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 (CgDAPDHBC621/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.
Highlights
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
A four-enzyme cascade pathway enabling the transformation of l-tyrosine to D-HPG was developed and the pathway was reconstructed in vivo
Cascade design and in vitro reconstruction of the D‐HPG‐biosynthesis pathway Comparison of the structures of l-tyrosine and D-HPG showed that the l-tyrosine side chain contained one more carbon than the D-HPG side chain, and that the CH2 subunit at the α position of the l-tyrosine side chain cannot be removed by natural enzymes
Summary
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. Introduction of the best variant generated by protein engineering (CgDAPDHBC621/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. This pathway shows great potential to produce D-HPG, there are two obvious disadvantages that remain to be solved: (1) the oxidation process catalyzed by Hmo accumulates cytotoxic H2O2, which requires additional catalase for consumption of H2O2, complicating the reaction process; and (2) the transamination processes catalyzed by HpgT or HpgAT require an amino donor as the cosubstrate, resulting in a large increase in the total reaction costs Considering these limitations, it is urgent to develop suitable catalytic enzymes. DAPDH could be employed to support the reductive amination of 4-hydroxyphenylglyoxalate (HPGA) to D-HPG
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