Generation of Oxidoreductases with Dual Alcohol Dehydrogenase and Amine Dehydrogenase Activity

Abstract

The l‐lysine‐ϵ‐dehydrogenase (LysEDH) from Geobacillus stearothermophilus naturally catalyzes the oxidative deamination of the ϵ‐amino group of l‐lysine. We previously engineered this enzyme to create amine dehydrogenase (AmDH) variants that possess a new hydrophobic cavity in their active site such that aromatic ketones can bind and be converted into α‐chiral amines with excellent enantioselectivity. We also recently observed that LysEDH was capable of reducing aromatic aldehydes into primary alcohols. Herein, we harnessed the promiscuous alcohol dehydrogenase (ADH) activity of LysEDH to create new variants that exhibited enhanced catalytic activity for the reduction of substituted benzaldehydes and arylaliphatic aldehydes to primary alcohols. Notably, these novel engineered dehydrogenases also catalyzed the reductive amination of a variety of aldehydes and ketones with excellent enantioselectivity, thus exhibiting a dual AmDH/ADH activity. We envisioned that the catalytic bi‐functionality of these enzymes could be applied for the direct conversion of alcohols into amines. As a proof‐of‐principle, we performed an unprecedented one‐pot “hydrogen‐borrowing” cascade to convert benzyl alcohol to benzylamine using a single enzyme. Conducting the same biocatalytic cascade in the presence of cofactor recycling enzymes (i.e., NADH‐oxidase and formate dehydrogenase) increased the reaction yields. In summary, this work provides the first examples of enzymes showing “alcohol aminase” activity.

Engineered Corynebacterium glutamicum as the Platform for the Production of Aromatic Aldehydes

“…In this study, 27 proteins were systematically selected based on literature search and bioinformatics analysis ( Supplementary Table S5 ) and a comprehensive in vivo screening was performed using the model strain, the 4-HB alcohol-producing C. glutamicum . Due to enzyme promiscuity with broad substrate spectrum ( Tseliou et al, 2021 ), we hypothesized that each knockout of the 20 genes in GAS355 would lead to selection of several promising genes responsible for reduction of 4-HB aldehyde to 4-HB alcohol. Surprisingly, only the NCgl0324 knockout mutant accumulated 2.9-fold more 4-HB aldehyde and 4.8-fold less 4-HB alcohol than the control strain, indicating that the NCgl0324 gene product plays a pivotal role in the conversion of 4-HB aldehyde to 4-HB alcohol.…”

Reductive aminations by imine reductases: from milligrams to tons

“…The simplicity of this route prompted protein engineering of LeuDH, targeted at altering the carboxylate binding pocket of the enzyme, and yielded a mutant for the enantioselective reductive amination of the aliphatic methyl isobutyl ketone (MIBK, 7) to give (R)-1,3-dimethylbutylamine 8 with 99.8% e.e. 23 There are now numerous reports detailing the subsequent evolution and application of engineered AmDHs, [24][25][26] which make use of other AADH scaffolds including PheDH and also an 3-deaminating L-lysine dehydrogenase (LE-AmDH), 27,28 for the transformation of non-native substrates. More recently, a family of naturally occurring AmDHs was identied, 29,30 that could transform simple ketones to amines using ammonia.…”

Engineering the activity of amine dehydrogenase in the asymmetric reductive amination of hydroxyl ketones

“…9 f Recently, ε-deaminating l -lysine dehydrogenase as a scaffold was engineered to catalyze the transformation of ketones to amines in high stereo- and chemo-selectivity. 10 In addition, implementation of AmDH in E. coli host cells has also expanded its applications. 11 Apart from ketone substrates, it is of interest to note that the engineered AmDHs are also capable of converting hydroxyl ketones to chiral amino alcohols in the most recent studies.…”