Simultaneously enhanced magnomechanical cooling and entanglement assisted by an auxiliary microwave cavity.

We propose a mechanism to simultaneously enhance quantum cooling and entanglement via coupling an auxiliary microwave cavity to a magnomechanical cavity. The auxiliary cavity acts as a dissipative cold reservoir that can efficiently cool multiple localized modes in the primary system via beam-splitter interactions, which enables us to obtain strong quantum cooling and entanglement. We analyze the stability of the system and determine the optimal parameter regime for cooling and entanglement under the auxiliary-microwave-cavity-assisted (AMCA) scheme. The maximum cooling enhancement rate of the magnon mode can reach 98.53%, which clearly reveals that the magnomechanical cooling is significantly improved in the presence of the AMCA. More importantly, the dual-mode entanglement of the system can also be significantly enhanced by AMCA in the full parameter region, where the initial magnon-phonon entanglement can be maximally enhanced by a factor of about 11. Another important result of the AMCA is that it also increases the robustness of the entanglement against temperature. Our approach provides a promising platform for the experimental realization of entanglement and quantum information processing based on cavity magnomechanics.

Revisiting the critical roles of reactive astrocytes in neurodegeneration.

Astrocytes, an integral component of the central nervous system (CNS), contribute to the maintenance of physiological homeostasis through their roles in synaptic function, K+ buffering, blood-brain barrier (BBB) maintenance, and neuronal metabolism. Reactive astrocytes refer to astrocytes undergoing morphological, molecular and functional remodelling in response to pathological stimuli. The activation and differentiation of astrocytes are implicated in the pathogenesis of multiple neurodegenerative diseases. However, there are still controversies regarding their subset identification, function and nomenclature in neurodegeneration. In this review, we revisit the multidimensional roles of reactive astrocytes in Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Furthermore, we propose a precise linkage between astrocyte subsets and their functions based on single-cell sequencing analyses.

Advances in nucleic acid aptamer-based detection of respiratory virus and bacteria: a mini review.

Respiratory pathogens infecting the human respiratory system are characterized by their diversity, high infectivity, rapid transmission, and acute onset. Traditional detection methods are time-consuming, have low sensitivity, and lack specificity, failing to meet the needs of rapid clinical diagnosis. Nucleic acid aptamers, as an emerging and innovative detection technology, offer novel solutions with high specificity, affinity, and broad target applicability, making them particularly promising for respiratory pathogen detection. This review highlights the progress in the research and application of nucleic acid aptamers for detecting respiratory pathogens, discussing their selection, application, potential in clinical diagnosis, and future development. Notably, these aptamers can significantly enhance the sensitivity and specificity of detection when combined with detection techniques such as fluorescence, colorimetry and electrochemistry. This review offers new insights into how aptamers can address the limitations of traditional diagnostic methods and advance clinical diagnostics. It also highlights key challenges and future research directions for the clinical application of nucleic acid aptamers.

Improving the catalytic performance of carbonyl reductase based on the functional loops engineering.

Vibegron functions as a potent and selective ß3-adrenergic receptor agonist, with its chiral precursor (2S,3R)-aminohydroxy ester (1b) being crucial to its synthesis. In this study, loop engineering was applied to the carbonyl reductase (EaSDR6) from Exiguobacterium algae to achieve an asymmetric reduction of the (rac)-aminoketone ester 1a. The variant M5 (A138L/A190V/S193A/Y201F/N204A) was obtained and demonstrated an 868-fold increase in catalytic efficiency (kcat/Km = 260.3 s-1 mM-1) and a desirable stereoselectivity (>99% enantiomeric excess, e.e.; >99% diastereomeric excess, d.e.) for the target product 1b in contrast to the wild-type EaSDR6 (WT). Structural alignment with WT indicated that loops 137-154 and 182-210 potentially play vital roles in facilitating catalysis and substrate binding. Moreover, molecular dynamics (MD) simulations of WT-1a and M5-1a complex illustrated that M5-1a exhibits a more effective nucleophilic attack distance and more readily adopts a pre-reaction state. The interaction analysis unveiled that M5 enhanced hydrophobic interactions with substrate 1a on cavities A and B while diminishing unfavorable hydrophilic interactions on cavity C. Computational analysis of binding free energies indicated that M5 displayed heightened affinity towards substrate 1a compared to the WT, aligning with its decreased Km value. Under organic-aqueous biphasic conditions, the M5 mutant showed >99% conversion within 12 h with 300 g/L substrate 1a (highest substrate loading as reported). This study enhanced the catalytic performance of carbonyl reductase through functional loops engineering and established a robust framework for the large-scale biosynthesis of the vibegron intermediate.

