Multidimensional combinatorial screening for high-level production of erythritol in Yarrowia lipolytica.

Yarrowia lipolytica was successfully engineered to synthesize erythritol from crude glycerol, a cheap by-product of biodiesel production, but the yield remained low. Here, a biosensor-guided adaptive evolution screening platform was constructed to obtain mutant strains which could efficiently utilize crude glycerol to produce erythritol. Erythrose reductase D46A (M1) was identified as a key mutant through whole-genome sequencing of the strain G12, which exhibited higher catalytic activity (1.6-fold of the wild-type). M1 was further modified to obtain a combinatorial mutant with 4.1-fold enhancement of catalytic activity. Finally, the metabolic network was reconfigured to redirect carbon fluxes toward erythritol synthesis. The erythritol titer of the engineered strain G31 reached 220.5 g/L with a productivity of 1.8 g/L/h in a 5-L bioreactor. The study provides valuable guidance for biosensor-based ultra-high-throughput screening strategies in Y. lipolytica, as well as presenting a new paradigm for the sustainable valorization of crude glycerol.

Production of 17α-hydroxyprogesterone using an engineered biocatalyst with efficient electron transfer and improved 5-aminolevulinic acid synthesis coupled with a P450 hydroxylase.

17α-Hydroxyprogesterone (17α-OH-PROG) is an important intermediate with a wide range of applications in the pharmaceutical industry. Strategies based on efficient electron transfer and cofactor regeneration were used for the production of 17α-OH-PROG. Here, CYP260A1, Fpr and Adx were expressed using a double plasmid system, resulting in higher biotransformation efficiency. Further optimization of reaction conditions and addition of polymyxin B increased the production of 17α-OH-PROG from 12.52 mg/L to 102.37 mg/L after 12 h of biotransformation. To avoid the addition of external 5-aminolevulinic acid (ALA) as a heme precursor for the P450 enzyme, a modified C5 pathway was introduced into the engineered strain, further reducing the overall process cost. The resulting whole-cell biocatalyst achieved the highest biotransformation yield of 17α-OH-PROG reported to date, offering a promising strategy for commercial application of P450 enzymes in industrial production of hydroxylated intermediates.

The synergistic anti-nociceptive effects of nefopam and gabapentinoids in inflammatory, osteoarthritis, and neuropathic pain mouse models.

Pain is a common public health problem and remains as an unmet medical need. Currently available analgesics usually have limited efficacy or are accompanied by many adverse side effects. To achieve satisfactory pain relief by multimodal analgesia, new combinations of nefopam and gabapentinoids (pregabalin/gabapentin) were designed and assessed in inflammatory, osteoarthritis and neuropathic pain. Isobolographic analysis was performed to analyze the interactions between nefopam and gabapentinoids in carrageenan-induced inflammatory pain, mono-iodoacetate-induced osteoarthritis pain and paclitaxel-induced peripheral neuropathic pain in mice. The anti-inflammatory effect and motor performance of monotherapy or their combinations were evaluated in the carrageenan-induced inflammatory responses and rotarod test, respectively. Nefopam (1, 3, 5, 10, 30 mg/kg, p.o.), pregabalin (3, 6, 12, 24 mg/kg, p.o.) or gabapentin (25, 50, 75, 100 mg/kg, p.o.) dose-dependently reversed mechanical allodynia in three pain models. Isobolographic analysis indicated that the combinations of nefopam and gabapentinoids exerted synergistic anti-nociceptive effects in inflammatory, osteoarthritis, and neuropathic pain mouse models, as evidenced by the experimental ED50 (median effective dose) falling below the predicted additive line. Moreover, the combination of nefopam-pregabalin/gabapentin alleviated carrageenan-induced inflammation and edema, and also prevented gabapentinoids-related sedation or ataxia by lowering their effective doses. Collectively, the co-administration of nefopam and gabapentinoids showed synergistic analgesic effects and may result in improved therapeutic benefits for treating pain.

Novel drug-drug salt crystals of metformin with ibuprofen or naproxen: Improved solubility, dissolution rate, and synergistic antinociceptive effects.

