The meta-selective C–H functionalization of aromatic substrates lacking meta-directing electron-withdrawing groups remains challenging. To engineer the regioselective enzymatic conversion of biphenyl (BP) to 3,3′-dihydroxybiphenyl (33DHBP), an industrial precursor to high-performance polymers, substrate positioning within the active site must be precisely controlled. Here, we report the regioselective catalytic promiscuity engineering of toluene/o-xylene monooxygenase (ToMO) to achieve sequential, meta-selective hydroxylation of the non-natural biaryl BP, where distinct ToMO variants were engineered to catalyze meta-selective hydroxylation of BP, 3-hydroxybiphenyl (3HBP), or both. Variants to catalyze the meta-selective hydroxylation of BP were designed using an automated docking workflow, with predicted binding poses consistent with the observed regioselectivity; this resulted in the development of the I100V-E103V-F205G variant that hydroxylated BP with 100% apparent meta-selectivity. Additional substitutions, especially L268A and L402A, were introduced to widen the long hydrophobic active-site access channel, further improving meta-selective BP hydroxylation in Pseudomonas putida. Variants to catalyze the meta-selective hydroxylation of 3HBP were selected through a structure-based residue scan of 437 active-site substitutions, resulting in the identification of the I100V-E103V-F176H variant that hydroxylated 3HBP with over 90% apparent meta-selectivity and no activity toward BP. In addition, the combined I100V-E103V-I162Y-F205G variant hydroxylated 3HBP with over 90% apparent meta-selectivity and improved hydroxylation of BP. This study demonstrates structural and computational monooxygenase engineering for the regioselective hydroxylation of non-natural biaryls, enabling the production of 33DHBP as a valuable precursor to specialized polymers.
C. Vavricka, Takeshi Matsui, Satoshi Yuzawa et al.· ACS Catalysis· 0 citations
Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) is a biodegradable copolymer whose mechanical properties can be tuned by the 3-hydroxyhexanoate (3HHx) fraction. However, current industrial production largely relies on established hosts and plant oil-based feedstocks. Here, we developed Rhodobacter capsulatus SB1003 as a new PHBH-producing platform by focusing on the two key determinants of copolymer formation: polyhydroxyalkanoate (PHA) synthase substrate specificity and intracellular monomer supply. A PHA synthase with broad-substrate specificity was integrated into the native phaC locus to generate a heterologous phaC strain. All PHA production experiments were performed under anaerobic photoheterotrophic conditions in 8-mL screw-cap tubes containing 7.6 mL of medium and illuminated with continuous white light. During butyrate cultivation under these conditions, the engineered strain accumulated polymer up to 41.5% of cell dry weight and incorporated detectable 3HHx, whereas the wild type showed no 3HHx incorporation. To increase 3HHx-CoA availability from butyrate, we introduced C4-to-C6 precursor-supply modules involving β-ketothiolase (BktB)/β-ketoacyl-CoA reductase (PhaB) and crotonyl-CoA carboxylase/reductase (Ccr)/ethylmalonyl-CoA decarboxylase (Emd), but these modifications led to only marginal improvements in the 3HHx fraction. In contrast, supplying C6 or longer fatty acids under the same conditions markedly increased 3HHx incorporation; cultivation on hexanoate yielded PHBH containing 32.4 mol% 3HHx. Collectively, this study demonstrates PHBH biosynthesis in R. capsulatus and indicates that limited 3HHx-CoA supply rather than polymerization capacity is the primary bottleneck, providing a foundation for further pathway and host optimization toward flexible PHBH production from diverse substrates.
Kako Miura, Takayuki Shimizu, Tomohisa Hasunuma et al.· Journal of Bioscience and Bi...· 0 citations
