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As most teleosts are unable to synthesize vitamin C, supplemental diets containing vitamin C diets play a crucial role in fish health. The aim of this study was to investigate the effect of dietary vitamin C on the intestinal enzyme activity and intestinal microbiota of silver pomfre (Pampus argenteus). Four experimental diets were supplemented with basic diets containing 300 mg of vitamin C/kg (group tjl3), 600 mg of vitamin C/kg (group tjl6), and 1200 mg of vitamin C/kg (group tjl12), as well as vitamin C-free supplemental basic diet (group tjl0), respectively. The four diets were fed to juvenile P. argenteus (average initial weight: 4.68 ± 0.93 g) for 6 weeks. The results showed that the activity of SOD (superoxide dismutase) and CAT (catalase) increased significantly while that of MDA (malondialdehyde) decreased significantly in group tjl3 compared to vitamin group tjl0. At the genus level, groups tjl0, tjl6, and tjl12 contained the same dominant microbial community, Stenotrophomonas, Photobacterium, and Vibrio, whereas group tjl3 was dominated by Stenotrophomonas, Delftia, and Bacteroides. Among the fish fed with a basic diet containing 300 mg of vitamin C/kg, the intestines exhibited a notable abundance of probiotic bacteria, including lactic acid bacteria (Lactobacillus) and Bacillus. The abundance of Aeromonas in groups tjl3 and tjl6 was lower than that of the vitamin C-free supplemental basic diet group, whereas Aeromonas was not detected in group tjl12. In addition, a causative agent of the disease outbreak in cultured P. argenteus, Photobacterium damselae subsp. Damselae (PDD) was the dominant microbiota community in groups tjl0, tjl6 and tjl12, whereas the abundance of PDD in group tjl3 was the lowest among the diets. Taken together, the diets supplied with vitamin C could influence the composition microbial community of P. argenteus. The low level of vitamin C (300 mg of vitamin C/kg per basic diet) supplementation could not only improve the antioxidant capacity but also resist the invasion of pathogenic bacteria.
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Antioxidantes , Ácido Ascórbico , Suplementos Nutricionais , Microbioma Gastrointestinal , Animais , Ácido Ascórbico/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Perciformes/microbiologia , Ração Animal/análise , Superóxido Dismutase/metabolismo , Bactérias/efeitos dos fármacos , Bactérias/isolamento & purificação , Dieta/veterinária , Catalase/metabolismoRESUMO
Chiral alcohols are essential building blocks of numerous pharmaceuticals and fine chemicals. Aldo-keto reductases (AKRs) constitute a superfamily of oxidoreductases that catalyze the reduction of aldehydes and ketones to their corresponding alcohols using NAD(P)H as a coenzyme. Knowledge about the crucial roles of AKRs immobilization in the biocatalytic synthesis of chiral alcohols is expanding. Herein, we reviewed the characteristics of various AKRs immobilization approaches, the applications of different immobilization materials, and the prospects of continuous flow bioreactor construction by employing these immobilized biocatalysts for synthesizing chiral alcohols. Finally, the opportunities and ongoing challenges for AKR immobilization are discussed and the outlook for this emerging area is analyzed.
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Álcoois , Aldo-Ceto Redutases , Biocatálise , Enzimas Imobilizadas , Aldo-Ceto Redutases/metabolismo , Aldo-Ceto Redutases/genética , Aldo-Ceto Redutases/química , Álcoois/química , Álcoois/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Estereoisomerismo , Reatores BiológicosRESUMO
Energy metabolism is highly interdependent with adaptive cell migration in vivo. Mechanical confinement is a critical physical cue that induces switchable migration modes of the mesenchymal-to-amoeboid transition (MAT). However, the energy states in distinct migration modes, especially amoeboid-like stable bleb (A2) movement, remain unclear. In this report, we developed multivalent DNA framework-based nanomachines to explore strategical mitochondrial trafficking and differential ATP levels during cell migration in mechanically heterogeneous microenvironments. Through single-particle tracking and metabolomic analysis, we revealed that fast A2-moving cells driven by biomimetic confinement recruited back-end positioning of mitochondria for powering highly polarized cytoskeletal networks, preferentially adopting an energy-saving mode compared with a mesenchymal mode of cell migration. We present a versatile DNA nanotool for cellular energy exploration and highlight that adaptive energy strategies coordinately support switchable migration modes for facilitating efficient metastatic escape, offering a unique perspective for therapeutic interventions in cancer metastasis.