As the monotherapy of available analgesics is usually accompanied by serious side effects or limited efficacy in the management of chronic pain, multimodal analgesia is widely used to achieve improved benefit-to-risk ratios in clinic. Drug-drug salts are extensively researched to optimize the physicochemical properties of active pharmaceutical ingredients (APIs) and achieve clinical benefits compared with individual APIs or their combination. New drug-drug salt crystals metformin-ibuprofen (MET-IBU) and metformin-naproxen (MET-NAP) were prepared from metformin (MET) and two poorly water-soluble anti-inflammatory drugs (IBU and NAP) by the solvent evaporation method. The structures of these crystals were confirmed by single crystal and powder X-ray diffraction, Hirshfeld surface, Fourier transform infrared spectroscopy and thermal analysis. Both MET-IBU and MET-NAP showed significantly improved solubility and intrinsic dissolution rate than the pure IBU or NAP. The stability test indicated that MET-IBU and MET-NAP have excellent physical stability under stressing test (10 days) and accelerated conditions (3 months). Moreover, isobolographic analysis suggested that MET-IBU and MET-NAP exerted potent and synergistic antinociceptive effects in λ-Carrageenan-induced inflammatory pain in mice, and both of them had an advantage in rapid pain relief. These results demonstrated the potential of MET-IBU and MET-NAP to achieve synergistic antinociceptive effects by developing drug-drug salt crystals.

Growth-Coupled Evolutionary Pressure Improving Epimerases for D-Allulose Biosynthesis Using a Biosensor-Assisted In Vivo Selection Platform.

Fast screening strategies that enable high-throughput evaluation and identification of desired variants from diversified enzyme libraries are crucial to tailoring biocatalysts for the synthesis of D-allulose, which is currently limited by the poor catalytic performance of ketose 3-epimerases (KEases). Here, the study designs a minimally equipment-dependent, high-throughput, and growth-coupled in vivo screening platform founded on a redesigned D-allulose-dependent biosensor system. The genetic elements modulating regulator PsiR expression levels undergo systematic optimization to improve the growth-responsive dynamic range of the biosensor, which presents ≈30-fold facilitated growth optical density with a high signal-to-noise ratio (1.52 to 0.05) toward D-allulose concentrations from 0 to 100 mm. Structural analysis and evolutionary conservation analysis of Agrobacterium sp. SUL3 D-allulose 3-epimerase (ADAE) reveal a highly conserved catalytic active site and variable hydrophobic pocket, which together regulate substrate recognition. Structure-guided rational design and directed evolution are implemented using the growth-coupled in vivo screening platform to reprogram ADAE, in which a mutant M42 (P38N/V102A/Y201L/S207N/I251R) is identified with a 6.28-fold enhancement of catalytic activity and significantly improved thermostability with a 2.5-fold increase of the half-life at 60 °C. The research demonstrates that biosensor-assisted growth-coupled evolutionary pressure combined with structure-guided rational design provides a universal route for engineering KEases.

Esketamine accelerates emergence from isoflurane general anaesthesia by activating the paraventricular thalamus glutamatergic neurones in mice.

BACKGROUND: Delayed emergence from general anaesthesia poses a significant perioperative safety hazard. Subanaesthetic doses of ketamine not only deepen anaesthesia but also accelerate recovery from isoflurane anaesthesia; however, the mechanisms underlying this phenomenon remain elusive. Esketamine exhibits a more potent receptor affinity and fewer adverse effects than ketamine and exhibits shorter recovery times after brief periods of anaesthesia. As the paraventricular thalamus (PVT) plays a pivotal role in regulating wakefulness, we studied its role in the emergence process during combined esketamine and isoflurane anaesthesia. METHODS: The righting reflex and cortical electroencephalography were used as measures of consciousness in mice during isoflurane anaesthesia with coadministration of esketamine. The expression of c-Fos was used to determine neuronal activity changes in PVT neurones after esketamine administration. The effect of esketamine combined with isoflurane anaesthesia on PVT glutamatergic (PVTGlu) neuronal activity was monitored by fibre photometry, and chemogenetic technology was used to manipulate PVTGlu neuronal activity. RESULTS: A low dose of esketamine (5 mg kg-1) accelerated emergence from isoflurane general anaesthesia (474 [30] s vs 544 [39] s, P=0.001). Esketamine (5 mg kg-1) increased PVT c-Fos expression (508 [198] vs 258 [87], P=0.009) and enhanced the population activity of PVTGlu neurones (0.03 [1.7]% vs 6.9 [3.4]%, P=0.002) during isoflurane anaesthesia (1.9 [5.7]% vs -5.1 [5.3]%, P=0.016) and emergence (6.1 [6.2]% vs -1.1 [5.0]%, P=0.022). Chemogenetic suppression of PVTGlu neurones abolished the arousal-promoting effects of esketamine (459 [33] s vs 596 [33] s, P<0.001). CONCLUSIONS: Our results suggest that esketamine promotes recovery from isoflurane anaesthesia by activating PVTGlu neurones. This mechanism could explain the rapid arousability exhibited upon treatment with a low dose of esketamine.

