Journal
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
Volume 88, Issue 9, Pages -Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/aem.00341-22
Keywords
isopropanol dehydrogenase; cofactor regeneration; thermostability evolution; cofactor specificity reversal; protein engineering
Categories
Funding
- National Key Research and Development Program of China [2019YFA0905000]
- National Natural Science Foundation of China [21536004, 21871085, 32071475]
- Natural Science Foundation of Shanghai [18ZR1408400]
- Fundamental Research Funds for the Central Universities [22221818014]
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This study improved the thermostability and catalytic activity of IPADH and successfully switched its cofactor preference from NAD(H) to NADP(H). The resulting variants showed great potential for cofactor regeneration and the engineering strategy could be applied to other oxidoreductases in the future.
Isopropanol dehydrogenase (IPADH) is one of the most attractive options for nicotinamide cofactor regeneration due to its low cost and simple downstream processing. However, poor thermostability and strict cofactor dependency hinder its practical application for bioconversions. In this study, we simultaneously improved the thermostability (433-fold) and catalytic activity (3.3-fold) of IPADH from Brucella suis via a flexible segment engineering strategy. Meanwhile, the cofactor preference of IPADH was successfully switched from NAD(H) to NADP(H) by 1.23 x 10(6)-fold. When these variants were employed in three typical bioredox reactions to drive the synthesis of important chiral pharmaceutical building blocks, they outperformed the commonly used cofactor regeneration systems (glucose dehydrogenase [GDH], formate dehydrogenase [FDH], and lactate dehydrogenase [LDH]) with respect to efficiency of cofactor regeneration. Overall, our study provides two promising IPADH variants with complementary cofactor specificities that have great potential for wide applications. IMPORTANCE Oxidoreductases represent one group of the most important biocatalysts for synthesis of various chiral synthons. However, their practical application was hindered by the expensive nicotinamide cofactors used. Isopropanol dehydrogenase (IPADH) is one of the most attractive biocatalysts for nicotinamide cofactor regeneration. However, poor thermostability and strict cofactor dependency hinder its practical application. In this work, the thermostability and catalytic activity of an IPADH were simultaneously improved via a flexible segment engineering strategy. Meanwhile, the cofactor preference of IPADH was successfully switched from NAD(H) to NADP(H). The resultant variants show great potential for regeneration of nicotinamide cofactors, and the engineering strategy might serve as a useful approach for future engineering of other oxidoreductases. Oxidoreductases represent one group of the most important biocatalysts for synthesis of various chiral synthons. However, their practical application was hindered by the expensive nicotinamide cofactors used.
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