Catechols have important applications in the pharmaceutical, food, cosmetic, and functional material industries. 4-hydroxyphenylacetate-3-hydroxylase (4HPA3H), a two-component enzyme system comprising HpaB (monooxygenase) and HpaC (FAD oxidoreductase), demonstrates significant potential for catechol production because it can be easily expressed, is highly active, and exhibits ortho-hydroxylation activity toward a broad spectrum of phenol substrates. HpaB determines the ortho-hydroxylation efficiency and substrate spectrum of the enzyme; therefore, studying its structure–activity relationship, improving its properties, and developing a robust HpaB-conducting system are of significance and value; indeed, considerable efforts have been made in these areas in recent decades. Here, we review the classification, molecular structure, catalytic mechanism, primary efforts in protein engineering, and industrial applications of HpaB in catechol synthesis. Current trends in the further investigation of HpaB are also discussed.

4-Hydroxyphenylacetate-3-hydroxylase (4HPA3H; EC 1.14.14.9) is a heterodimeric flavin-dependent monooxygenase complex that catalyzes the ortho-hydroxylation of resveratrol to produce piceatannol. Piceatannol has various health benefits and valuable applications in food, medicine, and cosmetics. Enhancing the catalytic activity of 4HPA3H toward resveratrol has the potential to benefit piceatannol production. In this study, the critical amino acid residues in the substrate pocket of 4HPA3H that affect its activity toward resveratrol were identified using semi-rational engineering. Two key amino acid sites (I157 and A211) were discovered and the simultaneous “best” mutant I157L/A211D enabled catalytic efficiency (Kcat/Km—resveratrol) to increase by a factor of 4.7-fold. Molecular dynamics simulations indicated that the increased flexibility of the 4HPA3H substrate pocket has the potential to improve the catalytic activity of the enzyme toward resveratrol. On this basis, we produced 3.78 mM piceatannol by using the mutant I157L/A211D whole cells. In this study, we successfully developed a highly active 4HPA3H variant for the hydroxylation of resveratrol to piceatannol.

γ-aminobutyric acid (GABA) has essential physiological functions in the human body. A novel method using glutamate decarboxylase (GAD) entrapped in polyvinyl alcohol (PVA)-sodium alginate (SA) capsules provides a green biological strategy for GABA synthesis. In this investigation, the stability range of immobilized GAD was effectively broadened, and immobilized GAD could be repeatedly used as a batch and fixed-bed column catalyst. The immobilized enzymes were stable and retained 89% of their activity in a pH range of 4.0–5.6, while there was an approximately 50% decrease in free GAD activity in the pH range of 4.8 ± 0.4. The immobilized GAD affinity to the substrate improved, and this was evidenced by the apparent decrease in K_m to 13.3 mmol/L from the 30.9 mmol/L for free GAD. The immobilized GAD retained >90.6% activity after eight cycles and a near-100% enzyme activity retention after 120 h of a continuous fixed-bed column catalyst operation. This study has thus presented an effective PVA–SA–GAD immobilization method that could be used to continuously scale-up GABA biosynthesis.

Salvianic acid A (SAA), as the main bioactive component of the traditional Chinese herb Salvia miltiorrhiza, has important application value in the treatment of cardiovascular diseases. In this study, a two-step bioprocess for the preparation of SAA from l-DOPA was developed. In the first step, l-DOPA was transformed to 3,4-dihydroxyphenylalanine (DHPPA) using engineered Escherichia coli cells expressing membrane-bound L-amino acid deaminase from Proteus vulgaris. After that, the unpurified DHPPA was directly converted into SAA by permeabilized recombinant E. coli cells co-expressing d-lactate dehydrogenase from Pediococcus acidilactici and formate dehydrogenase from Mycobacterium vaccae N10. Under optimized conditions, 48.3 mM of SAA could be prepared from 50 mM of l-DOPA, with a yield of 96.6%. Therefore, the bioprocess developed here was not only environmentally friendly, but also exhibited excellent production efficiency and, thus, is promising for industrial SAA production.

Salvianolic acid A (SAA) has an important application value for preventing and treating cardiovascular diseases. In this study, we developed a novel multienzyme cascade system for the efficient biosynthesis of SAA, utilizing l-tyrosine (l-Tyr) as a cost-effective and stable starting material. The cascade system incorporated four enzymes: membrane-bound l-amino acid deaminase from Proteus vulgaris (Pvml-AAD), d-lactate dehydrogenase from Pediococcus acidilactici (Pad-LDH), 4-hydroxyphenylacetate 3-hydroxylase from Escherichia coli (EcHpaBC), and formate dehydrogenase from Mycobacterium vaccae N10 (MvFDH). All reaction steps in the cascade system were thermodynamically favorable. In addition, to avoid generating an unstable intermediate (3,4-dihydroxyphenyl-pyruvate, DHPPA), which was produced owing to the promiscuity of EcHpaBC and Pad-LDH, we performed the cascade system according to the reaction sequence of deamination, chiral reduction, and ortho-hydroxylation. Under optimized conditions, the developed cascade system yielded 81.67 mM SAA from an initial concentration of 100 mM l-Tyr, corresponding to a yield of 81.67%.