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Recreating the natural evolutionary trend in key microdomains provides an effective strategy for engineering of a thermomicrobial N-demethylase.

ABSTRACT: N-demethylases have been reported to remove the methyl groups on primary or secondary amines, which could further affect the properties and functions of biomacromolecules or chemical compounds; however, the substrate scope and the robustness of N-demethylases have not been systematically investigated. Here we report the recreation of natural evolution in key microdomains of the Thermomicrobium roseum sarcosine oxidase (TrSOX), an N-demethylase with marked stability (melting temperature over 100 °C) and enantioselectivity, for enhanced substrate scope and catalytic efficiency on -C-N- bonds. We obtained the structure of TrSOX by crystallization and X-ray diffraction (XRD) for the initial framework. The natural evolution in the nonconserved residues of key microdomains-including the catalytic loop, coenzyme pocket, substrate pocket, and entrance site-was then identified using ancestral sequence reconstruction (ASR), and the substitutions that accrued during natural evolution were recreated by site-directed mutagenesis. The single and double substitution variants catalyzed the N-demethylation of N-methyl-L-amino acids up to 1800- and 6000-fold faster than the wild type, respectively. Additionally, these single substitution variants catalyzed the terminal N-demethylation of non-amino-acid compounds and the oxidation of the main chain -C-N- bond to a -C=N- bond in the nitrogen-containing heterocycle. Notably, these variants retained the enantioselectivity and stability of the initial framework. We conclude that the variants of TrSOX are of great potential use in N-methyl enantiomer resolution, main-chain Schiff base synthesis, and alkaloid modification or degradation.

SUBMITTER: Xin Y

PROVIDER: S-EPMC8892156 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Recreating the natural evolutionary trend in key microdomains provides an effective strategy for engineering of a thermomicrobial N-demethylase.

Xin Yu Y Shen Chen C Tang Mengwei M Guo Zitao Z Shi Yi Y Gu Zhenghua Z Shao Jun J Zhang Liang L

The Journal of biological chemistry 20220204 3

N-demethylases have been reported to remove the methyl groups on primary or secondary amines, which could further affect the properties and functions of biomacromolecules or chemical compounds; however, the substrate scope and the robustness of N-demethylases have not been systematically investigated. Here we report the recreation of natural evolution in key microdomains of the Thermomicrobium roseum sarcosine oxidase (TrSOX), an N-demethylase with marked stability (melting temperature over 100 ...[more]

PMID: 35124004

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Project description:Wine produced at low temperature is often considered to improve sensory qualities. However, there are certain drawbacks to low temperature fermentations: e.g. low growth rate, long lag phase, and sluggish or stuck fermentations. Selection and development of new Saccharomyces cerevisiae strains well adapted at low temperature is interesting for future biotechnological applications. This study aimed to select and develop wine yeast strains that well adapt to ferment at low temperature through evolutionary engineering, and to decipher the process underlying the obtained phenotypes. To this end, we used a pool of 27 commercial yeast strains and set up batch serial dilution experiments to mimic wine fermentation conditions at 12 ºC. Evolutionary engineering was accomplished by using the natural yeast mutation rate and mutagenesis procedures. One strain (P5) outcompeted the others under both experimental conditions and was able to impose after 200 generations. The evolved strains showed improved growth and low-temperature fermentation performance compared to the ancestral strain. This improvement was acquired only under inositol limitation. The transcriptomic comparison between the evolved and parental strains showed the greatest up-regulation in four mannoprotein coding genes, which belong to the DAN/TIR family (DAN1, TIR1, TIR4 and TIR3). Genome sequencing of the evolved strain revealed the presence of a SNP in the GAA1 gene and the construction of a site-directed mutant (GAA1Thr108) in a derivative haploid of the ancestral strain resulted in improved fermentation performance. GAA1 encodes a GPI transamidase complex subunit that adds GPI, which is required for inositol synthesis, to newly synthesized proteins, including mannoproteins. Thus we demonstrate the importance of inositol and mannoproteins in yeast adaptation at low temperature and the central role of the GAA1 gene by linking both metabolisms. The first aim of this study was to assess the most competitive strains that grow under wine fermentation conditions at low temperature. To this end, we performed a growth competition assay with 27 commercial wine strains inoculated at equal population size in synthetic grape must. In spite of the economical and industrial importance of these strains, their phenotypic variation in the main enological traits, particularly those related to optimum growth temperature, and their ability to adapt to low temperature fermentation have been poorly investigated. The second goal was to obtain an improved strain to grow and ferment at low temperature by evolutionary engineering. For this purpose, we maintained growth competition in synthetic grape must during 200 generations to select for the mutations that produce phenotypes with improved growth in this medium. One of these evolved cultures was previously treated with ethyl methanesulfonate (EMS) to increase the mutation rate. Finally, we aimed to decipher the molecular basis underlying this improvement by analyzing the genomic and transcriptional differences between the parental strain and the strain evolved at low temperature.

Dataset Information

Recreating the natural evolutionary trend in key microdomains provides an effective strategy for engineering of a thermomicrobial N-demethylase.

Publications

Recreating the natural evolutionary trend in key microdomains provides an effective strategy for engineering of a thermomicrobial N-demethylase.

Similar Datasets

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