Signalling cascades choreographing petal cell death: implications for postharvest quality.

Senescence is a multifaceted and dynamic developmental phase pivotal in the plant's lifecycle, exerting significant influence and involving intricate regulatory mechanisms marked by a variety of structural, biochemical and molecular alterations. Biochemical changes, including reactive oxygen species (ROS) generation, membrane deterioration, nucleic acid degradation and protein degradation, characterize flower senescence. The progression of senescence entails a meticulously orchestrated network of interconnected molecular mechanisms and signalling pathways, ensuring its synchronized and efficient execution. Within flowering plants, petal senescence emerges as a crucial aspect significantly impacting flower longevity and postharvest quality, emphasizing the pressing necessity of unravelling the underlying signalling cascades orchestrating this process. Understanding the complex signalling pathways regulating petal senescence holds paramount importance, not only shedding light on the broader phenomenon of plant senescence but also paving the way for the development of targeted strategies to enhance the postharvest longevity of cut flowers. Various signalling pathways participate in petal senescence, encompassing hormone signalling, calcium signalling, protein kinase signalling and ROS signalling. Among these, the ethylene signalling pathway is extensively studied, and the manipulation of genes associated with ethylene biosynthesis or signal transduction has demonstrated the potential to enhance flower longevity. A thorough understanding of these complex pathways is critical for effectively delaying flower senescence, thereby enhancing postharvest quality and ornamental value. Therefore, this review adopts a viewpoint that combines fundamental research into the molecular intricacies of senescence with a practical orientation towards developing strategies for improving the postharvest quality of cut flowers. The innovation of this review is to shed light on the pivotal signalling cascades underpinning flower senescence and offer insights into potential approaches for modulating these pathways to postpone petal senescence in ornamental plants.

Arabidopsis HECT and RING-type E3 Ligases Promote MAPKKK18 Degradation to Regulate Abscisic Acid Signaling.

Mitogen-activated protein kinase (MAPK) cascades are conserved signaling pathways that transduce extracellular signals into diverse cellular responses. Arabidopsis MAPKKK18 is a component of the MAPKKK17/18-MKK3-MPK1/2/7/14 cascades, which play critical roles in abscisic acid (ABA) signaling, drought tolerance and senescence. A very important aspect of MAP kinase signaling is both its activation and its termination, which must be tightly controlled to achieve appropriate biological responses. Recently, the ubiquitin-proteasome system (UPS) has received increasing attention as a key mechanism for maintaining the homeostasis of MAPK cascade components and other ABA signaling effectors. Previous studies have shown that the stability of MAPKKK18 is regulated by the UPS via the ABA core pathway. Here, using multiple proteomic approaches, we found that MAPKKK17/18 turnover is tightly controlled by three E3 ligases, UPL1, UPL4 and KEG. We also identified lysines 154 and 237 as critical for MAPKKK18 stability. Taken together, this study sheds new light on the mechanism that controls MAPKKK17/18 activity and function.

Combining Protein Phase Separation and Bio-orthogonal Linking to Coimmobilize Enzymes for Cascade Biocatalysis.

The designed and ordered co-immobilization of multiple enzymes for vectorial biocatalysis is challenging. Here, a combination of protein phase separation and bioorthogonal linking is used to generate a zeolitic imidazole framework (ZIF-8) containing co-immobilized enzymes. Zn2+ ions induce the clustering of minimal protein modules, such as 6-His tag, proline-rich motif (PRM) and SRC homology 3 (SH3) domains, and allow for phase separation of the coupled aldoketoreductase (AKR) and alcohol dehydrogenase (ADH) at low concentrations. This is achieved by fusing SpyCatcher and PRM-SH3-6His peptide fragments to the C and N termini of AKR, respectively, and the SpyTag to ADH. Addition of 2-methylimidazole results in droplet formation and enables in situ spatial embedding the recombinant AKR and ADH to generate the cascade biocalysis system encapsulated in ZIF-8 (AAE@ZIF). In synthesizing (S)-1-(2-chlorophenyl) ethanol, ater 6 cycles, the yield can still reach 91%, with 99.99% enantiomeric excess (ee) value for each cycle. However, the yield could only reach 72.9% when traditionally encapsulated AKR and ADH in ZIF-8 are used. Thus, this work demonstrates that a combination of protein phase separation and bio-orthogonal linking enables the in situ creation of a stable and spatially organized bi-enzyme system with enhanced channeling effects in ZIF-8.

