Structural and Functional Insights into a Nonheme Iron- and α-Ketoglutarate-Dependent Halogenase That Catalyzes Chlorination of Nucleotide Substrates.

Nonheme iron- and α-ketoglutarate (αKG)-dependent halogenases (NHFeHals), which catalyze the regio- and stereoselective halogenation of the unactivated C(sp3)-H bonds, exhibit tremendous potential in the challenging asymmetric halogenation. AdeV from Actinomadura sp. ATCC 39365 is the first identified carrier protein-free NHFeHal that catalyzes the chlorination of nucleotide 2'-deoxyadenosine-5'-monophosphate (2'-dAMP) to afford 2'-chloro-2'-deoxyadenosine-5'-monophosphate. Here, we determined the complex crystal structures of AdeV/FeII/Cl and AdeV/FeII/Cl/αKG at resolutions of 1.76 and 1.74 Å, respectively. AdeV possesses a typical ß-sandwich topology with H194, H252, αKG, chloride, and one water molecule coordinating FeII in the active site. Molecular docking, mutagenesis, and biochemical analyses reveal that the hydrophobic interactions and hydrogen bond network between the substrate-binding pocket and the adenine, deoxyribose, and phosphate moieties of 2'-dAMP are essential for substrate recognition. Residues H111, R177, and H192 might play important roles in the second-sphere interactions that control reaction partitioning. This study provides valuable insights into the catalytic selectivity of AdeV and will facilitate the rational engineering of AdeV and other NHFeHals for synthesis of halogenated nucleotides. IMPORTANCE Halogenated nucleotides are a group of important antibiotics and are clinically used as antiviral and anticancer drugs. AdeV is the first carrier protein-independent nonheme iron- and α-ketoglutarate (αKG)-dependent halogenase (NHFeHal) that can selectively halogenate nucleotides and exhibits restricted substrate specificity toward several 2'-dAMP analogues. Here, we determined the complex crystal structures of AdeV/FeII/Cl and AdeV/FeII/Cl/αKG. Molecular docking, mutagenesis, and biochemical analyses provide important insights into the catalytic selectivity of AdeV. This study will facilitate the rational engineering of AdeV and other carrier protein-independent NHFeHals for synthesis of halogenated nucleotides.

Structural investigation of a thermostable 1,2-ß-mannobiose phosphorylase from Thermoanaerobacter sp. X-514.

1,2-ß-Mannobiose phosphorylases (1,2-ß-MBPs) from glycoside hydrolase 130 (GH130) family are important bio-catalysts in glycochemistry applications owing to their ability in synthesizing oligomannans. Here, we report the crystal structure of a thermostable 1,2-ß-MBP from Thermoanaerobacter sp. X-514 termed Teth514_1789 to reveal the molecular basis of its higher thermostability and mechanism of action. We also solved the enzyme complexes of mannose, mannose-1-phosphate (M1P) and 1,4-ß-mannobiose to manifest the enzyme-substrate interaction networks of three main subsites. Notably, a Zn ion that should be derived from crystallization buffer was found in the active site and coordinates the phosphate moiety of M1P. Nonetheless, this Zn-coordination should reflect an inhibitory status as supplementing Zn severely impairs the enzyme activity. These results indicate that the effects of metal ions should be taken into consideration when applying Teth514_1789 and other related enzymes. Based on the structure, a reliable model of Teth514_1788 that shares 61.7% sequence identity to Teth514_1789 but displays a different substrate preference was built. Analyzing the structural features of these two closely related enzymes, we hypothesized that the length of a loop fragment that covers the entrance of the catalytic center might regulate the substrate selectivity. In conclusion, these information provide in-depth understanding of GH130 1,2-ß-MBPs and should serve as an important guidance for enzyme engineering for further applications.

Structural dissection of unnatural ginsenoside-biosynthetic UDP-glycosyltransferase Bs-YjiC from Bacillus subtilis for substrate promiscuity.

