High Catalytic Efficiency Research Articles

Recycling Valuable Phenol from Polycarbonate Plastic Waste Via Direct Depolymerization and Csp2-Csp3 Bond Cleavage Under Mild Conditions.

Upcycling plastic waste into commodity chemicals is recognized as an environmentally benign solution and beneficial for the sustained growth of humanity. Nevertheless, transition metal-free catalysts and energy-efficient conditions pose significant challenges due to the robust mechanical properties of plastics. Here, a strategy for selective production of phenol by upcycling polycarbonate waste via direct depolymerization and Csp2-Csp3 bond cleavage in an aqueous medium under mild conditions is reported. The commercial zeolites efficiently catalyze the depolymerization, Csp2-Csp3 bond hydrolysis, and direct Csp2-Csp3 bond scission at Cα of PC. Among all evaluated zeolites, HY (Si/Al=15) showed excellent catalytic performance, attributed to the ~75 % yield of phenol and ~15 % of acetone. The approach also employs different municipal waste PC for upcycling. Studies reveal that HY (15) exhibits high catalytic efficiency and phenol yield due to its optimum acid sites and textual properties. A scale-up experiment demonstrated that 3.1 g of phenol was produced from 5.0 g of PC, and the mass balance was 90 %. A combination of control experiments, NMR analysis, and DFT studies proposed the reaction pathway. Our findings present a sustainable avenue for upcycling PC waste and offer a new way to produce phenol, contributing to the advancement of a circular economy.

Multiple enzyme-mimic polypeptide based carbon nanoparticles by ROP and Fe coordination for ROS regulation and photo-thermal therapy against bacterial infection

Novel strategy is urgently needed to overcome the bacterial infection all over the world due to unreasonable use of biotics. In recent years, nanozymes have attracted great interests of researchers for their high catalytic efficiency and biocompatibility. In this study, a novel multiple enzyme-mimic polypeptide-based carbon nanoparticle was synthesized by N-carboxyanhydride mediated ring opening polymerization (ROP) and Fe coordination for actualizing ROS regulation and photo-thermal therapy. The multiple enzyme-mimic activities of the nanozyme, such as peroxidase, oxidase, catalase, and glutathione peroxidase, were detailly explored in ROS regulation for potential utilization in bacterial inhibition. The photo-thermal effect of the nanozyme was investigated under 808 nm NIR irradiation. Enhanced inhibition rate of the as prepared nanozyme was observed against Gram-negative Escherichia coli (99.03 %) and Gram positive Staphylococcus aureus (99.78 %) planktonic bacteria. Methicillin-resistant Staphylococcus aureus (MRSA) was chosen as the drug resistant bacteria model to evaluate the efficiency in bacterial biofilm disruption. Improved healing efficacy of 99.05 % against MRSA wound infection and excellent biosafety were observed in mice model experiments for the as prepared nanozyme. In conclusion, the as synthesized nanozyme with ROS regulation, enhanced bacteria inhibition, and excellent biocompatibility could be potentially applied in clinic against bacterial infection.

Mn(I)-Catalyzed Site-Selective C-H Activation: Unlocking Access to 3-Arylated Succinimides from 2-Arylpyridines and Maleimides.

An efficient and cost-effective Mn(I)-catalyzed site-selective C-H activation of 2-arylpyridines with maleimides has been described. This approach facilitates the synthesis of 3-arylated succinimide derivatives with high site selectivity, chemoselectivity, catalytic efficiency, and outstanding tolerance to numerous functional groups. The practicality of this approach is further evidenced by its successful application in large-scale reactions and the conversion of the synthesized succinimide derivatives into other valuable compounds.

