The extensive use of polyethylene terephthalate (PET) has resulted in severe environmental pollution and ecological stress. Despite advances in PET recycling, current processes struggle to achieve high product value, as the complete conversion to terephthalic acid remains energetically demanding and economically inefficient. The catalytic promiscuity inherent in natural enzyme evolution holds great promise for providing novel candidates to accelerate PET biodegradation and upcycling. Herein, we report for the first time that the acyltransferase metA from Mycobacterium tuberculosis (MtMetA) catalyzes the conversion of bis(2-hydroxyethyl) terephthalate (BHET), an intermediate of PET hydrolysis, into the high-value monomer mono(2-hydroxyethyl) terephthalate (MHET). Guided by molecular dynamics (MD) simulations, we applied a catalytic barrier-minimization strategy to optimize the active-site environment of MtMetA, yielding engineered variants, notably ΔBarrier2 and ΔBarrier3. Specifically, ΔBarrier2 achieved a 3.1-fold increase in MHET yield, while ΔBarrier3 demonstrated a 3.2-fold enhancement in catalytic efficiency (kcat/KM) relative to the wild-type. The truncated MtMetA variants also exhibited enhanced robustness, showing improved thermostability (3.5-fold increase in residual activity at 60 °C for ΔBarrier3), as well as higher tolerance to metal ions and organic solvents. Specifically, ΔBarrier2 displayed a 7.2-fold increase in product yield in Ca2+-containing systems, while ΔBarrier3 retained 2.3-fold higher residual activity in the presence of 50 % (v/v) isopropanol. MD simulations revealed that an enlarged active pocket and a shortened nucleophilic attack distance synergistically govern the enhanced catalytic activity and robustness. This work expands the enzymatic toolbox for PET recycling and targeted BHET degradation, advancing sustainable plastic waste management through biocatalytic innovations.
The accumulation of poly(ethylene terephthalate) (PET) waste in the environment poses a severe ecological threat. While extensive research has focused on high-performance PET degradation by thermophilic enzymes, PET hydrolases are efficient under lower-temperature conditions, which would better align with green and energy-saving demands the energy-efficient centralized treatment of PET waste remains underexplored. Herein, based on our previously engineered mesophilic IsPETaseS121P/D186A, we performed rational design to improve its PET degradation activity at relatively low temperature. Through rational design methods including salt bridge construction and hydrophobic engineering, we obtained effective variant PADFL (IsPETaseS121P/D186A/N246D/Y87F/N233L), demonstrating an 8.37-fold activity of IsPETaseS121P/D186A in PET degradation efficiency (56.52-fold of IsPETase). Molecular dynamics (MD) simulations further revealed stronger PET binding affinity, enhanced hydrogen bonding network, and reduced acylation energy barrier. Overall, this work enhances the degradation activity of the PET hydrolase through energy-based rational design and obtained optimized variant PADFL, offering a promising candidate for future efficient PET degradation under mild temperature conditions.
Enantioselective transamination of prochiral ketones is an indispensable transformation in the pharmaceutical and fine chemical industries. Transaminases have emerged as valuable biocatalysts for the preparation of a wide range of chiral amines through either kinetic resolution or asymmetric synthesis. Here, we explored the synthetic potential of the engineered amine transaminase 3FCR‐4M through systematic evaluation of combinatorial variants in both kinetic resolution and asymmetric synthesis mode. This dual assessment not only revealed synergistic effects between individually beneficial mutations, but also offered insight into the limited predictive value of kinetic resolution activity for asymmetric synthesis efficiency. Notably, we report for the first time the transaminase‐catalyzed direct amination of a terminal alkyne‐bearing substrate. The transamination of 1‐(4‐ethynylphenyl)ethanone proceeded with >99% conversion and excellent enantioselectivity (>99%ee), opening a new perspective for the integration of transaminase catalysis into click chemistry‐based synthetic workflows.
Konstantin F G Weigmann, Nils Michels, Mark Doerr et al.· ChemCatChem· 0 citations
Fe(II)/α-ketoglutarate (αKG)-dependent halogenases that catalyze site-selective C-H halogenation of free substrates without carrier proteins are attractive biocatalysts for diversifying pharmaceuticals and agrochemicals. However, their application remains limited by the narrow diversity of natural halogenases, poor stability, and restricted substrate scope. Protein stabilization is a common strategy to enhance mutational tolerance during enzyme engineering; however, Fe(II)/αKG-dependent halogenases are structurally closely related to hydroxylases, and consensus-based stabilizing mutations risk shifting activity toward competing hydroxylation. To address this challenge, a workflow was designed to improve structural stability while preserving substrate and product specificity by combining computational identification of substrate-recognition residues with Rosetta-based stabilization. This approach was applied to the l-lysine 4-chlorinase BesD from Streptomyces cattleya as a model enzyme. The resulting variants exhibited a T50 increase of more than 45 °C with no loss of substrate specificity or regioselective chlorination activity and served as stable seed enzymes for subsequent substrate scope expansion. This strategy, which systematically excludes substrate recognition- and/or reaction-selectively related residues from the mutation space to preserve native enzyme function, may provide a versatile platform for stabilizing enzymes without substantially compromising catalytic activity.