Glycosylation catalyzed by uridine diphosphate-dependent glycosyltransferases (UGT) contributes to the chemical and functional diversity of a number of natural products. Bacillus subtilis Bs-YjiC is a robust and versatile UGT that holds potentials in the biosynthesis of unnatural bioactive ginsenosides. To understand the molecular mechanism underlying the substrate promiscuity of Bs-YjiC, we solved crystal structures of Bs-YjiC and its binary complex with uridine diphosphate (UDP) at resolution of 2.18 Å and 2.44 Å, respectively. Bs-YjiC adopts the classical GT-B fold containing the N-terminal and C-terminal domains that accommodate the sugar acceptor and UDP-glucose, respectively. Molecular docking indicates that the spacious sugar-acceptor binding pocket of Bs-YjiC might be responsible for its broad substrate spectrum and unique glycosylation patterns toward protopanaxadiol-(PPD) and PPD-type ginsenosides. Our study reveals the structural basis for the aglycone promiscuity of Bs-YjiC and will facilitate the protein engineering of Bs-YjiC to synthesize novel bioactive glycosylated compounds.

Structural insights into thebaine synthase 2 catalysis.

Thebaine synthase 2 (THS2) that can transform (7S)-salutaridinol 7-O-acetate to thebaine catalyzes the final step of thebaine biosynthesis in Papaver somniferum. Here, the crystal structures of THS2 and its complex with thebaine are reported, revealing the interaction network in the substrate-binding pocket. Subsequent docking and QM/MM studies was performed to further explore the catalytic mechanism of THS2. Our results suggest that T105 may abstract the proton of C4-OH from the substrate under the assistance of H89. The resulting C4-O- phenolate anion then attacks the nearby C5, and triggers intramolecular SN2' syn displacement with the elimination of O-acetyl group. Moreover, the latter SN2' reaction is the rate-determining step of the whole enzymatic reaction with an overall energy barrier of 18.8 kcal/mol. These findings are of pivotal importance to understand the mechanism of action of thebaine biosynthesis, and would guide enzyme engineering to enhance the production of opiate alkaloids via metabolic engineering.

Construction of Escherichia coli cell factories for crocin biosynthesis.

BACKGROUND: Crocin is a carotenoid-derived natural product found in the stigma of Crocus spp., which has great potential in medicine, food and cosmetics. In recent years, microbial production of crocin has drawn increasing attention, but there were no reports of successful implementation. Escherichia coli has been engineered to produce various carotenoids, including lycopene, ß-carotene and astaxanthin. Therefore, we intended to construct E. coli cell factories for crocin biosynthesis. RESULTS: In this study, a heterologous crocetin and crocin synthesis pathway was first constructed in E. coli. Firstly, the three different zeaxanthin-cleaving dioxygenases CsZCD, CsCCD2 from Crocus sativus, and CaCCD2 from Crocus ancyrensis, as well as the glycosyltransferases UGT94E5 and UGT75L6 from Gardenia jasminoides, were introduced into zeaxanthin-producing E. coli cells. The results showed that CsCCD2 catalyzed the synthesis of crocetin dialdehyde. Next, the aldehyde dehydrogenases ALD3, ALD6 and ALD9 from Crocus sativus and ALD8 from Neurospora crassa were tested for crocetin dialdehyde oxidation, and we were able to produce 4.42 mg/L crocetin using strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8). Glycosyltransferases from diverse sources were screened by in vitro enzyme activity assays. The results showed that crocin and its various derivatives could be obtained using the glycosyltransferases YjiC, YdhE and YojK from Bacillus subtilis, and the corresponding genes were introduced into the previously constructed crocetin-producing strain. Finally, crocin-5 was detected among the fermentation products of strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8,pET28a-YjiC-YdhE-YojK) using HPLC and LC-ESI-MS. CONCLUSIONS: A heterologous crocin synthesis pathway was constructed in vitro, using glycosyltransferases from the Bacillus subtilis instead of the original plant glycosyltransferases, and a crocetin and crocin-5 producing E. coli cell factory was obtained. This research provides a foundation for the large-scale production of crocetin and crocin in E. coli cell factories.