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Amoeba , Linhagem Celular Tumoral , Movimento Celular , Fenômenos FísicosRESUMO
Maintaining genomic stability is a prerequisite for proliferating NPCs to ensure genetic fidelity. Though histone arginine methylation has been shown to play important roles in safeguarding genomic stability, the underlying mechanism during brain development is not fully understood. Protein arginine N-methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase that plays a role in transcriptional regulation. Here, we identify PRMT5 as a key regulator of DNA repair in response to double-strand breaks (DSBs) during NPC proliferation. Prmt5F/F; Emx1-Cre (cKO-Emx1) mice show a distinctive microcephaly phenotype, with partial loss of the dorsal medial cerebral cortex and complete loss of the corpus callosum and hippocampus. This phenotype is resulted from DSBs accumulation in the medial dorsal cortex followed by cell apoptosis. Both RNA sequencing and in vitro DNA repair analyses reveal that PRMT5 is required for DNA homologous recombination (HR) repair. PRMT5 specifically catalyzes H3R2me2s in proliferating NPCs in the developing mouse brain to enhance HR-related gene expression during DNA repair. Finally, overexpression of BRCA1 significantly rescues DSBs accumulation and cell apoptosis in PRMT5-deficient NSCs. Taken together, our results show that PRMT5 maintains genomic stability by regulating histone arginine methylation in proliferating NPCs.
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Células-Tronco Neurais , Reparo de DNA por Recombinação , Animais , Camundongos , Arginina/metabolismo , Reparo do DNA , Instabilidade Genômica , Genômica , Histonas/genética , Histonas/metabolismo , Células-Tronco Neurais/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
The aldo-keto reductase (AKR) KdAKR from Kluyvermyces dobzhanskii can reduce t-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate ((5S)-CHOH) to t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate ((3R,5S)-CDHH), which is the key chiral intermediate of rosuvastatin. Herein, a computer-aided design that combined the use of PROSS platform and consensus design was employed to improve the stability of a previously constructed mutant KdAKRM6 . Experimental verification revealed that S196C, T232A, V264I and V45L produced improved thermostability and activity. The "best" mutant KdAKRM10 (KdAKRM6 -S196C/T232A/V264I/V45L) was constructed by combining the four beneficial mutations, which displayed enhanced thermostability. Its T50 15 and Tm values were increased by 10.2 and 10.0°C, respectively, and half-life (t1/2 ) at 40°C was increased by 17.6 h. Additionally, KdAKRM10 demonstrated improved resistance to organic solvents compared to that of KdAKRM6 . Structural analysis revealed that the increased number of hydrogen bonds and stabilized hydrophobic core contributed to the rigidity of KdAKRM10 , thus improving its stability. The results validated the feasibility of the computer-aided design strategy in improving the stability of AKRs.
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Aldeído Redutase , Caproatos , Aldo-Ceto Redutases/química , Aldo-Ceto Redutases/genética , Caproatos/químicaRESUMO
Background: Initial choices of antimicrobial therapy for most cases of community-acquired pneumonia (CAP) in children under 5 years of age are typically based on local epidemiology, risk factors assessment, and subsequent clinical parameters and positive cultures, which can lead to the underdiagnosis and underestimation of lung infections caused by uncommon pathogens. Contezolid, an orally administered oxazolidinone antibiotic, gained approval from the National Medical Products Administration (NMPA) of China in June 2021 for managing complicated skin and soft tissue infections (cSSTI) caused by staphylococcus aureus (SA), streptococcus pyogenes, or streptococcus agalactis. Owing to its enhanced safety profile and ongoing clinical progress, the scope of contezolid's clinical application continues to expand, benefiting a growing number of patients with Gram-positive bacterial infections. Case summary: In this report, we present the first use of contezolid in a toddler with severe CAP caused by SA, aiming to avoid potential adverse drug reactions (ADRs) associated with vancomycin and linezolid. Conclusion: Although contezolid has not been officially indicated for CAP, it has been shown to be effective and safe in the management of SA-induced severe CAP in this toddler, suggesting its potential as an alternative option in the dilemma, especially for patients who are susceptible or intolerant to ADRs associated with first-line anti-methicillin-resistant staphylococcus aureus (MRSA) antimicrobial agents.
