Metal-free directed sp2-C-H borylation.

Organoboron reagents are important synthetic intermediates that have a key role in the construction of natural products, pharmaceuticals and organic materials1. The discovery of simpler, milder and more efficient approaches to organoborons can open additional routes to diverse substances2-5. Here we show a general method for the directed C-H borylation of arenes and heteroarenes without the use of metal catalysts. C7- and C4-borylated indoles are produced by a mild approach that is compatible with a broad range of functional groups. The mechanism, which is established by density functional theory calculations, involves BBr3 acting as both a reagent and a catalyst. The potential utility of this strategy is highlighted by the downstream transformation of the formed boron species into natural products and drug scaffolds.

Identification of a novel thermostable transaminase and its application in L-phosphinothricin biosynthesis.

Transaminase (TA) is a crucial biocatalyst for enantioselective production of the herbicide L-phosphinothricin (L-PPT). The use of enzymatic cascades has been shown to effectively overcome the unfavorable thermodynamic equilibrium of TA-catalyzed transamination reaction, also increasing demand for TA stability. In this work, a novel thermostable transaminase (PtTA) from Pseudomonas thermotolerans was mined and characterized. The PtTA showed a high specific activity (28.63 U/mg) towards 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO), with excellent thermostability and substrate tolerance. Two cascade systems driven by PtTA were developed for L-PPT biosynthesis, including asymmetric synthesis of L-PPT from PPO and deracemization of D, L-PPT. For the asymmetric synthesis of L-PPT from PPO, a three-enzyme cascade was constructed as a recombinant Escherichia coli (E. coli G), by co-expressing PtTA, glutamate dehydrogenase (GluDH) and D-glucose dehydrogenase (GDH). Complete conversion of 400 mM PPO was achieved using only 40 mM amino donor L-glutamate. Furthermore, by coupling D-amino acid aminotransferase (Ym DAAT) from Bacillus sp. YM-1 and PtTA, a two-transaminase cascade was developed for the one-pot deracemization of D, L-PPT. Under the highest reported substrate concentration (800 mM D, L-PPT), a 90.43% L-PPT yield was realized. The superior catalytic performance of the PtTA-driven cascade demonstrated that the thermodynamic limitation was overcome, highlighting its application prospect for L-PPT biosynthesis. KEY POINTS: • A novel thermostable transaminase was mined for L-phosphinothricin biosynthesis. • The asymmetric synthesis of L-phosphinothricin was achieved via a three-enzyme cascade. • Development of a two-transaminase cascade for D, L-phosphinothricin deracemization.

Identification of hyperthermophilic D-allulose 3-epimerase from Thermotoga sp. and its application as a high-performance biocatalyst for D-allulose synthesis.

D-Allulose 3-epimerase (DAE) is a vital biocatalyst for the industrial synthesis of D-allulose, an ultra-low calorie rare sugar. However, limited thermostability of DAEs hinders their use at high-temperature production. In this research, hyperthermophilic TI-DAE (Tm = 98.4 ± 0.7 ℃) from Thermotoga sp. was identified via in silico screening. A comparative study of the structure and function of site-directed saturation mutagenesis mutants pinpointed the residue I100 as pivotal in maintaining the high-temperature activity and thermostability of TI-DAE. Employing TI-DAE as a biocatalyst, D-allulose was produced from D-fructose with a conversion rate of 32.5%. Moreover, TI-DAE demonstrated excellent catalytic synergy with glucose isomerase CAGI, enabling the one-step conversion of D-glucose to D-allulose with a conversion rate of 21.6%. This study offers a promising resource for the enzyme engineering of DAEs and a high-performance biocatalyst for industrial D-allulose production.

Development of an aminotransferase-driven biocatalytic cascade for deracemization of d,l-phosphinothricin.

