Mining and rational design of psychrophilic catalases using metagenomics and deep learning models

• Published in: Applied microbiology and biotechnology 2024-12, Vol.108 (1), p.31-31, Article 31
• Main Authors: Wu, Shuning, Xu, Guoshun, Su, Yongping, Huang, Huoqing, Xu, Xinxin, Zhang, Yuhong, Tian, Jian, Zhang, Wei, Zhang, Zhiwei, Liu, Bo
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acids; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Catalase; Catalase - genetics; Deep Learning; Design; Gene sequencing; Life Sciences; Metagenomics; Microbial Genetics and Genomics; Microbiology; Molecular evolution; Mutagenesis; Mutants; Site-directed mutagenesis; Soil
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-023-12926-1

A complete catalase-encoding gene, designated soiCat1 , was obtained from soil samples via metagenomic sequencing, assembly, and gene prediction. soiCat1 showed 73% identity to a catalase-encoding gene of Mucilaginibacter rubeus strain P1, and the amino acid sequence of soiCAT1 showed 99% similarity to the catalase of a psychrophilic bacterium, Pedobacter cryoconitis . soiCAT1 was identified as a psychrophilic enzyme due to the low optimum temperature predicted by the deep learning model Preoptem, which was subsequently validated through analysis of enzymatic properties. Experimental results showed that soiCAT1 has a very narrow range of optimum temperature, with maximal specific activity occurring at the lowest test temperature (4 °C) and decreasing with increasing reaction temperature from 4 to 50 °C. To rationally design soiCAT1 with an improved temperature range, soiCAT1 was engineered through site-directed mutagenesis based on molecular evolution data analyzed through position-specific amino acid possibility calculation. Compared with the wild type, one mutant, soiCAT1 S205K , exhibited an extended range of optimum temperature ranging from 4 to 20 °C. The strategies used in this study may shed light on the mining of genes of interest and rational design of desirable proteins. Key points • Numerous putative catalases were mined from soil samples via metagenomics. • A complete sequence encoding a psychrophilic catalase was obtained. • A mutant psychrophilic catalase with an extended range of optimum temperature was engineered through site-directed mutagenesis.