α-Amino-β-carboxymuconate-ε-semialdehyde decarboxylase catalyzes Enol/Keto tautomerization of oxaloacetate

ACMSD (α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase) is a key metalloenzyme critical for regulating de novo endogenous NAD+/NADH biosynthesis through the tryptophan-kynurenine pathway. This decarboxylase is a recognized target implicated in mitochondrial diseases and neurodegenerative disorders. However, unraveling its enzyme-substrate complex has been challenging due to its high catalytic efficiency. Here, we present a combined biochemical and structural study wherein we determined the crystal structure of ACMSD in complex with malonate. Our analysis revealed significant rearrangements in the active site, particularly in residues crucial for ACMS decarboxylation, including Arg51, Arg239* (a residue from an adjacent subunit), His228, and Trp194. Docking modeling studies proposed a putative ACMS binding mode. Additionally, we found that ACMSD catalyzes oxaloacetic acid (OAA) tautomerization at a rate of 6.51 ± 0.42 s-1 but not decarboxylation. The isomerase activity of ACMSD on OAA warrants further investigation in future biological studies. Subsequent mutagenesis studies and crystallographic analysis of W194A variant shed light on the roles of specific second-coordination sphere residues. Our findings indicate that Arg51 and Arg239* are crucial for OAA tautomerization. Moreover, our comparative analysis with related isomerase superfamily members underscores a general strategy employing arginine residues to promote OAA isomerization. Given the observed isomerase activity of ACMSD on OAA and its structural similarity to ACMS, we propose that ACMSD may facilitate isomerization to ensure ACMS is in the optimal tautomeric form for subsequent decarboxylation initiated by the zinc-bound hydroxide ion. Overall, these findings deepen the understanding of the structure and function of ACMSD, offering insights into potential therapeutic interventions.

The Biochemistry of Artificial CO2-Fixation Pathways: The Exploitation of Carboxylase Enzymes Alternative to Rubisco

The last decade has registered a rapid development of new artificial CO2-bioconversion processes mirroring natural CO2-fixation by carboxylation and/or reduction reactions. The development of artificial pathways has shown that we have sufficient tools to design and implement, both in vitro and in vivo, complex reaction sequences pointing to construct microbial cell-factories to produce target chemicals at scale. This review is aimed to focus on the most efficient artificial CO2-fixing autotrophic cycles based on the use of carboxylase enzymes that, similarly to Rubisco enzyme, build a C–CO2 bond by reacting an enediolate or an enolate anion with CO2. The development of artificial CO2-fixing autotrophic cycles encompasses the analysis of the complete library of natural carboxylase enzymes taking part in the so called “central” and “assimilation” metabolism to select only those enzymes characterized by high catalytic efficiency, great stability, high substrate affinity, and oxygen tolerability. The review analyzes the biochemistry of the most efficient artificial CO2-fixation pathways implemented up today, evidencing the biosynthetic strategies adopted, the development of replenishing routes, and their integration with cell metabolism.

CO2(OH)2CO3 and Fe doped Co2(OH)2CO3 prepared as efficient and stable catalysts for activation of PMS: Two systems comparison and key role of high-valent metal-oxo species in mechanism revealing

High-valent metal-oxo species [M(IV) = O] exhibited high selectivity and chemical efficiency in advanced oxidation processes for removing organic pollutants, making their manipulation essential in catalytic systems. However, the low and unsustainable efficiency of generating M(IV) = O posed significant challenges. In this research, Co2(OH)2CO3 were successfully prepared via a one-pot hydrothermal method to efficiently activate peroxymonosulfate (PMS) with high-valent cobalt-oxo species [Co (IV) = O] as the primary active oxidation species. Building upon this foundation, COC was modified through variations in metal types and elemental ratios, in which magnetically enriched FexCo2-x(OH)2CO3 was synthesized by gradient Fe doping with higher catalytic efficiency and stability. The insertion of Fe resulted in the lengthening of the Co-O bond and improved the valence distribution of cobalt on the COC surface. Besides, it enabled part of the electrons to be transferred from the Fe active center to the Co active center, breaking the limitation of the traditional two-electron transfer and realizing the stable generation of more high-valent metals. Both reaction systems achieved efficient degradation of sulfadimethoxine (SDM) within 30 min and were able to completely degrade SDM within 30 min even after three cycles. FexCo2-x(OH)2CO3 maintained excellent catalytic performance even after 30 days of exposure to air. In both systems, high-valence metals played a dominant role, while the contributions of hydroxyl radicals and sulfate radicals were negligible. Overall, the two groups of developed catalysts both showed remarkable stability and pollutant removal performance in anions, humic acid, Different antibiotics and real water systems, which provided a new perspective on antibiotic removal.