Metabolomic profiling of ocular tissues in rabbit myopia: Uncovering differential metabolites and pathways.

To investigate the metabolic difference among tissue layers of the rabbits' eye during the development of myopia using metabolomic techniques and explore any metabolic links or cascades within the ocular wall. Ultra Performance Liquid Chromatography - Mass Spectrometry (UPLC-MS) was utilized for untargeted metabolite screening (UMS) to identify the significant differential metabolites produced between myopia (MY) and control (CT) (horizontal). Subsequently, we compared those key metabolites among tissues (Sclera, Choroid, Retina) of MY for distribution and variation (longitudinal). A total of 6285 metabolites were detected in the three tissues. The differential metabolites were screened and the metabolic pathways of these metabolites in each myopic tissue were labeled, including tryptophan and its metabolites, pyruvate, taurine, caffeine metabolites, as well as neurotransmitters like glutamate and dopamine. Our study suggests that multiple metabolic pathways or different metabolites under the same pathway, might act on different parts of the eyeball and contribute to the occurrence and development of myopia by affecting the energy supply to the ocular tissues, preventing antioxidant stress, affecting scleral collagen synthesis, and regulating various neurotransmitters mutually.

Discrete-state models identify pathway specific B cell states across diseases and infections at single-cell resolution.

Oxygen (O2) regulated pathways modulate B cell activation, migration and proliferation during infection, vaccination, and other diseases. Modeling these pathways in health and disease is critical to understand B cell states and ways to mediate them. To characterize B cells by their activation of O2 regulated pathways we develop pathway specific discrete state models using previously published single-cell RNA-sequencing (scRNA-seq) datasets from isolated B cells. Specifically, Single Cell Boolean Omics Network Invariant-Time Analysis (scBONITA) was used to infer logic gates for known pathway topologies. The simplest inferred set of logic gates that maximized the number of "OR" interactions between genes was used to simulate B cell networks involved in oxygen sensing until they reached steady network states (attractors). By focusing on the attractors that best represented sequenced cells, we identified genes critical in determining pathway specific cellular states that corresponded to diseased and healthy B cell phenotypes. Specifically, we investigate the transendothelial migration, regulation of actin cytoskeleton, HIF1A, and Citrate Cycle pathways. Our analysis revealed attractors that resembled the state of B cell exhaustion in HIV+ patients as well as attractors that promoted anerobic metabolism, angiogenesis, and tumorigenesis in breast cancer patients, which were eliminated after neoadjuvant chemotherapy (NACT). Finally, we investigated the attractors to which the Azimuth-annotated B cells mapped and found that attractors resembling B cells from HIV+ patients encompassed a significantly larger number of atypical memory B cells than HIV- attractors. Meanwhile, attractors resembling B cells from breast cancer patients post NACT encompassed a reduced number of atypical memory B cells compared to pre-NACT attractors.

Functional traits of predators and decomposer prey determine context dependency in trophic control over ecosystems.