2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) is the essential precursor keto acid for the asymmetric biosynthesis of herbicide l-phosphinothricin (l-PPT). Developing a biocatalytic cascade for PPO production with high efficiency and low cost is highly desired. Herein, a d-amino acid aminotransferase from Bacillus sp. YM-1 (Ym DAAT) with high activity (48.95 U/mg) and affinity (Km = 27.49 mM) toward d-PPT was evaluated. To circumvent the inhibition of by-product d-glutamate (d-Glu), an amino acceptor (α-ketoglutarate) regeneration cascade was constructed as a recombinant Escherichia coli (E. coli D), by coupling Ym d-AAT, d-aspartate oxidase from Thermomyces dupontii (TdDDO) and catalase from Geobacillus sp. CHB1. Moreover, the regulation of the ribosome binding site was employed to overcome the limiting step of expression toxic protein TdDDO in E. coli BL21(DE3). The aminotransferase-driven whole-cell biocatalytic cascade (E. coli D) showed superior catalytic efficiency for the synthesis of PPO from d,l-phosphinothricin (d,l-PPT). It revealed the production of PPO exhibited high space-time yield (2.59 g L-1 h-1 ) with complete conversion of d-PPT to PPO at high substrate concentration (600 mM d,l-PPT) in 1.5 L reaction system. This study first provides the synthesis of PPO from d,l-PPT employing an aminotransferase-driven biocatalytic cascade.

Isomerase and epimerase: overview and practical application in production of functional sugars.

The biosynthesis of functional sugars has gained significant attention due to their potential health benefits and increasing demand in the food industry. Enzymatic synthesis has emerged as a promising approach, offering high catalytic efficiency, chemoselectivity, and stereoselectivity. However, challenges such as poor thermostability, low catalytic efficiency, and food safety concerns have limited the commercial production of functional sugars. Protein engineering, including directed evolution and rational design, has shown promise in overcoming these barriers and improving biocatalysts for large-scale production. Furthermore, enzyme immobilization has proven effective in reducing costs and facilitating the production of functional sugars. To ensure food safety, the use of food-grade expression systems has been explored. However, downstream technologies, including separation, purification, and crystallization, still pose challenges in terms of efficiency and cost-effectiveness. Addressing these challenges is crucial to optimize the overall production process. Despite the obstacles, the future outlook for functional sugars is promising, driven by increasing awareness of their health benefits and continuous technological advancements. With further research and technological breakthroughs, industrial-scale production of functional sugars through biosynthesis will become a reality, leading to their widespread incorporation in various industries and products.

Computer-aided design of novel cellobiose 2-epimerase for efficient synthesis of lactulose using lactose.

Cellobiose 2-epimerase (CE) is ideally suited to synthesize lactulose from lactose, but the poor thermostability and catalytic efficiency restrict enzymatic application. Herein, a non-characterized CE originating from Caldicellulosiruptor morganii (CmCE) was discovered in the NCBI database. Then, a smart mutation library was constructed based on FoldX ΔΔG calculation and modeling structure analysis, from which a positive mutant D226G located within the α8/α9 loop exhibited longer half-lives at 65-75 °C as well as lower Km and higher kcat/Km values compared with CmCE. Molecular modeling demonstrated that the improvement of D226G was largely attributed to the rigidification of the flexible loop, the compactness of the catalysis pocket and the increment of substrate-binding capability. Finally, the yield of synthesizing lactulose catalyzed by D226G reached 45.5%, higher than the 35.9% achieved with CmCE. The disclosed effect of the flexible loop on enzymatic stability and catalysis provides insight to redesign efficient CEs to biosynthesize lactulose.

Engineering Novel (R)-Selective Transaminase for Efficient Symmetric Synthesis of d-Alanine.