Highly asymmetrically configured single atoms anchored on flame-roasting deposited carbon black as cathode catalysts for ultrahigh power density Zn-air batteries

Iron group element-based single atom (SA) catalysts are highly regarded as promising alternatives to commercial Pt/C for catalysis of oxygen reduction reaction (ORR). For applications in rechargeable zinc-air batteries (ZABs), achieving the necessary high catalytic efficiency of the SAs toward oxygen evolution reaction (OER) however remains a significant challenge. Here, highly asymmetrically configured Fe SAs created with N,S co-coordination and anchored on flame-roasting deposited carbon black (CB), Fe-N3S1/CB, are developed, achieving outstanding bifunctional oxygen catalytic efficiency, with an ultra-small potential gap of 0.661 V at 10 mA cm-2 (ΔE10), outperforming the (Pt/C+RuO2) composite catalyst (0.697 V). With a newly proposed binder-free composite air cathode design, the Fe-N3S1/CB based ZAB achieves an ultrahigh power density of 365.7 mW cm-2 at a current density of 511.3 mA cm-2, largely outperforming the (Pt/C+RuO2) based ZAB (225.9 mW cm-2 at 344.7 mA cm-2). Furthermore, the Fe-N3S1/CB based ZAB demonstrates excellent long-term stability, with only 8.2% decay in round-trip efficiency over 1000 (333.3 h) charge-discharge cycles at 10 mA cm-2. Density functional theory calculations elucidate that incorporation of sulfur into the coordination sphere of Fe facilitates the electrochemical dehydroxylation step for ORR and accelerates the electrochemical O2 desorption step for OER, thereby reducing the corresponding free energy differences on Fe SAs for largely enhanced catalytic efficiency.1. A large size hetero-atom element, sulfur, is introduced to create highly asymmetrically configured Fe single atoms for enhancements in catalytic efficiency toward both oxygen reduction reaction and oxygen evolution reaction, and a binder-free composite air cathode design is proposed to improve electrochemical performances of zinc-air batteries.2. An ultra-small potential gap of 0.661 V at 10 mA cm-2 (ΔE10) is achieved for the air cathode, and an ultrahigh discharge power density of 365.7 mW cm-2 at a current density of 511.3 mA cm-2 is acquired for the zinc-air battery.

Nanostructured bi-metallic biochar: An innovative approach for arsenic (III) removal from contaminated water

Possessing variable valence states, the element Arsenic (As) is intimidating the quality of the ecology and human health severely. In this study, eliminating As (III) from water-based solutions with great efficiency was done using Bagasse-Mn-Al, a sugarcane bagasse-derived biochar impregnated with Mn and Al. The Bagasse-Mn-Al composite yielded higher removal efficiency towards As (III) than the biochar itself. About 89.53 % of As (III) was removed within 65 min maintaining the very first concentration of As (III) at 400 μg/L, initial pH at 2–2.5, and adsorbent dosage at 0.625 g/L. The Bagasse-Mn-Al composite showed an adsorption potential maximum of 54.945 mg/g which is superior to most of the cheaply synthesized metal-impregnated biochar reported. Results from a variety of characterization techniques indicated that the •OH free radical in the Bagasse-Mn-Al composite mainly contributed to the removal of As (III) where oxidation and complexation were the major mechanisms. With high catalytic efficiency, this cost effectively produced metal-coated biochar showed easy and effective separation of As (III) from aqueous solution. Further, this study focuses on the high potential of Bagasse-Mn-Al adsorbent in the treatment of both ground and wastewater.

Porous polylactide membranes with high piezo-catalytic efficiency for organic wastewater treatment