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Carbonyl reductases are useful for producing optically active alcohols from their corresponding prochiral ketones. Herein, we applied a computer-assisted strategy to increase the thermostability of a previously constructed carbonyl reductase, LsCRM4 (N101D/A117G/F147L/E145A), which showed an outstanding activity in the synthesis of the ticagrelor precursor (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol. The stability changes introduced by mutations at the flexible sites were predicted using the computational tools FoldX, I-Mutant 3.0, and DeepDDG, which demonstrated that 12 virtually screened mutants could be thermally stable; 11 of these mutants exhibited increased thermostability. Then a superior mutant LsCRM4-V99L/D150F was screened out from the library that was constructed by iteratively combining the beneficial sites, which showed a 78% increase in activity and a 17.4°C increase in melting temperature compared to LsCRM4. Our computer-assisted design and combinatorial strategy dramatically increased the efficiency of thermostable enzyme production.
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Oxirredutases do Álcool , Etanol , Ticagrelor , Estabilidade Enzimática , Oxirredutases do Álcool/genética , Temperatura , ComputadoresRESUMO
Nocardia seriolae is the primary aetiological agent of nocardiosis in fish, which causes mass mortality in freshwater and marine fish. ß-ketoacyl-ACP synthase (KAS) is one of the essential enzymes in the synthesis of mycolic acids (MASs) in Mycobacterium spp. and has been chosen as the target for therapeutic intervention in mycobacterial diseases. In the present study, a kasB homologue gene (kasB) was identified in the genome of N. seriolae, and the gene-deficient mutant (ΔkasB) was generated based on a clinical isolate, XSYC-Ns. Compared to the wild-type (WT) strain, the ΔkasB showed a measurably growth defect in vitro but retained the acid-fastness in acid-fast staining. Observation of the cell ultrastructure showed some alterations in the cell wall of the ΔkasB strain. Compared to its original strain, the cell wall lipid layer seemed sparser, and a wider electron-transparent zone was observed in the cell wall of ΔkasB strain. Moreover, the ΔkasB strain showed impaired ability of cell invasion as well as intracellular survival in the cell line originating from the head-kidney of the large yellow croaker (LYC-hK), compared to its original strain. In addition, the deficiency of ΔkasB significantly attenuated the virulence of N. seriolae in largemouth bass. The present study suggested that the ΔkasB gene might be involved in the synthesis of extracellular cell-wall lipids in N. seriolae and play a crucial role in its pathogenicity.
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Bass , Doenças dos Peixes , Nocardiose , Nocardia , Animais , Virulência/genética , Doenças dos Peixes/microbiologia , Nocardia/genética , Nocardiose/veterinária , Nocardiose/microbiologiaRESUMO
Atorvastatin, a lipid-lowering drug that is widely used in the treatment of cardiovascular diseases, has significant clinical significance. This article focuses on the synthetic procedures of atorvastatin, including Paal-Knorr synthesis and several new synthetic strategies. It also outlines chemical and chemo-enzymatic methods for synthesizing optically active side chain of atorvastatin. In addition, a comprehensive overview of the analytical monitoring techniques for atorvastatin and its metabolites and impurities is reported, alongside a discussion of their strengths and limitations.
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This study presents a novel method for producing acicular aragonite using argon oxygen decarburization (AOD) slag while controlling the reaction temperature, reaction time, stirring speed, and the magnesium-tocalcium stoichiometric ratio. This approach provides steel plants with an opportunity to decrease their CO2 emissions and promote efficient resource utilization and CO2 storage through the production of high-quality value-added products. The experimental results showed that reaction temperature was the most significant factor affecting the carbonation efficiency of AOD slag, followed by reaction time, stirring speed, CO2 partial pressure, and the liquid-to-solid ratio (L/S). The study also found that elevated temperature and prolonged reaction duration favored the preferential precipitation of aragonite. Additionally, raising the temperature and the magnesium-tocalcium stoichiometric ratio was shown to enhance the formation of aragonite, affecting its crystal growth orientation and dimensions. The optimal combination of reaction parameters for the preparation of acicular aragonite was found to be the reaction time of 8 h, the magnesium-tocalcium stoichiometric ratio of 0.8, the reaction temperature of 120 °C, and the stirring speed of 200 r·min-1. Under these conditions, the resulting acicular aragonite exhibited excellent overall uniformity, a large aspect ratio, and a smooth crystal surface, with a content of 91.49 %, a single crystal length ranging from 9.86 to 32.6 µm, and a diameter ranging from 0.63 to 2.15 µm. This study provides valuable insights into the efficient production of acicular aragonite from steel slag while reducing CO2 emissions and promoting the sustainable use of resources.
