer was observed. After optimization of the reaction conditions, the enzyme cocktail contained V461K converted 60 g/L D-xylose to 22.1 g/L BTO with a yield of 52.1 %. This work illustrated that protein engineering is a powerful tool for modifying the output of metabolic pathway.

Coevolutionary analysis reveals a distal amino acid residue pair affecting the catalytic activity of GH5 processive endoglucanase from Bacillus subtilis BS-5.

EG5C-1, processive endoglucanase from Bacillus subtilis, is a typical bifunctional cellulase with endoglucanase and exoglucanase activities. The engineering of processive endoglucanase focuses on the catalytic pocket or carbohydrate-binding module tailoring based on sequence/structure information. Herein, a computational strategy was applied to identify the desired mutants in the enzyme molecule by evolutionary-coupling analysis; subsequently, four residue pairs were selected as evolutionary mutational hotspots. Based on iterative-saturation mutagenesis and subsequent enzymatic activity analysis, a superior mutant K51T/L93T has been identified away from the active center. This variant had increased specific activity from 4170 U/µmol of wild-type (WT) to 5678 U/µmol towards carboxymethyl cellulose-Na and an increase towards the substrate Avicel from 320 U/µmol in WT to 521 U/µmol. In addition, kinetic measurements suggested that superior mutant K51T/L93T had a high substrate affinity (Km ) and a remarkable improvement in catalytic efficiency (kcat /Km ). Furthermore, molecular dynamics simulations revealed that the K51T/L93T mutation altered the spatial conformation at the active site cleft, enhancing the interaction frequency between active site residues and substrate, and improving catalytic efficiency and substrate affinity. The current studies provided some perspectives on the effects of distal residue substitution, which might assist in the engineering of processive endoglucanase or other glycoside hydrolases.

Enhancing the catalytic activity of a GH5 processive endoglucanase from Bacillus subtilis BS-5 by site-directed mutagenesis.

Processive endoglucanases possess both endo- and exoglucanase activity, making them attractive discovery and engineering targets. Here, a processive endoglucanase EG5C-1 from Bacillus subtilis was employed as the starting point for enzyme engineering. Referring to the complex structure information of EG5C-1 and cellohexaose, the amino acid residues in the active site architecture were identified and subjected to alanine scanning mutagenesis. The residues were chosen for a saturation mutagenesis since their variants showed similar activities to EG5C-1. Variants D70Q and S235W showed increased activity towards the substrates CMC and Avicel, an increase was further enhanced in D70Q/S235W double mutant, which displayed a 2.1- and 1.7-fold improvement in the hydrolytic activity towards CMC and Avicel, respectively. In addition, kinetic measurements showed that double mutant had higher substrate affinity (Km) and a significantly higher catalytic efficiency (kcat/Km). The binding isotherms of wild-type EG5C-1 and double mutant D70Q/S235W suggested that the binding capability of EG5C-1 for the insoluble substrate was weaker than that of D70Q/S235W. Molecular dynamics simulations suggested that the collaborative substitutions of D70Q and S235W altered the hydrogen bonding network within the active site architecture and introduced new hydrogen bonds between the enzyme and cellohexaose, thus enhancing both substrate affinity and catalytic efficiency.

Ca2+ assisted glycosylation of phenolic compounds by phenolic-UDP-glycosyltransferase from Bacillus subtilis PI18.

A phenolic-UDP-glycosyltransferase Bs-PUGT from Bacillus subtilis PI18 was cloned and expressed in Escherichia coli BL21 (DE3). The purified Bs-PUGT could catalyze the glycosylation of tyrosol, 4-hydroxybenzyl alcohol, 2-hydroxybenzyl alcohol, caffeic acid, cinnamic alcohol, ferulic acid, and so on. This enzyme showed a high activity and stability over a broad pH range and was sensitive to temperature. Studies on the kinetic parameters indicated that the affinity of Bs-PUGT to UDP-G (Km) and its catalytic efficiency (Kcat) increased by 1.5-fold and 1.7-fold, respectively, with the addition of 10 mM Ca2+. The most effective glycosylation of caffeic acid catalyzed by whole-cell E. coli/Bs-PUGT was achieved with a molar yield of 78.3% in a system with pH 8.0, 30 °C, 25 g/L sucrose, 10 mM Ca2+, and 0.5 g/L substrate concentration. The addition of Ca2+ increased the molar yield of caffeic acid glucosides and shortened the reaction. This work proposes a strategy for the efficient glycosylation of phenolic compounds by microbe-derived glycosyltransferase assisted by metal ions.