Adjustment of the main biosynthesis modules to enhance the production of l-homoserine in Escherichia coli W3110.

l-homoserine is an important platform compound of many valuable products. Construction of microbial cell factory for l-homoserine production from glucose has attracted a great deal of attention. In this study, l-homoserine biosynthesis pathway was divided into three modules, the glucose uptake and upstream pathway, the downstream pathway, and the energy supply module. Metabolomics of the chassis strain HS indicated that the supply of ATP was inadequate, therefore, the energy supply module was firstly modified. By balancing the ATP supply module, the l-homoserine production increased by 66% to 12.55 g/L. Further, the results indicated that the upstream pathway was blocked, and increasing the culture temperature to 37°C could solve this problem and the l-homoserine production reached 21.38 g/L. Then, the downstream synthesis pathways were further strengthened to balance the fluxes, and the l-homoserine production reached the highest reported level of 32.55 g/L in shake flasks. Finally, fed-batch fermentation in a 5-L bioreactor was conducted, and l-homoserine production could reach to 119.96 g/L after 92 h cultivation, with the yield of 0.41 g/g glucose and productivity of 1.31 g/L/h. The study provides a well research foundation for l-homoserine production by microbial fermentation with the capacity for industrial application.

Fine and combinatorial regulation of key metabolic pathway for enhanced ß-alanine biosynthesis with non-inducible Escherichia coli.

ß-Alanine is the only ß-amino acid in nature and one of the most important three-carbon chemicals. This work was aimed to construct a non-inducible ß-alanine producer with enhanced metabolic flux towards ß-alanine biosynthesis in Escherichia coli. First of all, the assembled E. coli endogenous promoters and 5'-untranslated regions (PUTR) were screened to finely regulate the combinatorial expression of genes panDBS and aspBCG for an optimal flux match between two key pathways. Subsequently, additional copies of key genes (panDBS K104S and ppc) were chromosomally introduced into the host A1. On these bases, dynamical regulation of the gene thrA was performed to reduce the carbon flux directed in the competitive pathway. Finally, the ß-alanine titer reached 10.25 g/L by strain A14-R15, 361.7% higher than that of the original strain. Under fed-batch fermentation in a 5-L fermentor, a titer of 57.13 g/L ß-alanine was achieved at 80 h. This is the highest titer of ß-alanine production ever reported using non-inducible engineered E. coli. This metabolic modification strategy for optimal carbon flux distribution developed in this work could also be used for the production of various metabolic products.

Two-Photon-Excited Fluorescent Tetrabranched Triphenylborane Featuring the Cooperative Effect of Branching in Two-Photon Absorption.

We here disclose a new type of two-photon-excited fluorescent triarylborane, tetrabranched triphenylborane 1, which contains four electron-donating [4-(N,N-diphenylamino)phenyl]ethynyl branches at 2,6-positions of two phenyl rings. The cross section of 1 reaches 275 GM (1 GM = 10-50 cm4 s photon-1) in tetrahydrofuran. Compared with dibranched triphenylborane 2, the 2-fold increase in the number of electron-donating branches induces a 3.6-fold increase in the two-photon absorption cross section, suggesting the great cooperative effect of branching in the enhancement of two-photon absorption.