Piezo-catalysis has been emerged as a green and promising wastewater treatment technology for efficient removal of persistent organic pollutants (POPs), such as 4-nitrophenol (4-NP). However, it remains challenging to achieve advanced materials with combined advantages of high piezo-catalytic efficiency, good hydrophilicity, and environmental friendliness. In this work, highly crystalline porous polylactide (PLA) membranes possessing such exceptional advantages have been successfully prepared from biodegradable poly(L-lactide)/poly(D-lactide)/polyethylene glycol/polyoxyethylene-polyoxypropylene block copolymer (PLLA/PDLA/PEG/PEO-PPO-PEO) blends by melt processing and subsequent water etching. During the melt-processing, the two PLA enantiomers co-crystallize into stereocomplex (SC) crystals and simultaneously the PEG used as a porogen is expelled from the crystalline regions. The results show that the prepared PLA membranes have not only abundant micro-porous structures but also good hydrophilicity due to the preferential localization of PEO-PPO-PEO copolymer on the pore walls, thus giving rise to an extremely high water flux of 2400 L/m2h. More interestingly, the PLA membranes exhibit remarkable piezoelectric activity and the piezo-catalytic degradation efficiency of 4-NP can reach as high as 96 % under the triggering of water flow involved in vacuum filtration. In addition, the membranes show excellent reusability and the high catalytic efficiency can be well-maintained after five repeated uses. These findings suggest broad application prospects of our porous SC-PLA membranes in organic wastewater treatment.

Molecular Identification and Engineering a Salt-Tolerant GH11 Xylanase for Efficient Xylooligosaccharides Production.

This study identified a salt-tolerant GH11 xylanase, Xynst, which was isolated from a soil bacterium Bacillus sp. SC1 and can resist as high as 4 M NaCl. After rational design and high-throughput screening of site-directed mutant libraries, a double mutant W6F/Q7H with a 244% increase in catalytic activity and a 10 °C increment in optimal temperature was obtained. Both Xynst and W6F/Q7H xylanases were stimulated by high concentrations of salts. In particular, the activity of W6F/Q7H was more than eight times that of Xynst in the presence of 2 M NaCl at 65 °C. Kinetic parameters indicated they have the highest affinity for beechwood xylan (Km = 0.30 mg mL-1 for Xynst and 0.18 mg mL-1 for W6F/Q7H), and W6F/Q7H has very high catalytic efficiency (Kcat/Km = 15483.33 mL mg-1 s-1). Molecular dynamic simulation suggested that W6F/Q7H has a more compact overall structure, improved rigidity of the active pocket edge, and a flexible upper-end alpha helix. Hydrolysis of different xylans by W6F/Q7H released more xylooligosaccharides and yielded higher proportions of xylobiose and xylotriose than Xynst did. The conversion efficiencies of Xynst and W6F/Q7H on all tested xylans exceeded 20%, suggesting potential applications in the agricultural and food industries.

Glycine as a stabilizing osmolyte for Renilla luciferase: A kinetic and molecular dynamics analysis

Renilla luciferase, a luminescent enzyme, is utilized in gene expression analysis and biosensor technology, and extensive research has been conducted on its structure and function. Glycine is an osmolyte that plays a key role in protein stabilization against denaturation. However, its impact on enzyme properties is unpredictable. This study aimed to investigate the effect of glycine on the kinetics and stability of Renilla luciferase. The data revealed that glycine at a concentration range of 0.1 to 0.6 M improved enzyme kinetics and thermal stability. The highest catalytic efficiency was observed at a concentration of 0.5 M. Molecular dynamics simulations demonstrated that in the presence of 0.5 M glycine, substrate access to the enzyme’s active site was enhanced while the root-mean-square fluctuation (RMSF) of the protein backbone was reduced. Additionally, the analysis of protein-water hydrogen bonding interactions showed an increase in the hydrogen bonding between water molecules and Renilla luciferase. The present study may be used for the formulation of Renilla luciferase for commercial purposes.

Mechanistic Insight into the Enantioselective Degradation of Esterase QeH to (R)/(S)-Quizalofop-Ethyl with Molecular Dynamics Simulation Using a Residue-Specific Force Field.