Crystal structure of LepI, a multifunctional SAM-dependent enzyme which catalyzes pericyclic reactions in leporin biosynthesis.

LepI is a novel multifunctional enzyme that catalyzes stereoselective dehydration, Diels-Alder reaction, and retro-Claisen rearrangement. Here we report the crystal structure of LepI in complex with its co-factor S-adenosyl methionine (SAM). LepI forms a tetramer via the N-terminal helical domain and binds to a SAM molecule in the C-terminal catalytic domain. The binding modes of various LepI substrates are investigated by docking simulations, which suggest that the substrates are bound via both hydrophobic and hydrophilic forces, as well as cation-π interactions with the positively charged SAM. The reaction starts with a dehydration step in which H133 possibly deprotonates the pyridone hydroxyl group of the substrate, while D296 might protonate an alkyl-chain hydroxyl group. Subsequent pericyclization may be facilitated by the correct fold of the substrate's alkyl chain and a thermodynamic driving force towards σ-bonds at the expense of π-bonds. These results provide structural insights into LepI catalysis and are important in understanding the mechanism of enzymatic pericyclization.

Oxidation of Cucurbitadienol Catalyzed by CYP87D18 in the Biosynthesis of Mogrosides from Siraitia grosvenorii.

Mogrosides, the principally bioactive compounds extracted from the fruits of Siraitia grosvenorii, are a group of glycosylated cucurbitane-type tetracyclic triterpenoid saponins that exhibit a wide range of notable biological activities and are commercially available worldwide as natural sweeteners. The biosynthesis of mogrosides involves initial cyclization of 2,3-oxidosqualene to the triterpenoid skeleton of cucurbitadienol, followed by a series of oxidation reactions catalyzed by Cyt P450s (P450s) and then glycosylation reactions catalyzed by UDP glycosyltransferases (UGTs). We previously reported the identification of a cucurbitadienol synthase (SgCbQ) and a mogrol C-3 hydroxyl glycosyltransferase (UGT74AC1). However, molecular characterization of further transformation of cucurbitadienol to mogrol by P450s remains unavailable. In this study, we report the successful identification of a multifunctional P450 (CYP87D18) as being involved in C-11 oxidation of cucurbitadienol. In vitro enzymatic activity assays showed that CYP87D18 catalyzed the oxidation of cucurbitadienol at C-11 to produce 11-oxo cucurbitadienol and 11-hydroxy cucurbitadienol. Furthermore, 11-oxo-24,25-epoxy cucurbitadienol as well as 11-oxo cucurbitadienol and 11-hydroxy cucurbitadienol were produced when CYP87D18 was co-expressed with SgCbQ in genetic yeast, and their structures were confirmed by liquid chromatography-solid-phase extraction-nuclear magnetic resonance-mass spectrometry coupling (LC-SPE-NMR-MS). Taken together, these results suggest a role for CYP87D18 as a multifunctional cucurbitadienol oxidase in the mogrosides pathway.

Functional Characterization of Cucurbitadienol Synthase and Triterpene Glycosyltransferase Involved in Biosynthesis of Mogrosides from Siraitia grosvenorii.

Mogrosides, the major bioactive components isolated from the fruits of Siraitia grosvenorii, are a family of cucurbitane-type tetracyclic triterpenoid saponins that are used worldwide as high-potency sweeteners and possess a variety of notable pharmacological activities. Mogrosides are synthesized from 2,3-oxidosqualene via a series of reactions catalyzed by cucurbitadienol synthase (CbQ), Cyt P450s (P450s) and UDP glycosyltransferases (UGTs) in vivo. However, the relevant genes have not been characterized to date. In this study, we report successful identification of SgCbQ and UGT74AC1, which were previously predicted via RNA-sequencing (RNA-seq) and digital gene expression (DGE) profile analysis of the fruits of S. grosvenorii. SgCbQ was functionally characterized by expression in the lanosterol synthase-deficient yeast strain GIL77 and was found to accumulate cucurbitadienol as the sole product. UGT74AC1 was heterologously expressed in Escherichia coli as a His-tag protein and it showed specificity for mogrol by transfer of a glucose moiety to the C-3 hydroxyl to form mogroside IE by in vitro enzymatic activity assays. This study reports the identification of CbQ and glycosyltransferase from S. grosvenorii for the first time. The results also suggest that RNA-seq, combined with DGE profile analysis, is a promising approach for discovery of candidate genes involved in biosynthesis of triterpene saponins.