Sequence- and Structure-Based Mining of Thermostable D-Allulose 3-Epimerase and Computer-Guided Protein Engineering To Improve Enzyme Activity.

D-Allulose, a functional sweetener, can be synthesized from fructose using D-allulose 3-epimerase (DAEase). Nevertheless, a majority of the reported DAEases have inadequate stability under harsh industrial reaction conditions, which greatly limits their practical applications. In this study, big data mining combined with a computer-guided free energy calculation strategy was employed to discover a novel DAEase with excellent thermostability. Consensus sequence analysis of flexible regions and comparison of binding energies after substrate docking were performed using phylogeny-guided big data analyses. TtDAE from Thermogutta terrifontis was the most thermostable among 358 candidate enzymes, with a half-life of 32 h at 70 °C. Subsequently, structure-guided virtual screening and a customized strategy based on a combinatorial active-site saturation test/iterative saturation mutagenesis were utilized to engineer TtDAE. Finally, the catalytic activity of the M4 variant (P105A/L14C/T63G/I65A) was increased by 5.12-fold. Steered molecular dynamics simulations indicated that M4 had an enlarged substrate-binding pocket, which enhanced the fit between the enzyme and the substrate. The approach presented here, combining DAEases mining with further rational modification, provides guidance for obtaining promising catalysts for industrial-scale production.

Enhanced Thermostability of an l-Rhamnose Isomerase for d-Allose Synthesis by Computation-Based Rational Redesign of Flexible Regions.

d-Allose is a low-calorie rare sugar with great application potential in the food and pharmaceutical industries. The production of d-allose has been accomplished using l-rhamnose isomerase (L-RI), but concomitantly increasing the enzyme's stability and activity remains challenging. Here, we rationally engineered an L-RI from Clostridium stercorarium to enhance its stability by comprehensive computation-aided redesign of its flexible regions, which were successively identified using molecular dynamics simulations. The resulting combinatorial mutant M2-4 exhibited a 5.7-fold increased half-life at 75 °C while also exhibiting improved catalytic efficiency. Especially, by combining structure modeling and multiple sequence alignment, we identified an α0 region that was universal in the L-RI family and likely acted as a "helix-breaker". Truncating this region is crucial for improving the thermostability of related enzymes. Our work provides a significantly stable biocatalyst with potential for the industrial production of d-allose.

Pregabalin can interact synergistically with Kv7 channel openers to exert antinociception in mice.

Chronic pain is a common public health problem and remains an unmet medical need. Currently available analgesics usually have limited efficacy for the treatment of chronic pain, including neuropathic pain and persistent inflammatory pain, or they are accompanied by many adverse side effects. The voltage-gated calcium channel blocker (pregabalin) and potassium channel openers (flupirtine and retigabine) have been widely used for the management of chronic pain, but their effectiveness in combination is unclear. In this research, we evaluated the antinociceptive effects of pregabalin in combination with flupirtine or retigabine in carrageenan-induced inflammatory pain and paclitaxel-induced peripheral neuropathy in mice using the von Frey test. Isobolographic analysis indicated that pregabalin exerted synergistic antinociceptive effects when combined with flupirtine or retigabine in neuropathic and inflammatory pain models. Furthermore, the antinociceptive effects of pregabalin, flupirtine/retigabine, and their combinations were significantly attenuated by the Kv7 channel blocker XE991. The favored dose ratio between pregabalin and flupirtine/retigabine in combinations was also investigated. Finally, we evaluated the motor coordination of their combinations using the rotarod test, and the outcomes underpinned their safety. Collectively, our results support the potential use of pregabalin in combination with flupirtine or retigabine to alleviate chronic pain.