Enhanced D-pantothenic acid biosynthesis by plasmid-free E. coli through sodium pyruvate addition combined with glucose and temperature control strategy.

AIMS: D-Pantothenic acid (D-PA) is an important vitamin widely used in the feed, pharmaceutical, and food industries. This study aims to enhance the D-PA production of a recombinant Escherichia coli without plasmid and inducer induction. METHODS AND RESULTS: The fermentation medium in shake flask was optimized, resulting in an 39.50% increased D-PA titer (3.32 g L-1). Subsequently, the fed-batch fermentation in a 5-L fermenter were specifically investigated. Firstly, a two-stage temperature control strategy led to a D-PA titer of 52.09 g L-1. Additionally, a two-stage glucose feeding was proposed and D-PA titer was increased to 65.29 g L-1. It was also found that appropriate amount of sodium pyruvate was beneficial to cell growth and D-PA synthesis. Finally, a two-stage glucose feeding combined with sodium pyruvate addition resulted in a substantially improved D-PA production with a titer of 72.90 g L-1. CONCLUSION: The D-PA synthesis was significantly improved through the fermentation process established in this work, that is sodium pyruvate addition combined with the temperature and glucose control strategy. The results of this study could provide significant reference for the industrial fermentation production of D-PA.

Substrate access tunnel engineering of a Fe-type nitrile hydratase from Pseudomonas fluorescens ZJUT001 for substrate preference adjustment and catalytic performance enhancement.

Substrate access tunnel engineering is a useful strategy for enzyme modification. In this study, we improved the catalytic performance of Fe-type Nitrile hydratase (Fe-type NHase) from Pseudomonas fluorescens ZJUT001 (PfNHase) by mutating residue Q86 at the entrance of the substrate access tunnel. The catalytic activity of the mutant PfNHase-αQ86W towards benzonitrile, 2-cyanopyridine, 3-cyanopyridine, and 4-hydroxybenzonitrile was enhanced by 9.35-, 3.30-, 6.55-, and 2.71-fold, respectively, compared to that of the wild-type PfNHase (PfNHase-WT). In addition, the mutant PfNHase-αQ86W showed a catalytic efficiency (kcat/Km) towards benzonitrile 17.32-fold higher than the PfNHase-WT. Interestingly, the substrate preference of PfNHase-αQ86W shifted from aliphatic nitriles to aromatic nitrile substrates. Our analysis delved into the structural changes that led to this altered substrate preference, highlighting an expanded entrance tunnel region, theenlarged substrate-binding pocket, and the increased hydrophobic interactions between the substrate and enzyme. Molecular dynamic simulations and dynamic cross-correlation Matrix (DCCM) further supported these findings, providing a comprehensive explanation for the enhanced catalytic activity towards aromatic nitrile substrates.

Glutamatergic neurons in ventral pallidum modulate heroin addiction via epithalamic innervation in rats.

Glutamatergic neurons in ventral pallidum (VPGlu) were recently reported to mediate motivational and emotional behavior, but its role in opioid addiction still remains to be elucidated. In this study we investigated the function of VPGlu in the context-dependent heroin taking and seeking behavior in male rats under the ABA renewal paradigm. By use of cell-type-specific fiber photometry, we showed that the calcium activity of VPGlu were inhibited during heroin self-administration and context-induced relapse, but activated after extinction in a new context. The drug seeking behavior was accompanied by the decreased calcium signal of VPGlu. Chemogenetic manipulation of VPGlu bidirectionally regulated heroin taking and seeking behavior. Anterograde tracing showed that the lateral habenula, one of the epithalamic structures, was the major output region of VPGlu, and its neuronal activity was consistent with VPGlu in different phases of heroin addiction and contributed to the motivation for heroin. VPGlu axon terminals in LHb exhibited dynamic activity in different phases of heroin addiction. Activation of VPGlu-LHb circuit reduced heroin seeking behavior during context-induced relapse. Furthermore, the balance of excitation/inhibition from VP to LHb was shifted to enhanced glutamate transmission after extinction of heroin seeking motivation. Overall, the present study demonstrated that the activity of VPGlu was involved in the regulation of heroin addiction and identified the VPGlu-LHb pathway as a potential intervention to reduce heroin seeking motivation.