Decreased production of higher alcohols by Saccharomyces cerevisiae for Chinese rice wine fermentation by deletion of Bat aminotransferases.

An appropriate level of higher alcohols produced by yeast during the fermentation is one of the most important factors influencing Chinese rice wine quality. In this study, BAT1 and BAT2 single- and double-gene-deletion mutant strains were constructed from an industrial yeast strain RY1 to decrease higher alcohols during Chinese rice wine fermentation. The results showed that the BAT2 single-gene-deletion mutant strain produced best improvement in the production of higher alcohols while remaining showed normal growth and fermentation characteristics. Furthermore, a BAT2 single-gene-deletion diploid engineered strain RY1-Δbat2 was constructed and produced low levels of isobutanol and isoamylol (isoamyl alcohol and active amyl alcohol) in simulated fermentation of Chinese rice wine, 92.40 and 303.31 mg/L, respectively, which were 33.00 and 14.20 % lower than those of the parental strain RY1. The differences in fermentation performance between RY1-Δbat2 and RY1 were minor. Therefore, construction of this yeast strain is important in future development in Chinese wine industry and provides insights on generating yeast strains for other fermented alcoholic beverages.

Structural insights into a novel nonheme iron-dependent oxygenase in selenoneine biosynthesis.

Selenoneine (SEN) is a natural histidine derivative with radical-scavenging activity and shows higher antioxidant potential than its sulfur-containing isolog ergothioneine (EGT). Recently, the SEN biosynthetic pathway in Variovorax paradoxus was reported. Resembling EGT biosynthesis, the committed step of SEN synthesis is catalyzed by a nonheme Fe-dependent oxygenase termed SenA. This enzyme catalyzes oxidative carbon­selenium (C-Se) bond formation to conjugate N-α-trimethyl histidine (TMH) and selenosugar to yield selenoxide; the process parallels the EGT biosynthetic route, in which sulfoxide synthases known as EgtB members catalyze the conjugation of TMH and cysteine or γ-glutamylcysteine to afford sulfoxides. Here, we report the crystal structures of SenA and its complex with TMH and thioglucose (SGlc), an analog of selenoglucose (SeGlc) at high resolution. The overall structure of SenA adopts the archetypical fold of EgtB, which comprises a DinB-like domain and an FGE-like domain. While the TMH-binding site is highly conserved to that of EgtB, a various substrate-enzyme interaction network in the selenosugar-binding site of SenA features a number of water-mediated hydrogen bonds. The obtained structural information is beneficial for understanding the mechanism of SenA-mediated C-Se bond formation.

Structural and functional insights into the self-sufficient flavin-dependent halogenase.

Flavin-dependent halogenases (FDHs) have tremendous applications in synthetic chemistry. A single-component FDH, AetF, exhibits both halogenase and reductase activities in a continuous polypeptide chain. AetF exhibits broad substrate promiscuity and catalyzes the two-step bromination of l-tryptophan (l-Trp) to produce 5-bromotryptophan (5-Br-Trp) and 5,7-dibromo-l-tryptophan (5,7-di-Br-Trp). To elucidate the mechanism of action of AetF, we solved its crystal structure in complex with FAD, FAD/NADP+, FAD/l-Trp, and FAD/5-Br-Trp at resolutions of 1.92-2.23 Å. The obtained crystal structures depict the unprecedented topology of single-component FDH. Structural analysis revealed that the substrate flexibility and dibromination capability of AetF could be attributed to its spacious substrate-binding pocket. In addition, highly-regulated interaction networks between the substrate-recognizing residues and 5-Br-Trp are crucial for the dibromination activity of AetF. Several Ala variants underwent monobromination with >98 % C5-regioselectivity toward l-Trp. These results reveal the catalytic mechanism of single-component FDH for the first time and contribute to efficient FDH protein engineering for biocatalytic halogenation.