State-of-the-art strategies and research advances for the biosynthesis of D-amino acids.

D-amino acids (D-AAs) are the enantiomeric counterparts of L-amino acids (L-AAs) and important functional factors with a wide variety of physiological activities and applications in the food manufacture industry. Some D-AAs, such as D-Ala, D-Leu, and D-Phe, have been favored by consumers as sweeteners and fragrances because of their unique flavor. The biosynthesis of D-AAs has attracted much attention in recent years due to their unique advantages. In this review, we comprehensively analyze the structure-function relationships, biosynthesis pathways, multi-enzyme cascade and whole-cell catalysis for the production of D-AAs. The state-of-the-art strategies, including immobilization, protein engineering, and high-throughput screening, are summarized. Future challenges and perspectives of strategies-driven by bioinformatics technologies and smart computing technologies, as well as enzyme immobilization, are also discussed. These new approaches will promote the commercial production and application of D-AAs in the food industry by optimizing the key enzymes for industrial biocatalysts.

Substantial Improvement of an Epimerase for the Synthesis of D-Allulose by Biosensor-Based High-Throughput Microdroplet Screening.

Biosynthesis of D-allulose has been achieved using ketose 3-epimerases (KEases), but its application is limited by poor catalytic performance. In this study, we redesigned a genetically encoded biosensor based on a D-allulose-responsive transcriptional regulator for real-time monitoring of D-allulose. An ultrahigh-throughput droplet-based microfluidic screening platform was further constructed by coupling with this D-allulose-detecting biosensor for the directed evolution of the KEases. Structural analysis of Sinorhizobium fredii D-allulose 3-epimerase (SfDAE) revealed that a highly flexible helix/loop region exposes or occludes the catalytic center as an essential lid conformation regulating substrate recognition. We reprogrammed SfDAE using structure-guided rational design and directed evolution, in which a mutant M3-2 was identified with 17-fold enhanced catalytic efficiency. Our research offers a paradigm for the design and optimization of a biosensor-based microdroplet screening platform.

Directional immobilization of D-allulose 3-epimerase using SpyTag/SpyCatcher strategy as a robust biocatalyst for synthesizing D-allulose.

D-Allulose, as low-calorie rare sugar, possessed several notable biological activities and was biosynthesized by D-allulose 3-epimerase (DAEase). Here, CcDAE from Clostridium cellulolyticum was successfully immobilization via covalent attachment (RI-CcDAE), and Resin-SpyCatcher/SpyTag-CcDAE modular (DI-CcDAE). Both immobilized CcDAEs exhibited higher thermal and pH stabilities than the free form, and they maintained 80.0 % of relative activity after 7 consecutive cycles and 25 days of storage. Predominantly, DI-CcDAE represented superior catalytic efficiency with a 2.4-fold increase of kcat/Km, compared with RI-CcDAE (0.75 s-1 mM-1 vs 0.31 s-1 mM-1). The RI-CcDAE and DI-CcDAE were then applied in mixed fruit Jiaosu to convert D-fructose into D-allulose, which exhibited the productivity of D-allulose 1.08 g/Lh-1 and 1.57 g/Lh-1, respectively. This research provided a promising directional immobilization strategy for DAEase, and robust biocatalyst for production of functional foodstuff containing D-allulose.

Engineering of Acid-Resistant d-Allulose 3-Epimerase for Functional Juice Production.

d-Allulose, a rare sugar and functional sweetener, can be biosynthesized by d-allulose 3-isomerase (DAE). However, most of the reported DAEs exhibit poor resistance under acidic conditions, which severely limited their application. Here, surface charge engineering and random mutagenesis were used to construct a mutant library of CcDAE from Clostridium cellulolyticum H10, combined with high-throughput screening to identify mutants with high activity and resistance under acidic conditions. The mutant M3 (I114R/K123E/H209R) exhibited high activity (3.36-fold of wild-type) and acid resistance (10.6-fold of wild-type) at pH 4.5. The structure-function relationship was further analyzed by molecular dynamics (MD) simulations, which indicated that M3 had a higher number of hydrogen bonds and negative surface charges than the wild type. A multienzyme cascade system including M3 was used to convert high-calorie sugars in acidic juices, and functional juices containing 7.8-15.4 g/L d-allulose were obtained. Our study broadens the manufacture of functional foods containing d-allulose.