Research Highlight: Piccoli, G. C. d. O., Antiqueira, P. A. P., Srivastava, D. S., & Romero, G. Q. (2024). Trophic cascades within and across ecosystems: The role of anti-predatory defences, predator type and detritus quality. Journal of Animal Ecology, 00, 1-14. https://doi.org/10.1111/1365-2656.14063. Ecosystem functioning is controlled by the interplay between bottom-up supply of limiting nutrients and top-down animal feedback effects. However, the degree of animal versus nutrient control is context-dependent. A key challenge lies in characterizing this context dependency which is hypothesized to depend on differences in animal functional traits. Reporting on an important experiment, Piccoli et al. (2014) evaluate how interactions among functionally different predators and decomposer prey create context dependency in top-down control of a model system-tropical bromeliad tank ecosystems. Bromeliad plants hold water in their tanks supporting microcosm ecosystems containing terrestrial and aquatic insect larvae and arachnids. The ecosystems are supported by nutrients in plant litter that rains down from forest canopies into the tanks. Nutrients are released after litter is decomposed by a functionally diverse community of larval insect decomposers that differ in feeding mode and antipredator defence strategy. This decomposer community is preyed upon by an exclusively narrowly ranging aquatic insect larval predator and widely ranging spider predator that crosses between the aquatic and surrounding terrestrial ecosystems. Experimental manipulation of the animal community to test for the degree of control by predators mediated by the functionally diverse prey community included four treatments: (i) a control with the detritivores composing different function groups but without predators, (ii) the cross-ecosystem spider predator added, (iii) the purely aquatic damselfly larvae predator added and (iv) both predator types added to capture their interacting effect on ecosystem function (decomposition, nutrient release, and plant growth). Notably, the study resolved the causal pathways and strengths of direct and indirect control using structural equation modelling. These findings reveal how context dependency arises due to different capacities of the predators alone and together to overcome prey defences and control their abundances, with attendant cascading effects that diminished as well as enhanced decomposition and nutrient release to support bromeliad plant production. The study reveals that predators have a decided, albeit qualitatively and quantitatively different, hand in shaping the degree of bottom-up control through feedback effect on the release of limiting nutrients. This ground-breaking study provides a way forward in understanding the mechanisms determining context dependency in the control over ecosystem functioning.

Insectivorous birds and bats outperform ants in the top-down regulation of arthropods across strata of a Japanese temperate forest.

Birds, bats and ants are recognised as significant arthropod predators. However, empirical studies reveal inconsistent trends in their relative roles in top-down control across strata. Here, we describe the differences between forest strata in the separate effects of birds, bats and ants on arthropod densities and their cascading effects on plant damage. We implemented a factorial design to exclude vertebrates and ants in both the canopy and understorey. Additionally, we separately excluded birds and bats from the understorey using diurnal and nocturnal exclosures. At the end of the experiments, we collected all arthropods and assessed herbivory damage. Arthropods responded similarly to predator exclusion across forest strata, with a density increase of 81% on trees without vertebrates and 53% without both vertebrates and ants. Additionally, bird exclusion alone led to an 89% increase in arthropod density, while bat exclusion resulted in a 63% increase. Herbivory increased by 42% when vertebrates were excluded and by 35% when both vertebrates and ants were excluded. Bird exclusion alone increased herbivory damage by 28%, while the exclusion of bats showed a detectable but non-significant increase (by 22%). In contrast, ant exclusion had no significant effect on arthropod density or herbivory damage across strata. Our results reveal that the effects of birds and bats on arthropod density and herbivory damage are similar between the forest canopy and understorey in this temperate forest. In addition, ants were not found to be significant predators in our system. Furthermore, birds, bats and ants appeared to exhibit antagonistic relationships in influencing arthropod density. These findings highlight, unprecedentedly, the equal importance of birds and bats in maintaining ecological balance across different strata of a temperate forest.

Stakeholder perceptions of and attitudes towards problematic polypharmacy and prescribing cascades: a qualitative study.