Cryo-EM structure and rational engineering of a superefficient ochratoxin A-detoxifying amidohydrolase.

Ochratoxin A (OTA) is among the most prevalent mycotoxins detected in agroproducts, posing serious threats to human and livestock health. Using enzymes to conduct OTA detoxification is an appealing potential strategy. The recently identified amidohydrolase from Stenotrophomonas acidaminiphila, termed ADH3, is the most efficient OTA-detoxifying enzyme reported thus far and can hydrolyze OTA to nontoxic ochratoxin α (OTα) and L-ß-phenylalanine (Phe). To elucidate the catalytic mechanism of ADH3, we solved the single-particle cryo-electron microscopy (cryo-EM) structures of apo-form, Phe- and OTA-bound ADH3 to an overall resolution of 2.5-2.7 Å. The role of OTA-binding residues was investigated by structural, mutagenesis and biochemical analyses. We also rationally engineered ADH3 and obtained variant S88E, whose catalytic activity was elevated by 3.7-fold. Structural analysis of variant S88E indicates that the E88 side chain provides additional hydrogen bond interactions to the OTα moiety. Furthermore, the OTA-hydrolytic activity of variant S88E expressed in Pichia pastoris is comparable to that of Escherichia coli-expressed enzyme, revealing the feasibility of employing the industrial yeast strain to produce ADH3 and its variants for further applications. These results unveil a wealth of information about the catalytic mechanism of ADH3-mediated OTA degradation and provide a blueprint for rational engineering of high-efficiency OTA-detoxifying machineries.

Complete bio-degradation of poly(butylene adipate-co-terephthalate) via engineered cutinases.

Poly(butylene adipate-co-terephthalate) (PBAT), a polyester made of terephthalic acid (TPA), 1,4-butanediol, and adipic acid, is extensively utilized in plastic production and has accumulated globally as environmental waste. Biodegradation is an attractive strategy to manage PBAT, but an effective PBAT-degrading enzyme is required. Here, we demonstrate that cutinases are highly potent enzymes that can completely decompose PBAT films in 48 h. We further show that the engineered cutinases, by applying a double mutation strategy to render a more flexible substrate-binding pocket exhibit higher decomposition rates. Notably, these variants produce TPA as a major end-product, which is beneficial feature for the future recycling economy. The crystal structures of wild type and double mutation of a cutinase from Thermobifida fusca in complex with a substrate analogue are also solved, elucidating their substrate-binding modes. These structural and biochemical analyses enable us to propose the mechanism of cutinase-mediated PBAT degradation.

The structural and functional investigation into an unusual nitrile synthase.

The biosynthesis of neurotoxin aetokthonotoxin (AETX) that features a unique structure of pentabrominated biindole nitrile involves a first-of-its-kind nitrile synthase termed AetD, an enzyme that shares very low sequence identity to known structures and catalyzes an unprecedented mechanism. In this study, we resolve the crystal structure of AetD in complex with the substrate 5,7-di-Br-L-Trp. AetD adopts the heme oxygenase like fold and forms a hydrophobic cavity within a helical bundle to accommodate the indole moiety. A diiron cluster comprising two irons that serves as a catalytic center binds to the carboxyl O and the amino N of the substrate. Notably, we demonstrate that the AetD-catalyzed reaction is independent of the bromination of the substrate and also solved crystal structures of AetD in complex with 5-Br-L-Trp and L-Trp. Altogether, the present study reveals the substrate-binding pattern and validates the diiron cluster-comprising active center of AetD, which should provide important basis to support the mechanistic investigations into this class of nitrile synthase.

Structural analysis and engineering of aldo-keto reductase from glyphosate-resistant Echinochloa colona.