Fine-Tuning of Carbon Flux and Artificial Promoters in Bacillus subtilis Enables High-Level Biosynthesis of d-Allulose.

d-Allulose is an attractive rare sugar that can be used as a low-calorie sweetener with significant health benefits. To meet the increasing market demands, it is necessary to develop an efficient and extensive microbial fermentation platform for the synthesis of d-allulose. Here, we applied a comprehensive systematic engineering strategy in Bacillus subtilis WB600 by introducing d-allulose 3-epimerase (DAEase), combined with the deactivation of fruA, levDEFG, and gmuE, to balance the metabolic network for the efficient production of d-allulose. This resulting strain initially produced 3.24 g/L of d-allulose with a yield of 0.93 g of d-allulose/g d-fructose. We further screened and obtained a suitable dual promoter combination and performed fine-tuning of its spacer region. After 64 h of fed-batch fermentation, the optimized engineered B. subtilis produced d-allulose at titers of 74.2 g/L with a yield of 0.93 g/g and a conversion rate of 27.6%. This d-allulose production strain is a promising platform for the industrial production of rare sugar.

Structural insight and engineering of a plastic degrading hydrolase Ple629.

Polyethylene terephthalate (PET) is one of the most abundantly produced synthetic polyesters. The vast number of waste plastics including PET has challenged the waste management sector while also posing a serious threat to the environment due to improper littering. Recently, enzymatic PET degradation has been shown to be a viable option for a circular plastic economy, which can mitigate the plastic pollution. While protein engineering studies on specific PET degradation enzymes such as leaf-branch compost cutinase (LCC), Thermobifida sp. cutinases and Ideonella sakaiensis PETase (IsPETase) have been extensively published, other homologous PET degrading enzymes have received less attention. Ple629 is a polyester hydrolase identified from marine microbial consortium having activity on PET and the bioplastic polybutylene adipate terephthalate (PBAT). In order to explore its catalytic mechanism and improve its potential for PET hydrolysis, we solved its crystal structure in complex with a PET monomer analogue, and validated its structural and mechanistic similarity to known PET hydrolases. By structural comparisons, we identified some hot spot positions described in previous research on protein engineering of PET hydrolases. We substitute these amino acid residues in Ple629, and obtained variants with improved activity and thermo-stability. The most promising variant D226A/S279A exhibited a more than 5.5-fold improved activity on PET nanoparticles than the wild-type enzyme, suggesting its potential applicability in the biotechnological plastic recycling.

Reshaping the binding channel of a novel GH113 family ß-mannanase from Paenibacillus cineris (PcMan113) for enhanced activity.

Endo-ß-mannanases are important enzymes for degrading lignocellulosic biomass to generate mannan, which has significant health effects as a prebiotic that promotes the development of gut microbiota. Here, a novel endo-ß-mannanase belonging to glycoside hydrolase (GH) family 113 from Paenibacillus cineris (PcMan113) was cloned, expressed and characterized, as one of only a few reported GH113 family ß-mannanases. Compared to other functionally and structurally characterized GH113 mannanases, recombinant PcMan113 showed a broader substrate spectrum and a better performance. Based on a structural homology model, the highly active mutant PcMT3 (F110E/N246Y) was obtained, with 4.60- and 5.53-fold increases of enzyme activity (towards KG) and catalytic efficiency (kcat/Km, against M5) compared with the WT enzyme, respectively. Furthermore, molecular dynamics (MD) simulations were conducted to precisely explore the differences of catalytic activity between WT and PcMT3, which revealed that PcMT3 has a less flexible conformation, as well as an enlarged substrate-binding channel with decreased steric hindrance and increased binding energy in substrate recognition. In conclusion, we obtained a highly active variant of PcMan113 with potential for commercial application in the manufacture of manno-oligosaccharides.