d-Alanine belongs to nonessential amino acids that have diverse applications in the fields of food and health care. (R)-transaminase [(R)-TA]-catalyzed asymmetric amination of pyruvate is a feasible alternative for the synthesis of d-alanine, but low catalytic efficiency and thermostability limit enzymatic utilization. In this work, several potential (R)-TAs were discovered using NCBI database mining synchronously with enzymatic structure-function analysis, among which Capronia epimyces TA (CeTA) showed the highest activity for amination of pyruvate using (R)-α-methylbenzylamine as the donor. Furthermore, enzymatic residues surrounding a large catalysis pocket were subjected to saturation and combinatorial mutagenesis, and positive mutant F113T showed dramatic improvement in activity and thermostability. Molecular modeling indicated that the substitution of phenylalanine with threonine afforded alleviation of steric hindrance in the pocket and induced formation of additional hydrogen bonds with neighboring residues. Finally, using recombinant cells containing F113T as a biocatalyst, the conversion yield of amination of 100 mM pyruvate to d-alanine achieved up to 95.2%, which seemed to be the highest level in the literature regarding synthesis of d-alanine using TAs. The inherent characteristics rendered CeTA F113T a promising platform for efficient preparation of d-alanine operating with high productivity. IMPORTANCE d-Alanine is an important compound with many valuable applications. Its asymmetric synthesis employing (R)-ω-TA is considered an attractive choice. According to the stereoselectivity, ω-TAs have either (R)- or (S)-enantiopreference. There has been a variety of literature regarding screening, characterizing, and molecular modification of (S)-ω-TAs; in contrast, the research about (R)-ω-TA has lagged behind. In this work, we identify several (R)-ω-TAs and succeeded in creating mutant F113T, which showed not only better efficiency toward pyruvate but also higher thermostability compared with the original enzyme. The obtained original enzymes and positive mutants displayed important application value for pushing symmetric synthesis of d-alanine to a higher level.

Structural insights into the thermostability mechanism of a nitrile hydratase from Caldalkalibacillus thermarum by comparative molecular dynamics simulation.

Nitrile hydratase (NHase), an excellent bio-catalyst for the synthesis of amide compounds, was composed of two heterologous subunits. A thermoalkaliphilic NHase NHCTA1 (Tm = 71.3°C) obtained by in silico screening in our study exhibited high flexibility of α-subunit but excellent thermostability, as opposed to previous examples. To gain a deeper structural insight into the thermostability of NHCTA1, comparative molecular dynamics simulation of NHCTA1 and reported NHases was carried out. By comparison, we speculated that ß-subunit played a key role in adjusting the flexibility of α-subunit and the different conformations of linker in "α5-helix-coil ring" supersecondary structure of ß-subunit can affect the interaction between ß-subunit and α-subunit. Mutant NHCTA1-α6 C with a random coil linker and mutant NHCTA1-αßγ with a truncated linker were therefore constructed to understand the impact on NHCTA1 thermostability by varying the supersecondary structure. The varied thermostability of NHCTA1-α6 C and NHCTA1-αßγ (Tmα6C = 74.4°C, Tmαßγ = 65.6°C) verified that the flexibility of α-subunit adjusted by ß-subunit was relevant to the stability of NHCTA1. This study gained an insight into the NNHCTA1 thermostability by virtual dynamics comparison and experimental studies without crystallization, and this approach could be applied to other industrial-important enzymes.

A Single-Transaminase-Catalyzed Biocatalytic Cascade for Efficient Asymmetric Synthesis of l-Phosphinothricin.

A single-transaminase-catalyzed biocatalytic cascade was developed by employing the desired biocatalyst, ATA-117-Rd11, that showed high activity toward 2-oxo-4-[(hydroxy)(methyl)phosphinoyl] butyric acid (PPO) and α-ketoglutarate, and low activity against pyruvate. The cascade successfully promotes a highly asymmetric amination reaction for the synthesis of l-phosphinothricin (l-PPT) with high conversion (>95 %) and>99 % ee. In a scale-up experiment, using 10 kg pre-frozen E. coli cells harboring ATA-117-Rd11 as catalyst, 80 kg PPO was converted to ≈70 kg l-PPT after 24 hours with a high ee value (>99 %).

Immobilization of recombinant Escherichia coli cells expressing glucose isomerase using modified diatomite as a carrier for effective production of high fructose corn syrup in packed bed reactor.