Glyphosate is a dominant organophosphate herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) of the shikimate pathway. Glyphosate is extensively applied since manufactured, which has led to the emergence of various glyphosate-resistant crops and weeds. However, the molecular mechanism of many glyphosate-resistance machineries remains unclear. Recently, the upregulated expression of two homologous aldo-keto reductases (AKRs), designated as AKR4C16 and AKR4C17, were found to contribute to the glyphosate resistance in Echinochloa colona. This represents the first naturally evolved glyphosate-degrading machinery reported in plants. Here, we report the three-dimensional structure of these two AKR enzymes in complex with cofactor by performing X-ray crystallography. Furthermore, the binding-mode of glyphosate were elucidated in a ternary complex of AKR4C17. Based on the structural information and the previous study, we proposed a possible mechanism of action of AKR-mediated glyphosate degradation. In addition, a variant F291D of AKR4C17 that was constructed based on structure-based engineering showed a 70% increase in glyphosate degradation. In conclusion, these results demonstrate the structural features and glyphosate-binding mode of AKR4C17, which increases our understanding of the enzymatic mechanism of glyphosate bio-degradation and provides an important basis for the designation of AKR-based glyphosate-resistance for further applications.

Structure-based rational design of a short-chain dehydrogenase/reductase for improving activity toward mycotoxin patulin.

Patulin is a fatal mycotoxin that is widely detected in drinking water and fruit-derived products contaminated by diverse filamentous fungi. CgSDR from Candida guilliermondii represents the first NADPH-dependent short-chain dehydrogenase/reductase that catalyzes the reduction of patulin to the nontoxic E-ascladiol. To elucidate the catalytic mechanism of CgSDR, we solved its crystal structure in complex with cofactor and substrate. Structural analyses indicate that patulin is situated in a hydrophobic pocket adjacent to the cofactor, with the hemiacetal ring orienting toward the nicotinamide moiety of NADPH. In addition, we conducted structure-guided engineering to modify substrate-binding residue V187 and obtained variant V187F, V187K and V187W, whose catalytic activity was elevated by 3.9-, 2.2- and 1.7-fold, respectively. The crystal structures of CgSDR variants suggest that introducing additional aromatic stacking or hydrogen-bonding interactions to bind the lactone ring of patulin might account for the observed enhanced activity. These results illustrate the catalytic mechanism of SDR-mediated patulin detoxification for the first time and provide the upgraded variants that exhibit tremendous potentials in industrial applications.

Structural insights to a bi-functional isoprenyl diphosphate synthase that can catalyze head-to-tail and head-to-middle condensation.

Isoprenoids represent the largest group of natural products, whose basal skeletons are synthesized by various isoprenyl diphosphate synthases (IDSs). As majority of IDSs catalyze head-to-tail reaction to produce linear form isoprenoids, some catalyze head-to-middle reaction to produce branched form products. In a previous study, an IDS termed MA1831 from Methanosarcina acetivorans was found to be capable of catalyzing both types of reaction. In addition to the canonical linear product of C35 in length, MA1831 also catalyzes head-to-middle condensation of farnesyl diphosphate (FPP) and dimethylallyl diphosphate (DMAPP) to produce geranyllavandulyl diphosphate. In order to investigate the mechanism of action of MA1831, we determined its crystal structures in apo-form and in complex with substrates and analogues. The complex structures that contain isopentenyl S-thiolodiphosphate and DMAPP as homoallylic substrates were also reported, which should represent the reaction modes of MA1831-mediated head-to-tail and head-to-middle reaction, respectively. Based on the structural information, the mechanism of MA1831 catalyze head-to-tail and head-to-middle condensation reaction was proposed.

Crystal structure and biochemical analysis of the specialized deoxynivalenol-detoxifying glyoxalase SPG from Gossypium hirsutum.