The enantioselective mechanism of the esterase QeH against the two enantiomers of quizalofop-ethyl (QE) has been primitively studied using computational and experimental approaches. However, it is still unclear how the esterase QeH adjusts its conformation to adapt to substrate binding and promote enzyme-substrate interactions in the catalytic kinetics. The equilibrium processes of enzyme-substrate interactions and catalytic dynamics were reproduced by performing independent molecular dynamics (MD) runs on the QeH-(R)/(S)-QE complexes with a newly developed residue-specific force field (RSFF2C). Our results indicated that the benzene ring of the (R)-QE structure can simultaneously form anion-π and cation-π interactions with the side-chain group of Glu328 and Arg384 in the binding cavity of the QeH-(R)-QE complex, resulting in (R)-QE being closer to its catalytic triplet system (Ser78-Lys81-Tyr189) with the distances measured for the hydroxyl oxygen atom of the catalytic Ser78 of QeH and the carbonyl carbon atom of (R)-QE of 7.39 Å, compared to the 8.87 Å for (S)-QE, whereas the (S)-QE structure can only form an anion-π interaction with the side chain of Glu328 in the QeH-(S)-QE complex, being less close to its catalytic site. The computational alanine scanning mutation (CAS) calculations further demonstrated that the π-π stacking interaction between the indole ring of Trp351 and the benzene ring of (R)/(S)-QE contributed a lot to the binding stability of the enzyme-substrate (QeH-(R)/(S)-QE). These results facilitate the understanding of their catalytic processes and provide new theoretical guidance for the directional design of other key enzymes for the initial degradation of aryloxyphenoxypropionate (AOPP) herbicides with higher catalytic efficiencies.

Interface potential-induced natural antioxidant mimic system for the treatment of Alzheimer’s disease

Although the pathogenesis of Alzheimer’s disease (AD) is still unknown, the molecular pathological phenomena is clear, mainly due to mitochondrial dysfunction and central nervous system inflammation caused by imbalanced antioxidant capacity and synaptic dysfunction, so antioxidant therapy is still the preferred treatment for AD. However, although antioxidant enzymes have high catalytic efficiency, the substrate spectrum is narrow; Antioxidants have wider range of effects, but their efficiency is low. Since the antioxidant defense system in high-grade organisms is composed of both enzymatic and non-enzymatic systems, therefore we synthesized a metal-organic framework (MOF) with superoxide dismutase activity, and depending on the interface potential effect, curcumin was loaded to construct a synergistic antioxidant treatment system. More importantly, due to the complementary surface electrostatic potential between MOF and curcumin, the system exhibited both good antioxidant activity and efficient β-amyloid plaque scavenging ability, which slowed down the cognitive dysfunction in the brain of AD mice.

Iron porphyrin as a cytochrome P450 model for the oxidation of xanthene

Cytochrome P450 enzyme is a superfamily of heme-containing enzyme, which play an important part in the metabolism of xenobiotics and biosynthesis of natural products. Cytochrome P450 enzyme is able to catalyze a series of challenging oxidation reactions, thus, it has attracted increased attention as a promising biocatalyst for synthetic chemistry. Transition-metal complexes of porphyrins have been successfully synthesized as models for the cytochrome P450 enzyme, and used to the oxidation of organic compounds such as hydrocarbons. In the current study, an iron porphyrin, 5,10,15,20-tetra (4-methoxyphenyl) porphine ferric chloride (III) (Fe(III)TCPP-OMe) is used as the catalyst for the oxidation of xanthene with peroxide oxidants (cumyl hydroperoxide (CmOOH) and tert‑butyl hydroperoxide (t-BuOOH)). Fe(III)TCPP-OMe shows high catalytic oxidation efficiency of xanthene and high selectivity for the formation of 9-H-xanthene-9-ol. This work provides a feasible method for the oxidation of hydrocarbons.

L-Lactate dehydrogenase from Cyanidioschyzon merolae shows high catalytic efficiency for pyruvate reduction and is inhibited by ATP

l-Lactate is a commodity chemical used in various fields. Microorganisms have produced l-lactate via lactic fermentation using saccharides derived from crops as carbon sources. Recently, l-lactate production using microalgae, whose carbon source is carbon dioxide, has been spotlighted because the prices of the crops have increased. A red alga Cyanidioschyzon merolae produce l-lactate via lactic fermentation under dark anaerobic conditions. The l-lactate titer of C. merolae is higher than those of other microalgae but lower than those of heterotrophic bacteria. Therefore, an increase in the l-lactate titer is required in C. merolae. l-Lactate dehydrogenase (l-LDH) catalyzes the reduction of pyruvate to l-lactate during lactic fermentation. C. merolae possesses five isozymes of l-LDH. The results of previous transcriptome analysis suggested that l-LDHs are the key enzymes in the lactic fermentation of C. merolae. However, their biochemical characteristics, such as catalytic efficiency and tolerance for metabolites, have not been revealed. We compared the amino acid sequences of C. merolael-LDHs (CmLDHs) and characterized one of the isozymes, CmLDH1. BLAST analysis revealed that the sequence similarities of CmLDH1 and the other isozymes were above 99%. The catalytic efficiency of CmLDH1 under its optimum conditions was higher than those of l-LDHs of other organisms. ATP decreased the affinity and turnover number of CmLDH1 for NADH. These findings contribute to understanding the characteristics of l-LDHs of microalgae and the regulatory mechanisms of lactic fermentation in C. merolae.