INTRODUCTION: Problematic polypharmacy is the prescribing of five or more medications potentially inappropriately. Unintentional prescribing cascades represent an under-researched aspect of problematic polypharmacy and occur when an adverse drug reaction (ADR) is misinterpreted as a new symptom resulting in the initiation of a new medication. The aim of this study was to elicit key stakeholders' perceptions of and attitudes towards problematic polypharmacy, with a focus on prescribing cascades. METHODS: qualitative one-to-one semi-structured interviews were conducted with predefined key stakeholder groups. Inductive thematic analysis was employed. RESULTS: Thirty-one stakeholders were interviewed: six patients, two carers, seven general practitioners, eight pharmacists, four hospital doctors, two professional organisation representatives and two policymakers. Three main themes were identified: (i) ADRs and prescribing cascades-a necessary evil. Healthcare professionals (HCPs) expressed concern that experiencing an ADR would negatively impact patients' confidence in their doctor. However, patients viewed ADRs pragmatically as an unpredictable risk. (ii) Balancing the risk/benefit tipping point. The complexity of prescribing decisions in the context of polypharmacy made balancing this tipping point challenging. Consequently, HCPs avoided medication changes. (iii) The minefield of medication reconciliation. Stakeholders, including patients and carers, viewed medication reconciliation as a perilous activity due to systemic communication deficits. CONCLUSION: Stakeholders believed that at a certain depth of polypharmacy, the risk that a new symptom is being caused by an existing medication becomes incalculable. Therefore, in the absence of harm, medication changes were avoided. However, medication reconciliation post hospital discharge compelled prescribing decisions and was seen as a high-risk activity by stakeholders.

Developmental cascades from early childhood attachment security to adolescent level of personality functioning among high-risk youth.

This study examines associations between early childhood attachment security and adolescent personality functioning in a high-risk sample within a developmental psychopathology framework. Data from 2,268 children (1165 male; 1103 female) and caregivers participating in Future of Families and Child Well-Being Study (FFCWS) were used to examine (1) effects of genetic polymorphisms of the serotonin transporter (5-HTTLPR) and dopamine D4 receptor (DRD4) genes and adverse childhood experiences (ACEs) on attachment security and emotional and behavioral dysregulation in early childhood and (2) longitudinal associations and transactional relationships among attachment security, dysregulation, negative parenting attitudes and behaviors, social competence, and adolescent personality functioning. Results revealed that ACEs predicted attachment security over and above sex or the genetic risk, and gene × environment interactions did not increment prediction. Results of cascade models showed that greater early childhood attachment security predicted higher adolescent level of personality functioning via pathways through intermediary variables. Limitations and future research directions are discussed.

Recurrence of macular edema in patients with branch retinal vein occlusion: a proteomic study.

BACKGROUND: Branch retinal vein occlusion (BRVO) is a common retinal vascular disease leading to severe vision loss and blindness. This study aimed to investigate and reveal the pathophysiological mechanisms underlying macular edema (ME) recurrence in patients with BRVO through a proteomic approach. METHODS: We detected proteins in the aqueous humor of 14 untreated, four refractory, and four post-operative patients with BRVO-ME and 12 age-matched cataract controls using four-dimensional label-free proteomic and bioinformatics analyses. RESULTS: In total, 84 proteins exhibited significant differential expression between the BRVO and control samples (fold change [FC] ≥ 1.2 and adjusted p-value < 0.05). Compared to the control group, 43 and 41 proteins were upregulated and downregulated, respectively, in the BRVO group. These proteins were involved in cell adhesion, visual perception, retina homeostasis, and platelet activation. Several significantly enriched signaling pathways included complement and coagulation cascades and platelet activation. In the protein-protein interaction networks generated using the search tool for retrieval of interacting genes (STRING), the fibrinogen alpha chain and fibrinogen beta chain constituted a tightly connected cluster. Many common protein expression trends, such as the fibrinogen alpha chain and fibrinogen beta chain, were observed in both the recurrent and refractory groups. Differentially expressed proteins in the two groups were involved in complement activation, acute-phase response, platelet activation, and platelet aggregation. Important signaling pathways include the complement and coagulation cascades, and platelet activation. Protein-protein interaction analysis suggested that the fibrinogen alpha chain and fibrinogen beta chain constituted a tightly connected cluster. The expression of some differentially expressed proteins shared by the BRVO and the recurrent and refractory groups was reversed in the post-operative group. CONCLUSIONS: Our study is the first to analyze the proteomics of recurrent, refractory, and post-operative groups treated for BRVO-ME, and may potentially provide novel therapeutic interventions for the recurrence of ME.

