Semirational Design of 4-Hydroxyphenylpyruvate Dioxygenase for Efficient and Regioselective Production of 3-Hydroxy-3-methylbutyrate

3-Hydroxy-3-methylbutyrate (HMB), an important dietary supplement, can enhance the quality of life for sedentary and elderly people. However, the catalytic efficiency of the pathway enzymes limited the biosynthesis of HMB. Here, we identified an efficient 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) from <i>Bacteroides barnesiae</i> (<i>Bb</i>HPPD) that converted l-Leucine (l-Leu) to HMB together with an efficient L-amino acid deaminase (L-AAD) in lab-preserved <i>Proteus mirabilis</i> (<i>Pm</i>L-AAD^Q92A). The regioselective hydroxylation mechanism of the decarboxylated intermediate of 4-methyl-2-oxopentanoic acid (α-KIC) catalyzed by <i>Bb</i>HPPD was elucidated for the first time. Structure-guided semirational design of <i>Bb</i>HPPD generated a mutant M4. Compared to the wild type, the titer of mutant M4 increased by 254% when using α-KIC as a substrate. Finally, the engineered <i>Escherichia coli</i> 06 strain achieved the HMB titer of 11.6 g/L, a molar conversion of 65%, and a space-time yield of 0.64 g/(L·h) (the highest to date) in 18 h. This study lays the foundation for industrial-scale biosynthesis of HMB.