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.

Rerouting phytosterol degradation pathway for directed androst-1,4-diene-3,17-dione microbial bioconversion.

Steroid-based drugs are now mainly produced by the microbial transformation of phytosterol, and a two-step bioprocess is adopted to reach high space-time yields, but byproducts are frequently observed during the bioprocessing. In this study, the catabolic switch between the C19- and C22-steroidal subpathways was investigated in resting cells of Mycobacterium neoaurum NRRL B-3805, and a dose-dependent transcriptional response toward the induction of phytosterol with increased concentrations was found in the putative node enzymes including ChoM2, KstD1, OpccR, Sal, and Hsd4A. Aldolase Sal presented a dominant role in the C22 steroidal side-chain cleavage, and the byproduct was eliminated after sequential deletion of opccR and sal. Meanwhile, the molar yield of androst-1,4-diene-3,17-dione (ADD) was increased from 59.4 to 71.3%. With the regard of insufficient activity of rate-limiting enzymes may also cause byproduct accumulation, a chromosomal integration platform for target gene overexpression was established supported by a strong promoter L2 combined with site-specific recombination in the engineered cell. Rate-limiting steps of ADD bioconversion were further characterized and overcome. Overexpression of the kstD1 gene further strengthened the bioconversion from AD to ADD. After subsequential optimization of the bioconversion system, the directed biotransformation route was developed and allowed up to 82.0% molar yield with a space-time yield of 4.22 g·L-1·day-1. The catabolic diversion elements and the genetic overexpression tools as confirmed and developed in present study offer new ideas of M. neoaurum cell factory development for directed biotransformation for C19- and C22-steroidal drug intermediates from phytosterol. KEY POINTS: • Resting cells exhibited a catabolic switch between the C19- and C22-steroidal subpathways. • The C22-steroidal byproduct was eliminated after sequential deletion of opccR and sal. • Rate-limiting steps were overcome by promoter engineering and chromosomal integration.

Transcriptome analysis of Kluyveromyces marxianus under succinic acid stress and development of robust strains.

Kluyveromyces marxianus has become an attractive non-conventional yeast cell factory due to its advantageous properties such as high thermal tolerance and rapid growth. Succinic acid (SA) is an important platform molecule that has been applied in various industries such as food, material, cosmetics, and pharmaceuticals. SA bioproduction may be compromised by its toxicity. Besides, metabolite-responsive promoters are known to be important for dynamic control of gene transcription. Therefore, studies on global gene transcription under various SA concentrations are of great importance. Here, comparative transcriptome changes of K. marxianus exposed to various concentrations of SA were analyzed. Enrichment and analysis of gene clusters revealed repression of the tricarboxylic acid cycle and glyoxylate cycle, also activation of the glycolysis pathway and genes related to ergosterol synthesis. Based on the analyses, potential SA-responsive promoters were investigated, among which the promoter strength of IMTCP2 and KLMA_50231 increased 43.4% and 154.7% in response to 15 g/L SA. In addition, overexpression of the transcription factors Gcr1, Upc2, and Ndt80 significantly increased growth under SA stress. Our results benefit understanding SA toxicity mechanisms and the development of robust yeast for organic acid production. KEY POINTS: • Global gene transcription of K. marxianus is changed by succinic acid (SA) • Promoter activities of IMTCP2 and KLMA_50123 are regulated by SA • Overexpression of Gcr1, Upc2, and Ndt80 enhanced SA tolerance.

Preparation of (S)-epichlorohydrin using a novel halohydrin dehalogenase by selective conformation adjustment.