Positioning adolescence in the developmental timeline.

Adolescence, the second decade of life, bridges childhood and adulthood, but also represents a host of unique experiences that impact health and well-being. Lifespan theories often emphasize the continuity of individual characteristics and their contexts from childhood to adolescence, underscoring the distal influence of childhood experiences. Yet, adolescence is marked by transitions that may provoke discontinuities, particularly within individuals, their contexts, and their interactions within those contexts. These discontinuities occur at varied times, orders, and intensities for individual youth, suggesting that adolescence may be a developmental turning point where earlier life experiences may be mediated, reversed, or transformed by proximal events. This perspective piece emphasizes the importance of considering transitions, discontinuities, and developmental turning points in adolescence as well as their potential to explain heterogeneity in adolescent and adult outcomes. We explore one biological and one contextual transition in adolescence and highlight innovative theories and methods for investigating continuity and discontinuity dynamics across development, which could lead to new insights related to the adolescent period and its importance in shaping future life trajectories.

Family cumulative risk, life satisfaction, and anxiety and depression in adolescents: A developmental cascades model.

INTRODUCTION: Family cumulative risk (FCR) is predominantly regarded as an antecedent for adolescent mental health, as the prevailing perspective continues to emphasize the influential role of parents, despite recognizing the child's influence. To identify the interplay between family adversity (FCR, process-related FCR, and sociodemographic-related FCR), life satisfaction (LS), and anxiety and depression (AD), this study examined the cascade effects among these constructs. METHOD: Participants (N = 707; 52.9% male; grades 10 and 11) from four high schools in Wuhan, China, were recruited to participate, and they completed the measures in October 2018, April 2019, and November 2019. Family sociodemographic risk (e.g., single parenthood) and family process risk (e.g., low family cohesion) were simulated in the models for FCR, sociodemographic-related FCR, and process-related FCR. RESULTS: The random intercept cross-lagged panel models (RI-CLPMs) revealed a lagged effect from LS to FCR; lagged effects from LS and AD to process-related FCR at the within-person level; and significant associations between LS, AD, and family adversity at the between-person level. CONCLUSIONS: The lagged effects provide evidence for the influential child perspective and suggest that FCR and family process risk are sensitive to adolescent well-being and psychopathological symptoms. School mental health prevention and intervention programs that take a complete mental health approach to enhance children's well-being and alleviate symptoms would help prevent increases in family risk.

An Escherichia coli-Based Phosphorylation System for Efficient Screening of Kinase Substrates.

Posttranslational modifications (PTMs), particularly phosphorylation, play a pivotal role in expanding the complexity of the proteome and regulating diverse cellular processes. In this study, we present an efficient Escherichia coli phosphorylation system designed to streamline the evaluation of potential substrates for Arabidopsis thaliana plant kinases, although the technology is amenable to any. The methodology involves the use of IPTG-inducible vectors for co-expressing kinases and substrates, eliminating the need for radioactive isotopes and prior protein purification. We validated the system's efficacy by assessing the phosphorylation of well-established substrates of the plant kinase SnRK1, including the rat ACETYL-COA CARBOXYLASE 1 (ACC1) and FYVE1/FREE1 proteins. The results demonstrated the specificity and reliability of the system in studying kinase-substrate interactions. Furthermore, we applied the system to investigate the phosphorylation cascade involving the A. thaliana MKK3-MPK2 kinase module. The activation of MPK2 by MKK3 was demonstrated to phosphorylate the Myelin Basic Protein (MBP), confirming the system's ability to unravel sequential enzymatic steps in phosphorylation cascades. Overall, this E. coli phosphorylation system offers a rapid, cost-effective, and reliable approach for screening potential kinase substrates, presenting a valuable tool to complement the current portfolio of molecular techniques for advancing our understanding of kinase functions and their roles in cellular signaling pathways.