Teppei Niide, Keita Miyawaki, Hyuga Miyamoto et al.· ACS Chemical Biology· 0 citations
We report the discovery and engineering of a new (R)-selective transaminase (RTA-223, UniProt W9Z089), identified from Capronia coronata. The wild-type enzyme was found to display broad activity on a range of bulky aryl and alkyl ketone substrates, with high enantioselectivity using both d-alanine and isopropyl amine as amine donors. We then investigated activity towards pro-sitagliptin ketone, a well-known challenging pharmaceutical target of industrial significance. Although no forward amination of pro-sitagliptin was initially detected by wild-type RTA-223, low-level activity in the reverse deamination reaction enabled engineering without the need for truncated sitagliptin analogues. Adopting this reverse screening strategy, two active-site mutations unlocked forward amination activity, and subsequent rounds of directed evolution delivered a quadruple mutant (H53L/V60G/F113A/V148A) capable of converting pro-sitagliptin to (R)-sitagliptin with high levels of stereoselectivity (e.r. 93 : 7). We further performed a kinetic analysis of the candidates from across the evolution process, using a previously reported coupled assay system linked to d-alanine oxidation. This work validates reverse screening as an effective approach for evolving transaminases toward sterically hindered substrates and highlights RTA-223 as a promising candidate for further biocatalyst development.
Matthew Treadell, Gareth R E Surman, G. Ford et al.· RSC Chemical Biology· 0 citations
Rhamnosyltransferases are remarkable biocatalysts for the synthesis of rhamnosylated natural products with valuable physicochemical properties and bioactivities. However, their application is hindered by poor stability and low catalytic efficiency. Here, we achieved simultaneous enhancement of catalytic efficiency and stability of a 1,2-rhamnosyltransferase by a distal mutational engineering strategy. The variant M9 exhibited a 589.43-fold extension in half-life, a 2.5 °C increase in Tm, a 13.6 °C increase in T50, and an 8- to 763-fold increase in activity toward diverse flavonoids compared with the wild type. Molecular dynamics simulations provided insights into enhanced thermostability and catalytic efficiency. To demonstrate its synthetic utility, a whole-cell biocatalytic system was constructed in E. coli by coexpressing M9 and UDP-rhamnose synthase, enabling a neohesperidin titer of 1.14 g L–1 without exogenous sugar donor supplementation. This study presents a practical enzyme engineering strategy for simultaneous activity–stability enhancement in glycosyltransferases and provides a promising biocatalyst for rhamnosylated natural product biosynthesis.
Wenjuan Dai, Chaorong Guo, Hongyan Yang et al.· Journal of Agricultural and...· 0 citations
Poly(ethylene terephthalate) (PET) is a widely used plastic whose persistence and improper disposal pose serious environmental and health risks. In this study, three novel PET hydrolases TbPETase, AbPETase, and AfPETase were identified from Thermoanaerobacterales, Acidimicrobiales, and Actinokineospora fastidiosa, respectively. Among these, TbPETase exhibited the highest enzymatic activity and thermostability. Based on structural analysis, we performed semirational truncations targeting the intrinsically disordered N- and C-terminal regions of TbPETase, generating two improved variants ΔN36 and ΔC4. The double mutant, TbPETaseΔN36/ΔC4, demonstrated a 2.3-fold increase in overall enzymatic activity and a 2.6-fold improvement in catalytic efficiency (kcat/Km) compared to the wild-type enzyme, along with significantly enhanced thermal stability. Molecular dynamics simulations revealed that the removal of flexible terminal regions increased the overall structural rigidity of TbPETaseΔN36/ΔC4. This structural stabilization was associated with the formation of a hydrogen bond at T215 and a π–π stacking interaction at W193. In a 100 mL one-pot reaction system, the combination of TbPETaseΔN36/ΔC4 with an engineered BMHETase variant, BMHETase6M, achieved 81.2% degradation of semicrystalline PET powder at 60 °C over 60 h, yielding terephthalic acid as the major product. These findings demonstrate the potential of TbPETaseΔN36/ΔC4 as a highly efficient and industrially applicable biocatalyst for PET degradation.
Lin Zhang, Keyan Chen, Zhiwen Xi et al.· ACS Synthetic Biology· 0 citations