To improve the operational stability of glucose isomerase in E. coli TEGI-W139F/V186T, the immobilized cells were prepared with modified diatomite as a carrier and 74.1% activity of free cells was recovered after immobilization. Results showed that the immobilized cells still retained 86.2% of the initial transformational activity after intermittent reused 40 cycles and the yield of D-fructose reached above 42% yield at 60 °C. Moreover, the immobilized cells were employed in the continuous production of High Fructose Corn Syrup (HFCS) in a recirculating packed bed reactor for 603 h at a constant flow rate. It showed that the immobilized cells exhibited good operational stability and the yield of D-fructose retained above 42% within 603 h. The space-time yield of high fructose corn syrup reached 3.84 kg L-1 day-1. The investigation provided an efficient immobilization method for recombinant cells expressing glucose isomerase with higher stability, and the immobilized cells are a promising biocatalyst for HFCS production.

A chromatography-free and aqueous waste-free process for thioamide preparation with Lawesson's reagent.

After completing the thio-substitution with Lawesson's reagent, ethanol was found to be effective in the decomposition of the inherent stoichiometric six-membered-ring byproduct from the Lawesson's reagent to a highly polarized diethyl thiophosphonate. The treatment significantly simplified the following chromatography purification of the desired thioamide in a small scale preparation. As scaling up the preparation of two pincer-type thioamides, we have successfully developed a convenient process with ethylene glycol to replace ethanol during the workup, including a traditional phase separation, extraction, and recrystallization. The newly developed chromatography-free procedure did not generate P-containing aqueous waste, and only organic effluents were discharged. It is believed that the optimized procedure offers the great opportunity of applying the Lawesson's reagent for various thio-substitution reactions on a large scale.

Proteome sequencing and analysis of Ophiocordyceps sinensis at different culture periods.

BACKGROUND: Ophiocordyceps sinensis is an important traditional Chinese medicine for its comprehensive active ingredients, such as cordycepin, cordycepic acid, and Cordyceps polysaccharide. O. sinensis zjut, a special strain isolated from O. sinensis, has similar pharmacological functions to wild O. sinensis. Currently, O. sinensis with artificial cultivation has been widely studied, but systematic fundamental research at protein levels has not been determined. RESULTS: Proteomes of O. sinensis zjut at different culture periods (growth period, 3rd day; pre-stable period, 6th day; and stable period, 9th day) were relatively quantified by relative isotope markers and absolute quantitative technology. In total, 4005 proteins were obtained and further annotated with Gene Ontology, Kyoto Encyclopedia of Genes and Genomes database. Based on the result of the annotations, metabolic pathways of active ingredients, amino acids and fatty acid were constructed, and the related enzymes were exhibited. Subsequently, comparative proteomics of O. sinensis zjut identified the differentially expressed proteins (DEPs) by growth in different culture periods, to find the important proteins involved in metabolic pathways of active ingredients. 605 DEPs between 6d-VS-3d, 1188 DEPs between 9d-VS-3d, and 428 DEPs between 9d-VS-6d were obtained, respectively. CONCLUSION: This work provided scientific basis to study protein profile and comparison of protein expression levels of O. sinensis zjut, and it will be helpful for metabolic engineering works to active ingredients for exploration, application and improvement of this fungus.

Tandem Synthesis of 2-Carboxybenzofurans via Sequential Cu-Catalyzed C-O Coupling and Mo(CO)6-Mediated Carbonylation Reactions.

A modular tandem synthesis of 2-carboxybenzofurans from 2-gem-dibromovinylphenols has been established based on a sequence of Cu-catalyzed intramolecular C-O coupling and Mo(CO)6-mediated intermolecular carbonylation reactions. This protocol allowed one-step access to a broad variety of functionalized benzofuran-2-carboxylic acids, esters, and amides in good to excellent yields under Pd- and CO gas-free conditions.

Enhancement of protoplast preparation and regeneration of Hirsutella sinensis based on process optimization.

OBJECTIVE: To explore the optimal methods for the protoplast preparation and regeneration of Hirsutella sinensis by optimizing the limiting factors. RESULTS: During the treatment of enzymatic protoplast preparation, mycelium cultured for 7 days was the optimal start material. The maximum protoplast preparation rate of 4.3 × 107 protoplasts/g fresh weight (FW) was obtained after 0.5 h treatment of 1 mg/ml mixed lytic enzymes in KH2PO4-K2HPO4 buffer (pH 5.5) with 0.6 M KCl at 18 °C. As for the protoplast regeneration, the maximum protoplast regeneration rate reached 12.32% through 5 × 103 protoplasts mL-1 cultivated for 20 days in the regeneration medium with 0.6 M mannitol and 1.5% agar. CONCLUSIONS: The preparation and regeneration of H. sinensis protoplasts was firstly established based on process optimization and it provided a foundation for the study of H. sinensis mutagenesis.