OsWRKY78 regulates panicle exsertion via gibberellin signaling pathway in rice.

Panicle exsertion is one of the crucial agronomic traits in rice (Oryza sativa). Shortening of panicle exsertion often leads to panicle enclosure and severely reduces seed production. Gibberellin (GA) plays important roles in regulating panicle exsertion. However, the underlying mechanism and the relative regulatory network remain elusive. Here, we characterized the oswrky78 mutant showing severe panicle enclosure, and found that the defect of oswrky78 is caused by decreased bioactive GA contents. Biochemical analysis demonstrates that OsWRKY78 can directly activate GA biosynthesis and indirectly suppress GA metabolism. Moreover, we found OsWRKY78 can interact with and be phosphorylated by mitogen-activated protein kinase (MAPK) kinase OsMAPK6, and this phosphorylation can enhance OsWRKY78 stability and is necessary for its biological function. Taken together, these results not only reveal the critical function of OsWRKY78, but also reveal its mechanism via mediating crosstalk between MAPK and the GA signaling pathway in regulating panicle exsertion.

Longitudinal Associations Between Reactive and Proactive Aggression and Depression in Early Adolescence: Between- and Within-Person Effects.

Depressive symptoms and aggression often co-occur, and previous studies have found different bidirectional links between depressive symptoms and aggression, suggesting inconsistent developmental cascades. Moreover, it is unclear whether different functions of aggression are differentially associated with depressive symptoms over time. The present study examined the longitudinal associations of reactive and proactive aggression with depressive symptoms in early adolescence. Adolescents (n = 942, 50.7% girls; mean age = 12.54 years, SD = 0.42) were surveyed annually over three years (2019-2021). Random-intercept cross-lagged panel models were used to disentangle between- and within-person effects. The results showed moderate between-person associations of depressive symptoms with the two aggressive functions. And depressive symptoms were more highly associated with reactive aggression than with proactive aggression. However, the state-level bidirectional cross-lagged associations between reactive and proactive aggression and depressive symptoms were not significant. This study highlights the stable trait-like association between depressive symptoms and reactive aggression, and the absence of state-level bidirectional cross-lagged associations challenges previous developmental cascades in adolescents.

Refractory IgA Nephropathy: A Challenge for Future Nephrologists.

IgA nephropathy (IgAN) represents the most prevalent form of primary glomerulonephritis, and, on a global scale, it ranks among the leading culprits behind end-stage kidney disease (ESKD). Presently, the primary strategy for managing IgAN revolves around optimizing blood pressure and mitigating proteinuria. This is achieved through the utilization of renin-angiotensin system (RAS) inhibitors, namely, angiotensin-converting enzyme inhibitors (ACEi) and angiotensin receptor blockers (ARBs). As outlined by the KDIGO guidelines, individuals who continue to show a persistent high risk of progressive ESKD, even with comprehensive supportive care, are candidates for glucocorticoid therapy. Despite these therapies, some patients have a disease refractory to treatment, defined as individuals that present a 24 h urinary protein persistently >1 g after at least two rounds of regular steroids (methylprednisolone or prednisone) and/or immunosuppressant therapy (e.g., mycophenolate mofetil), or who do not tolerate regular steroids and/or immunosuppressant therapy. The aim of this Systematic Review is to revise the current literature, using the biomedical database PubMed, to investigate possible therapeutic strategies, including SGLT2 inhibitors, endothelin receptor blockers, targeted-release budesonide, B cell proliferation and differentiation inhibitors, fecal microbiota transplantation, as well as blockade of complement components.

Hand preference trajectories as predictors of language outcomes above and beyond SES: Infant patterns explain more variance than toddler patterns at 5 years of age.

