Enhanced Thermostability of Pseudomonas nitroreducens Isoeugenol Monooxygenase by the Combinatorial Strategy of Surface Residue Replacement and Consensus Mutagenesis

Vanillin has many applications in industries. Isoeugenol monooxygenase (IEM) can catalyze the oxidation of isoeugenol to vanillin in the presence of oxygen under mild conditions. However, the low thermal stability of IEM limits its practical application in the biosynthesis of natural vanillin. Herein, two rational strategies were combined to improve the thermostability of IEM from Pseudomonas nitroreducens Jin1. Two variants (K83R and K95R) with better thermostability and one mutant (G398A) with higher activity were identified from twenty candidates based on the Surface Residue Replacement method. According to the Consensus Mutagenesis method, one mutant (I352R) with better thermostability and another mutant (L273F) with higher activity were also identified from nine candidates. After combinatorial mutation, a triple mutant K83R/K95R/L273F with the best thermostability and catalytic efficiency was generated. Compared with the wild-type IEM, the thermal inactivation half-lives (t1/2) of K83R/K95R/L273F at 25 °C, 30 °C, and 35 °C increased 2.9-fold, 11.9-fold, and 24.7-fold, respectively. Simultaneously, it also exhibited a 4.8-fold increase in kcat, leading to a 1.2-fold increase in catalytic efficiency (kcat/Km). When the whole cell of K83R/K95R/L273F was applied to the biotransformation of isoeugenol on preparative scale, the vanillin concentration reached 240.1 mM with space-time yield of 109.6 g/L/d, and vanillin was achieved in 77.6% isolated yield and >99% purity.

Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.