Covalent immobilization of recombinant Citrobacter koseri transaminase onto epoxy resins for consecutive asymmetric synthesis of L-phosphinothricin.

Transaminase responsible for alienating prochiral ketone compound is applicable to asymmetric synthesis of herbicide L-phosphinothricin (L-PPT). In this work, the covalent immobilization of recombinant transaminase from Citrobacter koseri (CkTA) was investigated on different epoxy resins. Using optimum ES-105 support, a higher immobilized activity was obtained via optimizing immobilization process in terms of enzyme loading, coupling time and initial PLP concentration. Crucially, due to blocking unreacted epoxy groups on support surface with amino acids, the reaction temperature of blocked immobilized biocatalyst was enhanced from 37 to 57 °C. Its thermostability at 57 °C was also found to be superior to that of free CkTA. The Km value was shifted from 36.75 mM of free CkTA to 39.87 mM of blocked immobilized biocatalyst, demonstrating that the affinity of enzyme to the substrate has not been apparently altered. Accordingly, the biocatalyst performed the consecutive synthesis of L-PPT for 11 cycles (yields>91%) with retaining more than 91.13% of the initial activity. The seemingly the highest reusability demonstrates this biocatalyst has prospective for reducing the costs of consecutive synthesis of L-PPT with high conversion.

Aminoazanium of DABCO: An Amination Reagent for Alkyl and Aryl Pinacol Boronates.

The aminoazanium of DABCO (H2 N-DABCO) has been developed as a general and practical amination reagent for the direct amination of alkyl and aryl pinacol boronates. This compound is stable and practical for use as a reagent. Various primary, secondary. and tertiary alkyl-Bpin and aryl-Bpin substrates were aminated to give the corresponding amine derivatives. The amination is stereospecific. The anti-Markovnikov hydroamination of olefins was easily achieved by catalytic hydroboration with HBpin and in subsequent situ amination using H2 N-DABCO. Moreover, the combination of 1,2-diboration of olefins, using B2 pin2 , with this amination process achieved the unprecedented 1,2-diamination of olefins. The amination protocol was also successfully extended to aryl pinacol boronates.

Structure-Based Engineering of Amidase from Pantoea sp. for Efficient 2-Chloronicotinic Acid Biosynthesis.

2-Chloronicotinic acid is a key intermediate of pharmaceuticals and pesticides. Amidase-catalyzed hydrolysis provides a promising enzymatic method for 2-chloronicotinic acid production from 2-chloronicotinamide. However, biocatalytic hydrolysis of 2-chloronicotinamide is difficult due to the strong steric and electronic effect caused by 2-position chlorine substituent of the pyridine ring. In this study, an amidase from a Pantoea sp. (Pa-Ami) was designed and engineered to have improved catalytic properties. Single mutant G175A and double mutant G175A/A305T strains exhibited 3.2- and 3.7-fold improvements in their specific activity for 2-chloronicotinamide, and the catalytic efficiency was significantly increased, with kcat/Km values 3.1 and 10.0 times higher than that of the wild type, respectively. Structure-function analysis revealed that the distance between Oγ of Ser177 (involved in the catalytic triad) and the carbonyl carbon of 2-chloronicotinamide was shortened in the G175A mutant, making the nucleophilic attack on the Oγ of Ser177 easier by virtue of proper orientation. In addition, the A305T mutation contributed to a suitable tunnel formation to facilitate the substrate entry and product release, resulting in improved catalytic efficiency. With the G175A/A305T double mutant as a biocatalyst, a maximum of 1,220 mM 2-chloronicotinic acid was produced with a 94% conversion, and the space-time yield reached as high as 575 gproduct liter-1 day-1 These results provide not only a novel robust biocatalyst for the production of 2-chloronicotinic acid but also new insights into amidase structure-function relationships.IMPORTANCE In recent years, the demand for 2-chloronicotinic acid has been greatly increased. To date, several chemical methods have been used for the synthesis of 2-chloronicotinic acid, but all include tedious steps and/or drastic reaction conditions, resulting in both economic and environmental issues. It is requisite to develop an efficient and green synthesis route. We recently screened Pa-Ami and demonstrated its potential for synthesis of 2-chloronicotinic acid from 2-chloronicotinamide. However, chlorine substitution on the pyridine ring of nicotinamide significantly affected the activity of Pa-Ami. Especially for 2-chloronicotinamide, the enzyme activity and catalytic efficiency were relatively low. In this study, based on structure-function analysis, we succeeded in engineering the amidase by structure-guided saturation mutagenesis. The engineered Pa-Ami exhibited quite high catalytic activity toward 2-chloronicotinamide and could serve as a promising biocatalyst for the biosynthesis of 2-chloronicotinic acid.