Molecular and biochemical characterization of a plant-like iota-class glutathione S-transferase from the halotolerant cyanobacterium Halothece sp. PCC7418.

This study identifies a unique glutathione S-transferase (GST) in extremophile using genome, phylogeny, bioinformatics, functional characterization, and RNA sequencing analysis. Five putative GSTs (H0647, H0729, H1478, H3557, and H3594) were identified in Halothece sp. PCC7418. Phylogenetic analysis suggested that H0647, H1478, H0729, H3557, and H3594 are distinct GST classes. Of these, H0729 was classified as an iota-class GST, encoding a high molecular mass GST protein with remarkable features. The protein secondary structure of H0729 revealed the presence of a glutaredoxin (Grx) Cys-Pro-Tyr-Cys (C-P-Y-C) motif that overlaps with the N-terminal domain and harbors a topology similar to the thioredoxin (Trx) fold. Interestingly, recombinant H0729 exhibited a high catalytic efficiency for both glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB), with catalytic efficiencies that were 155- and 32-fold higher, respectively compared to recombinant H3557. Lastly, the Halothece gene expression profiles suggested that antioxidant and phase II detoxification encoding genes are crucial in response to salt stress. Iota-class GST was identified in cyanobacteria. This GST exhibited a high catalytic efficiency toward xenobiotic substrates. Our findings shed light a diversified evolution of GST in cyanobacteria and provide functional dynamics of the genes encoding the enzymatic antioxidant and detoxification systems under abiotic stresses.

Biochemical characterization of a flavonoid O-methyltransferase from Artemisia annua and its application in tamarixetin production

The O-position methylation of flavonoids is widely recognized as a key modification that substantially boosts their bioactivity and bioavailability, leading to enhanced absorption. The use of specific O-methyltransferases is frequently necessary for various substrate modifications. Here, a novel Artemisia annua O-methyltransferase (AaOMT) gene was cloned, heterologously expressed in Escherichia coli, and its biochemical properties against various flavonoids were characterized. Amino acid and phylogenic analyses indicated that AaOMT is a new flavonoid O-methyltransferase that belongs to the Class II O-methyltransferases family. It could specifically methylate most flavonoids at the 4′-OH position unless flavanones. The optimal temperature and pH for in vitro reactions were 35 °C and 8.0, respectively. Kinetic analysis revealed that AaOMT exhibits the highest affinity and relatively high catalytic efficiency towards quercetin. Consequently, recombinant strains harboring AaOMT were used to convert quercetin into tamarixetin in vivo. Additionally, different vectors with promoters and E. coli hosts were screened to enhance tamarixetin production. By optimizing cultivation conditions and scaling up fermentation capacity, we achieved a maximum tamarixetin titer of 706.9 mg/L (∼2.24 mM) finally. This study provides a novel, efficient, and regioselective 4′-O-methyltransferase (4′-OMT) and a strategy for the mass production of those rare and high-value 4′-methoxyflavonoids through metabolic engineering in vivo.

Characterization of the 2ODD genes of DOXC subfamily and its members involved in flavonoids biosynthesis in Scutellaria baicalensis