Deoxynivalenol (DON) and its acetylated derivatives such as 3-acetyldeoxynivalenol (3A-DON) and 15-acetyldeoxynivalenol (15A-DON) are notorious mycotoxins in Fusarium contaminated cereals, which pose a great threat to human and livestock health. The specialized glyoxalase I from Gossypium hirsutum (SPG) can lower the toxicity of 3A-DON by conducting isomerization to transfer C8 carbonyl to C7 and double bond from C9-C10 to C8-C9. Here we report that the substrate-flexible SPG can also recognize 15A-DON and DON, probably following the same isomerization mechanism as that for 3A-DON. The crystallographic, mutagenesis, and biochemical analyses revealed that SPG provides a hydrophobic pocket to accommodate the substrate and residue E167 might serve as the catalytic base. A variant SPGY62A that was constructed based on structure-based protein engineering exhibited elevated catalytic activity towards DON, 3A-DON, and 15A-DON by >70%. Furthermore, variant SPGY62A was successfully expressed in Pichia pastoris, whose catalytic activity was also compared to that produced in Escherichia coli. These results provide a blueprint for further protein engineering of SPG and reveal the potential applications of the enzyme in detoxifying DON, 3A-DON and 15A-DON.

Catalytically inactive lytic polysaccharide monooxygenase PcAA14A enhances the enzyme-mediated hydrolysis of polyethylene terephthalate.

The massive accumulation of polyethylene terephthalate (PET) in the global ecosystem is a growing environmental crisis. Development of environmental friendly strategies to achieve enzyme-catalyzed PET degradation has attracted tremendous attention. In this study, we demonstrated the synergistic effects of combining a specific PET-degrading enzyme IsPETaseEHA variant from PET-assimilating bacterium Ideonella sakaiensis and a lytic polysaccharide monooxygenase from a white-rot fungus Pycnoporus coccineus (PcAA14A) in PET degradation. We found that the presence of PcAA14A alone did not result in PET hydrolysis, but its presence could stimulate IsPETaseEHA-mediated hydrolytic efficiency by up to 1.3-fold. Notably, the stimulatory effects of PcAA14A on IsPETaseEHA-catalyzed PET hydrolysis were found to be independent of monooxygenase activity. Dose-effects of IsPETaseEHA and PcAA14A on PET hydrolysis were observed, with the optimal concentrations being determined to 25 µg/mL and 0.25 µg/mL, respectively. In the 5-day PET hydrolysis experiment, 1097 µM hydrolysis products were produced by adding the optimized concentrations of IsPETaseEHA and PcAA14A, which was 27.7% higher than those were produced by IsPETaseEHA alone. Our study reports the first time that PcAA14A could stimulate the IsPETaseEHA-mediated PET hydrolysis through a monooxygenase activity independent manner.

Enhancing PET hydrolytic enzyme activity by fusion of the cellulose-binding domain of cellobiohydrolase I from Trichoderma reesei.

The large amounts of polyethylene terephthalate (PET) that enter and accumulate in the environment have posed a serious threat to global ecosystems and human health. A PET hydrolase from PET-assimilating bacterium Ideonella sakaiensis (IsPETase) that exhibits superior PET hydrolytic activity at mild conditions is attracting enormous attention in development of plastic biodegrading strategies. In order to enhance the PET hydrolysis capacity of IsPETase, we selected several polymer-binding domains that can adhere to a hydrophobic polymer surface and fused these to a previously engineered IsPETaseS121E/D186H/R280A (IsPETaseEHA) variant. We found that fusing a cellulose-binding domain (CBM) of cellobiohydrolase I from Trichoderma reesei onto the C-terminus of IsPETaseEHA showed a stimulatory effect on enzymatic hydrolysis of PET. Compared to the parental enzyme, IsPETaseEHA_CBM exhibited 71.5 % and 44.5 % higher hydrolytic activity at 30 ℃ and 40 ℃, respectively. The catalytic activity of IsPETaseEHA_CBM was increased by 86 % when the protein concentration was increased from 2.5 µg/mL to 20 µg/mL. These findings suggest that the fusion of polymer-binding module to IsPETase is a promising strategy to stimulate the enzymatic hydrolysis of PET.