Chiral epichlorohydrin (ECH) is an attractive intermediate for chiral pharmaceuticals and chemicals preparation. The asymmetric synthesis of chiral ECH using 1,3-dicholoro-2-propanol (1,3-DCP) catalyzed by a haloalcohol dehalogenase (HHDH) was considered as a feasible approach. However, the reverse ring opening reaction caused low optical purity of chiral ECH, thus severely restricts the industrial application of HHDHs. In the present study, a novel selective conformation adjustment strategy was developed with an engineered HheCPS to regulate the kinetic parameters of the forward and reverse reactions, based on site saturation mutation and molecular simulation analysis. The HheCPS mutant E85P was constructed with a markable change in the conformation of (S)-ECH in the substrate pocket and a slight impact on the interaction between 1,3-DCP and the enzyme, which resulted in the kinetic deceleration of the reverse reactions. Compared with HheCPS, the catalytic efficiency (kcat(S)-ECH/Km(S)-ECH) of the reversed reaction dropped to 0.23-fold (from 0.13 to 0.03 mM-1 s-1), while the catalytic efficiency (kcat(1,3-DCP)/Km(1,3-DCP)) of the forward reaction only reduced from 0.83 to 0.71 mM-1 s-1. With 40 mM 1,3-DCP as substrate, HheCPS E85P catalyzed the synthesis of (S)-ECH with the yield up to 55.35% and the e.e. increased from 92.54 to >99%. Our work provided an effective approach for understanding the stereoselective catalytic mechanism as well as the green manufacturing of chiral epoxides.

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.

Ultrasound evaluation of gastric emptying of high-energy semifluid solid beverage in parturients during labor at term: a randomized controlled trial.

PURPOSE: What to intake during labor is controversial. The purpose of this study was to compare the gastric emptying of high-energy semifluid solid beverage (HESSB) versus that of carbohydrate (CHO) solution of equal calories and volume by evaluating the gastric antral cross-sectional area (CSA) using ultrasonography in parturients during labor at term. METHODS: The study was conducted at a maternity and infant hospital between June and October 2020. Forty parturients scheduled for epidural labor analgesia during labor at term were randomly assigned to receive HESSB (300 mL, n = 20) or CHO (300 mL, n = 20). Gastric antral CSA was measured at baseline and 5, 30, 60, 90, and 120 min after consumption of the drink. The primary outcome was gastric antral CSA at 120 min in the HESSB group and CHO group. RESULTS: The gastric antral CSA between the HESSB group and CHO group at 120 min was not statistically significant (2.73 cm2 ± 0.55 vs. 2.55 cm2 ± 0.72, P = 0.061). All patients returned to baseline at 120 min after intake of 300 mL isocaloric HESSB and CHO, confirmed by evaluation of gastric antral CSA. The visual analog scale score for satiety was higher in the HESSB group (P < 0.001), with better taste satisfaction (7[5-8] vs. 5[4-6], P < 0.001). CONCLUSION: The change of gastric antral cross-sectional area after HESSB is similar to the corresponding calories and volume of CHO and the gastric emptying of HESSB can be emptied within 2 h with better taste satisfaction and satiety in pregnant women under labor analgesia.

A novel positive modulator of GABAA receptor exhibiting antidepressive properties.

γ-Aminobutyric acid (GABA) neurotransmission alterations have been implicated to play a role in depression pathogenesis. While GABAA receptor positive allosteric modulators are emerging as promising in clinical practice, their precise antidepressant mechanism remains to be further elucidated. The aim of the present study was to investigate the effects of LY-02, a novel compound derived from the metabolite of timosaponin, on depression in animals and its mechanism. The results of behavioral tests showed that LY-02 exhibited better antidepressant effects in both male C57BL/6 mice and Sprague Dawley (SD) rats. The results of cellular voltage clamp experiments showed that LY-02 enhanced GABA-mediated currents in HEK293T cells expressing recombinant α6ß3δ subunit-containing GABAA receptors. Electrophysiological recording from brain slices showed that LY-02 decreased the amplitude of spontaneous inhibitory postsynaptic current (sIPSC) and increased action potentials of pyramidal neurons in the medial prefrontal cortex (mPFC) of C57BL/6 mice. Western blot results showed that LY-02 dose-dependently up-regulated the protein expression levels of brain-derived neurotrophic factor (BDNF), tropomyosin related kinase B (TrkB) and postsynaptic density protein 95 (PSD-95) in mPFC of mice. The above results suggest that LY-02, as a positive modulator of GABAA receptors, reduces inhibitory neurotransmission in pyramidal neurons. It further activates the BDNF/TrkB signaling pathway, thus exerting antidepressant effects. It suggests that LY-02 is a potential novel therapeutic agent for depression treatment.