Background2-oxoglutarate-dependent dioxygenase (2ODD) superfamily is the second largest enzyme family in the plant genome and plays diverse roles in secondary metabolic pathways. The medicinal plant Scutellaria baicalensis Georgi contains various flavonoids, which have the potential to treat coronavirus disease 2019 (COVID-19), such as baicalein and myricetin. Flavone synthase I (FNSI) and flavanone 3-hydroxylase (F3H) from the 2ODDs of DOXC subfamily have been reported to participate in flavonoids biosynthesis. It is certainly interesting to study the 2ODD members involved in the biosynthesis of flavonoids in S. baicalensis.ResultsWe provided a genome-wide analysis of the 2ODDs of DOXC subfamily in S. baicalensis, a total of 88 2ODD genes were identified, 82 of which were grouped into 25 distinct clades based on phylogenetic analysis of At2ODDs. We then performed a functional analysis of Sb2ODDs involved in the biosynthesis of flavones and dihydroflavonols. Sb2ODD1 and Sb2ODD2 from DOXC38 clade exhibit the activity of FNSI (Flavone synthase I), which exclusively converts pinocembrin to chrysin. Sb2ODD1 has significantly higher transcription levels in the root. While Sb2ODD7 from DOXC28 clade exhibits high expression in flowers, it encodes a F3H (flavanone 3-hydroxylase). This enzyme is responsible for catalyzing the conversion of both naringenin and pinocembrin into dihydrokaempferol and pinobanksin, kinetic analysis showed that Sb2ODD7 exhibited high catalytic efficiency towards naringenin.ConclusionsOur experiment suggests that Sb2ODD1 may serve as a supplementary factor to SbFNSII-2 and play a role in flavone biosynthesis specifically in the roots of S. baicalensis. Sb2ODD7 is mainly responsible for dihydrokaempferol biosynthesis in flowers, which can be further directed into the metabolic pathways of flavonols and anthocyanins.

Efficient 2,3-Butanediol Production from Ethanol by a Modified Four-Enzyme Synthetic Biosystem.

2,3-butanediol (2,3-BD) is a versatile bio-based platform chemical. An artificial four-enzyme synthetic biosystem composed of ethanol dehydrogenase, NADH oxidase, formolase and 2,3-butanediol dehydrogenase was designed for upgrading ethanol to 2,3-BD in our previous study. However, a key challenge in developing in vitro enzymatic systems for 2,3-BD synthesis is the relatively sluggish catalytic efficiency of formolase, which catalyzes the rate-limiting step in such systems. Herein, this study reports how engineering the tunnel and substrate binding pocket of FLS improved its catalytic performance. A series of single-point and combinatorial variants were successfully obtained which displayed both higher catalytic efficiency and better substrate tolerance than wild-type FLS. Subsequently, a cell-free biosystem based on the FLS:I28V/L482E enzyme was implemented for upgrading ethanol to 2,3-BD. Ultimately, this system achieved efficient production of 2,3-BD from ethanol by the fed-batch method, reaching a concentration of 1.39 M (124.83 g/L) of the product and providing both excellent productivity and yield values of 5.94 g/L/h and 92.7%, respectively. Taken together, this modified enzymatic catalysis system provides a highly promising alternative approach for sustainable and cost-competitive production of 2,3-BD.

Evolving dual-trait EPSP synthase variants using a synthetic yeast selection system

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) functions in the shikimate pathway which is responsible for the production of aromatic amino acids and precursors of other essential secondary metabolites in all plant species. EPSPS is also the molecular target of the herbicide glyphosate. While some plant EPSPS variants have been characterized with reduced glyphosate sensitivity and have been used in biotechnology, the glyphosate insensitivity typically comes with a cost to catalytic efficiency. Thus, there exists a need to generate additional EPSPS variants that maintain both high catalytic efficiency and high glyphosate tolerance. Here, we create a synthetic yeast system to rapidly study and evolve heterologous EPSP synthases for these dual traits. Using known EPSPS variants, we first validate that our synthetic yeast system is capable of recapitulating growth characteristics observed in plants grown in varying levels of glyphosate. Next, we demonstrate that variants from mutagenesis libraries with distinct phenotypic traits can be isolated depending on the selection criteria applied. By applying strong dual-trait selection pressure, we identify a notable EPSPS mutant after just a single round of evolution that displays robust glyphosate tolerance (Ki of nearly 1 mM) and improved enzymatic efficiency over the starting point (~2.5 fold). Finally, we show the crystal structure of corn EPSPS and the top resulting mutants and demonstrate that certain mutants have the potential to outperform previously reported glyphosate-resistant EPSPS mutants, such as T102I and P106S (denoted as TIPS), in whole-plant testing. Altogether, this platform helps explore the trade-off between glyphosate resistance and enzymatic efficiency.