Continuous Spectrophotometric Assay for High-Throughput Screening of Predominant d-Allulose 3-Epimerases.

d-Allulose is an attractive noncaloric sugar substitute with numerous health benefits, which can be biosynthesized by d-allulose 3-epimerases (DAEases). However, enzyme instability under harsh industrial reaction conditions hampered its practical applications. Here, we developed a continuous spectrophotometric assay (CSA) for the efficient analysis of d-allulose in a mixture. Furthermore, a high-throughput screening strategy for DAEases was developed using CSA by coupling DAEase with a NADH-dependent ribitol dehydrogenase, enabling high-throughput screening of DAEase variants with desired properties. The variant M15S/P40N/S209N exhibited a half-life of 22 h at 60 °C and an 8.7 °C increase of the T5060 value, with a 1.2-fold increase of activity. Structural modeling and molecular dynamics simulations indicated that the improvement of thermostability and activity was due to some new hydrogen bonds between chains at the dimer interface and between the residue and the substrate d-fructose. This work offers a robust tool and theoretical basis for the improvement of DAEases, which will benefit the enzymatic biosynthesis of d-allulose and promote its industrial application.

Structural Basis of Salicylic Acid Decarboxylase Reveals a Unique Substrate Recognition Mode and Access Channel.

Salicylic acid (SA) decarboxylase from Trichosporon moniliiforme (TmSdc), which reversibly catalyses the decarboxylation of SA to yield phenol, is of significant interest because of its potential for the production of benzoic acid derivatives under environmentally friendly reaction conditions. TmSdc showed a preference for C2 hydroxybenzoate derivatives, with kcat/Km of SA being 3.2 × 103 M-1 s-1. Here, we presented the first crystal structures of TmSdc, including a complex with SA. The three conserved residues of Glu8, His169, and Asp298 are the catalytic residues within the TIM-barrel domain of TmSdc. Trp239 forms a unique hydrophobic recognition site by interacting with the phenyl ring of SA, while Arg235 is responsible for recognizing the hydroxyl group at the C2 of SA via hydrogen bond interactions. Using a semi-rational combinatorial active-site saturation test, we obtained the TmSdc mutant MT3 (Y64T/P191G/F195V/E302D), which exhibited a 26.4-fold increase in kcat/Km with SA, reaching 8.4 × 104 M-1 s-1. Steered molecular dynamics simulations suggested that the structural changes in MT3 relieved the steric hindrance within the substrate access channel and enlarged the substrate-binding pocket, leading to the increased activity by improving substrate access. Our data elucidate the unique substrate recognition mode and the substrate entrance tunnel of SA decarboxylase.

Biochemical and structural characterization of a novel thermophilic and acidophilic ß-mannanase from Aspergillus calidoustus.

ß-Mannanases hydrolyze lignocellulosic biomass with the release of mannan oligosaccharides, which are considered as renewable resource in higher plants. Here, we cloned, expressed and characterized a novel endo-ß-mannanase (ManAC) from Aspergillus calidoustus. Homology alignment analysis indicated that ManAC belonged to glycosyl hydrolase (GH) 5 family members. The analysis of structural homologous model revealed that five residues, Arg116, Asn231, His305, Tyr307, and Trp370, constituted the active site of ManAC. Glu232 and Glu340, proton donor and nucleophile, formed the catalytic residues of ManAC. The recombinant ManAC exhibited maximal activity at pH 2.5 and 70 °C, and it was acid tolerant at a pH range of 2.0-6.0 and thermostable under 60 °C. Meanwhile, the activity of ManAC was not significantly affected by various metal ions, except for Mg2+ and Ag2+. The recombinant ManAC exhibited the highest ß-mannanase activity towards locust bean gum (669.7 U/mg) with the Km and Vmax values of 3.4 mg/mL and 982.4 µmol/min/mg, respectively. These thermophilic and acidophilicc characteristics is better than most extreme ß-mannanase. As the first reported mannanse from Aspergillus calidoustus (ManAC), these excellent properties of ManAC strongly promote the synthesis of mannooligosaccharides which have potential for food and feed industrial applications.