Social isolation reinforces aging-related behavioral inflexibility by promoting neuronal necroptosis in basolateral amygdala.

Aging is characterized with a progressive decline in many cognitive functions, including behavioral flexibility, an important ability to respond appropriately to changing environmental contingencies. However, the underlying mechanisms of impaired behavioral flexibility in aging are not clear. In this study, we reported that necroptosis-induced reduction of neuronal activity in the basolateral amygdala (BLA) plays an important role in behavioral inflexibility in 5-month-old mice of the senescence-accelerated mice prone-8 (SAMP8) line, a well-established model with age-related phenotypes. Application of Nec-1s, a specific inhibitor of necroptosis, reversed the impairment of behavioral flexibility in SAMP8 mice. We further observed that the loss of glycogen synthase kinase 3α (GSK-3α) was strongly correlated with necroptosis in the BLA of aged mice and the amygdala of aged cynomolgus monkeys (Macaca fascicularis). Moreover, genetic deletion or knockdown of GSK-3α led to the activation of necroptosis and impaired behavioral flexibility in wild-type mice, while the restoration of GSK-3α expression in the BLA arrested necroptosis and behavioral inflexibility in aged mice. We further observed that GSK-3α loss resulted in the activation of mTORC1 signaling to promote RIPK3-dependent necroptosis. Importantly, we discovered that social isolation, a prevalent phenomenon in aged people, facilitated necroptosis and behavioral inflexibility in 4-month-old SAMP8 mice. Overall, our study not only revealed the molecular mechanisms of the dysfunction of behavioral flexibility in aged people but also identified a critical lifestyle risk factor and a possible intervention strategy.

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.

Efficient biosynthesis of Vibegron intermediate using a novel carbonyl reductase based on molecular modification of hydrogen bonding network regulation.

Vibegron is a novel, potent, highly selective ß3-adrenergic receptor agonist for the treatment of overactive bladder with higher therapeutic capacity and lower side effects. Methyl(2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-phenylpropanoate ((2S,3R)-aminohydroxy ester) is a key chiral intermediate for the synthesis of Vibegron. A novel carbonyl reductase from Exiguobacterium sp. s126 (EaSDR6) was isolated using data mining technology from GenBank database with preferable catalytic activity. Hydrogen bond network regulation was performed using site-directed saturation mutagenesis and combination mutagenesis. The mutant EaSDR6A138L/S193A was obtained with the activity improvement by 4.58 folds compared with the wild type EaSDR6. The Km of EaSDR6A138L/S193A was decreased from 1.57 mM to 0.67 mM, kcat was increased by 2.17 folds, and the overall catalytic efficiency kcat/Km was increased by 5.07 folds. The organic-aqueous biphasic bioreaction system for the asymmetric synthesis of (2S,3R)-aminohydroxy ester was constructed for the first time. Under the substrate concentration of 150 g/L, the yield of (2S,3R)-aminohydroxy ester was > 99.99%, the e.e. was > 99.99%, and the spatiotemporal yield was 1.55 g/(L·h·g DCW) after 12 h reaction. While the substrate concentration was increased to 200 g/L and the reaction lasted for 36 h, the yield of (2S,3R)-aminohydroxy ester was > 99.99%, the e.e. was > 99.99% and the spatiotemporal yield was 1.05 g/(L·h·g DCW). The substrate concentration and spatiotemporal yield were higher than ever reported.