Promoter engineering strategies for the overproduction of valuable metabolites in microbes.

Promoter engineering is an enabling technology in metabolic engineering and synthetic biology. As an indispensable part of synthetic biology, the promoter is a key factor in regulating genetic circuits and in coordinating multi-gene biosynthetic pathways. In this review, we summarized the recent progresses in promoter engineering in microbes. Specifically, the endogenous promoters are firstly discussed, followed by the statement of the influence of nucleotides exchange on the strength of promoters explored by site-selective mutagenesis. We then introduced the promoter libraries with a wide range of strength, which are constructed focusing on core promoter regions and upstream activating sequences by rational designs. Finally, the application of promoter libraries in the optimization of multi-gene metabolic pathways for high-yield production of metabolites was illustrated with a couple of recent examples.

Biocatalytic hydrolysis of chlorinated nicotinamides by a superior AS family amidase and its application in enzymatic production of 2-chloronicotinic acid.

2-Chloronicotinic acid (2-CA) is an important building block for a series of agrochemicals and pharmaceuticals. Amidase-catalyzed hydrolysis of 2-chloronicotinamide is one of the most attractive approaches for 2-CA production. However, development of the bioprocess was plagued by low activity of amidase for 2-chloronicotinamide. In this work, an amidase signature (AS) family amidase from Pantoea sp. (Pa-Ami), with superior activity for nicotinamide and its chlorinated derivatives, was exploited and characterized. Kinetic analysis and molecular docking clearly indicated that chlorine substitution in the pyridine ring of nicotinamide, especially the substitution at 2-position led to a dramatic decrease of Pa-Ami activity. The productivity of the bioprocess was significantly improved using fed-batch mode at low reaction temperature and 2-CA was produced as high as 370 mM with a substrate conversion of 94.2%. These results imply that Pa-Ami is potentially promising biocatalyst for industrial production of 2-CA.

Enhanced production of xylose from corncob hydrolysis with oxalic acid as catalyst.

The acid-catalyzed treatment was a conventional process for xylose production from corncob. To increase the release of xylose and to reduce the by-products formation and water usage, the oxalic acid was used as catalyst to hydrolyze the corncob and the hydrolytic conditions were investigated. The highest xylose yield of 32.7 g L-1, representing 96.1% of total theoretical xylose yield, was obtained using 1.2% oxalic acid after hydrolysis for 120 min at 130 °C, which was more than 10% higher than that of sulfuric acid-catalyzed hydrolysis. Mixed acids-catalyzed hydrolysis performed a synergistic effect for xylose production and 31.7 g L-1 of xylose was reached after reacting for 90 min with oxalic acid and sulfuric acid at a ratio of 1:4 (w/w). A kinetic model was developed to elucidate the competitive reaction between xylose formation and its degradation in the hydrolysis process, and the experimental data obtained in this study were perfectly in agreement with that of predicted from the model. Furthermore, the final xylose yield of 85% was achieved after purification and crystallization. It was demonstrated that xylose production from the corncob hydrolysis with oxalic acid as the catalyst was an effective alternative to the traditional sulfuric acid-based hydrolysis.