A multi-enzyme cascade for efficient production of d-p-hydroxyphenylglycine from l-tyrosine

Abstract

In this study, a four-enzyme cascade pathway was developed and reconstructed in vivo for the production of d-p-hydroxyphenylglycine (D-HPG), a valuable intermediate used to produce β-lactam antibiotics and in fine-chemical synthesis, from l-tyrosine. In this pathway, catalytic conversion of the intermediate 4-hydroxyphenylglyoxalate by meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum (CgDAPDH) was identified as the rate-limiting step, followed by application of a mechanism-guided “conformation rotation” strategy to decrease the hydride-transfer distance d_{(C6HDAP−C4NNADP)} and increase CgDAPDH activity. Introduction of the best variant generated by protein engineering (CgDAPDH^{BC621/D120S/W144S/I169P} with 5.32 ± 0.85 U·mg⁻¹ specific activity) into the designed pathway resulted in a D-HPG titer of 42.69 g/L from 50-g/L l-tyrosine in 24 h, with 92.5% conversion, 71.5% isolated yield, and > 99% enantiomeric excess in a 3-L fermenter. This four-enzyme cascade provides an efficient enzymatic approach for the industrial production of D-HPG from cheap amino acids.

Keywords

d-p-hydroxyphenylglycine')">d-p-hydroxyphenylglycine / meso-diaminopimelate dehydrogenase / Hydride transfer distance / l-tyrosine')">l-tyrosine / Protein engineering

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Funding

Fundamental Research Funds for the Central Universities(JUSRP21915)

Provincial Natural Science Foundation of Jiangsu Province(BK20200622)

Youth Program of National Natural Science Foundation of China(22008089)

General Program of National Natural Science Foundation of China(21878126)

Key Technologies R & D Program of Jiangsu Province(BE2018623)

National First-Class Discipline Program of Light Industry Technology and Engineering(LITE2018-20)