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Aldo-keto reductases (AKRs) are important biocatalysts that can be used to synthesize chiral pharmaceutical alcohols. In this study, the catalytic activity and stereoselectivity of a NADPH-dependent AKR from Kluyveromyces dobzhanskii (KdAKR) toward t-butyl 6-chloro (5S)-hydroxy-3-oxohexanoate ((5S)-CHOH) were improved by mutating its residues in the loop regions around the substrate-binding pocket. And the thermostability of KdAKR was improved by a consensus sequence method targeted on the flexible regions. The best mutant M6 (Y28A/L58I/I63L/G223P/Y296W/W297H) exhibited a 67-fold higher catalytic efficiency compared to the wild-type (WT) KdAKR, and improved R-selectivity toward (5S)-CHOH (dep value from 47.6% to >99.5%). Moreover, M6 exhibited a 6.3-fold increase in half-life (t1/2 ) at 40°C compared to WT. Under the optimal conditions, M6 completely converted 200 g/L (5S)-CHOH to diastereomeric pure t-butyl 6-chloro-(3R, 5S)-dihydroxyhexanoate ((3R, 5S)-CDHH) within 8.0 h, with a space-time yield of 300.7 g/L/day. Our results deepen the understandings of the structure-function relationship of AKRs, providing a certain guidance for the modification of other AKRs.
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Caproatos , Kluyveromyces , Aldo-Ceto Redutases/genética , Aldo-Ceto Redutases/química , Catálise , Aldeído Redutase/genéticaRESUMO
Structural information can help engineer enzymes. Usually, specific amino acids in particular regions are targeted for functional reconstruction to enhance the catalytic performance, including activity, stereoselectivity, and thermostability. Appropriate selection of target sites is the key to structure-based design, which requires elucidation of the structure-function relationships. Here, we summarize the mutations of residues in different specific regions, including active center, access tunnels, and flexible loops, on fine-tuning the catalytic performance of enzymes, and discuss the effects of altering the local structural environment on the functions. In addition, we keep up with the recent progress of structure-based approaches for enzyme engineering, aiming to provide some guidance on how to take advantage of the structural information.
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Aminoácidos , Engenharia de Proteínas , Biocatálise , Catálise , Estabilidade EnzimáticaRESUMO
The emerging outbreak of monkeypox is closely associated with the viral infection and spreading, threatening global public health. Virus-induced cell migration facilitates viral transmission. However, the mechanism underlying this type of cell migration remains unclear. Here we investigate the motility of cells infected by vaccinia virus (VACV), a close relative of monkeypox, through combining multi-omics analyses and high-resolution live-cell imaging. We find that, upon VACV infection, the epithelial cells undergo epithelial-mesenchymal transition-like transformation, during which they lose intercellular junctions and acquire the migratory capacity to promote viral spreading. After transformation, VACV-hijacked RhoA signaling significantly alters cellular morphology and rearranges the actin cytoskeleton involving the depolymerization of robust actin stress fibers, leading-edge protrusion formation, and the rear-edge recontraction, which coordinates VACV-induced cell migration. Our study reveals how poxviruses alter the epithelial phenotype and regulate RhoA signaling to induce fast migration, providing a unique perspective to understand the pathogenesis of poxviruses.