Prior studies found hand preference trajectories predict preschool language outcomes. However, this approach has been limited to examining bimanual manipulation in toddlers. It is not known whether hand preference during infancy for acquiring objects (i.e., reach-to-grasp) similarly predicts childhood language ability. The current study explored this motor-language developmental cascade in 90 children. Hand preference for acquiring objects was assessed monthly from 6 to 14 months and language skill was assessed at 5 years. Latent class growth analysis identified three infant hand preference classes: left, early right, and late right. Infant hand preference classes predicted 5-year language skills. Children in the left and early right classes, who were categorized as having a consistent hand preference, had higher expressive and receptive language scores relative to children in the inconsistent late right class. Consistent classes did not differ from each other on language outcomes. Infant hand preference patterns explained more variance for expressive and receptive language relative to previously reported toddler hand preference patterns, above and beyond socioeconomic status (SES). Results suggest that hand preference, measured at different time points across development using a trajectory approach, is reliably linked to later language.

Peptide and Enzyme Catalysts Work in Concert in Stereoselective Cascade Reactions-Oxidation followed by Conjugate Addition.

Enzymes and peptide catalysts consist of the same building blocks but require vastly different environments to operate best. Herein, we show that an enzyme and a peptide catalyst can work together in a single reaction vessel to catalyze a two-step cascade reaction with high chemo- and stereoselectivity. Abundant linear alcohols, nitroolefins, an alcohol oxidase, and a tripeptide catalyst provided chiral γ-nitroaldehydes in aqueous buffer. High yields (up to 92 %) and stereoselectivities (up to 98 % ee) were achieved for the cascade through the rational design of the peptide catalyst and the identification of common reaction conditions.

Precision Engineering of the Co-immobilization of Enzymes for Cascade Biocatalysis.

The design and orderly layered co-immobilization of multiple enzymes on resin particles remain challenging. In this study, the SpyTag/SpyCatcher binding pair was fused to the N-terminus of an alcohol dehydrogenase (ADH) and an aldo-keto reductase (AKR), respectively. A non-canonical amino acid (ncAA), p-azido-L-phenylalanine (p-AzF), as the anchor for covalent bonding enzymes, was genetically inserted into preselected sites in the AKR and ADH. Employing the two bioorthogonal counterparts of SpyTag/SpyCatcher and azide-alkyne cycloaddition for the immobilization of AKR and ADH enabled sequential dual-enzyme coating on porous microspheres. The ordered dual-enzyme reactor was subsequently used to synthesize (S)-1-(2-chlorophenyl)ethanol asymmetrically from the corresponding prochiral ketone, enabling the in situ regeneration of NADPH. The reactor exhibited a high catalytic conversion of 74 % and good reproducibility, retaining 80 % of its initial activity after six cycles. The product had 99.9 % ee, which that was maintained in each cycle. Additionally, the double-layer immobilization method significantly increased the enzyme loading capacity, which was approximately 1.7 times greater than that of traditional single-layer immobilization. More importantly, it simultaneously enabled both the purification and immobilization of multiple enzymes on carriers, thus providing a convenient approach to facilitate cascade biocatalysis.

Modularization of Immobilized Multienzyme Cascades for Continuous-Flow Enantioselective C-H Amination.

Multienzyme cascades (MECs) have gained much attention in synthetic chemistry but remain far from being a reliable synthetic tool. Here we report a four-enzyme cascade comprising a cofactor-independent and a cofactor self-sustaining bienzymatic modules for the enantioselective benzylic C-H amination of arylalkanes, a challenging transformation from bulk chemicals to high value-added chiral amines. The two modules were subsequently optimized by enzyme co-immobilization with microenvironmental tuning, and finally integrated in a gas-liquid segmented flow system, resulting in simultaneous improvements in enzyme performance, mass transfer, system compatibility, and productivity. The flow system enabled continuous C-H amination of arylalkanes (up to 100 mM) utilizing the sole cofactor NADH (0.5 mM) in >90 % conversion, achieving a high space-time yield (STY) of 3.6 g ⋅ L-1 ⋅ h-1, which is a 90-fold increase over the highest value previously reported.