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Mpox , Vaccinia virus , Humanos , Movimento Celular , Surtos de Doenças , Células EpiteliaisRESUMO
BACKGROUND: A positive resection margin is a major risk factor for local breast cancer recurrence after breast-conserving surgery (BCS). Preoperative imaging examinations are frequently employed to assess the surgical margin. AIM: To investigate the role and value of preoperative imaging examinations [magnetic resonance imaging (MRI), molybdenum target, and ultrasound] in evaluating margins for BCS. METHODS: A retrospective study was conducted on 323 breast cancer patients who met the criteria for BCS and consented to the procedure from January 2014 to July 2021. The study gathered preoperative imaging data (MRI, ultrasound, and molybdenum target examination) and intraoperative and postoperative pathological information. Based on their BCS outcomes, patients were categorized into positive and negative margin groups. Subsequently, the patients were randomly split into a training set (226 patients, approximately 70%) and a validation set (97 patients, approximately 30%). The imaging and pathological information was analyzed and summarized using R software. Non-conditional logistic regression and LASSO regression were conducted in the validation set to identify factors that might influence the failure of BCS. A column chart was generated and applied to the validation set to examine the relationship between pathological margin range and prognosis. This study aims to identify the risk factors associated with failure in BCS. RESULTS: The multivariate non-conditional logistic regression analysis demonstrated that various factors raise the risk of positive margins following BCS. These factors comprise non-mass enhancement (NME) on dynamic contrast-enhanced MRI, multiple focal vascular signs around the lesion on MRI, tumor size exceeding 2 cm, type III time-signal intensity curve, indistinct margins on molybdenum target examination, unclear margins on ultrasound examination, and estrogen receptor (ER) positivity in immunohistochemistry. LASSO regression was additionally employed in this study to identify four predictive factors for the model: ER, molybdenum target tumor type (MT Xmd Shape), maximum intensity projection imaging feature, and lesion type on MRI. The model constructed with these predictive factors exhibited strong consistency with the real-world scenario in both the training set and validation set. Particularly, the outcomes of the column chart model accurately predicted the likelihood of positive margins in BCS. CONCLUSION: The proposed column chart model effectively predicts the success of BCS for breast cancer. The model utilizes preoperative ultrasound, molybdenum target, MRI, and core needle biopsy pathology evaluation results, all of which align with the real-world scenario. Hence, our model can offer dependable guidance for clinical decision-making concerning BCS.
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The design and development of high-performance, low-cost catalysts with long-term durability are crucial for hydrogen generation from water electrolysis. Interfacial engineering is an appealing strategy to boost the catalytic performance of electrode materials toward hydrogen evolution reaction (HER). Herein, we report a simple phosphidation followed by sulfidation treatment to construct heterogeneous cobalt phosphide-cobalt sulfide nanowire arrays on carbon cloth (CoP/CoS2/CC). When evaluated as catalysts toward the HER, the resultant CoP/CoS2/CC exhibits efficient pH-universal hydrogen production due to the heterostructure, synergistic contribution of CoP and CoS2, and conductive substrate. To attain a current density of 10 mA cm-2, overpotentials of only 111.2, 58.1, and 182.9 mV for CoP/CoS2/CC are required under alkaline, acidic, and neutral conditions, respectively. In particular, the as-prepared CoP/CoS2/CC shows markedly improved HER electroactivity in 1.0 M KOH, even outperforming commercial Pt-C/CC at a current density of >50 mA cm-2. In addition, the self-assembled CoP/CoS2||NiFe layered double hydroxide electrolyzer demonstrates efficient catalytic performance and long-time stability, excelling the benchmark Pt-C||IrO2. These findings indicate an effective pathway for the fabrication of high-performance heterogeneous electrocatalysts for hydrogen production in the future.
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Cell migration occurs in confined microenvironments, which plays a vital role in the process of tumor metastasis. However, it is challenging to study their behaviors in vivo. Here we developed a cell squeeze system that can be scaled down to micrometers to mimic native physical confined microenvironments, wherein degrees of surface adhesion and mechanical constraints could be manipulated in order to investigate cell-migrating behaviors. Based on the microscale cell squeeze system, we found the synergistic role of lamin A/C and vimentin in cell transition and migration under strong confinement. The dynamic variations in lamin A/C and vimentin expression establish a positive feedback loop in response to confinement, effectively promoting amoeboid migration by modulating nuclear deformability while ensuring cell viability. This work shed light on modulating cell response to microenvironments by altering the expression of lamin A/C and/or vimentin, which may be a more efficient way of inhibiting cancer metastasis.
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Movimento Celular , Lamina Tipo A , Núcleo Celular/metabolismo , Filamentos Intermediários , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Vimentina/metabolismo , Humanos , Células HeLaAssuntos
Carcinoma Papilar , Neoplasias da Glândula Tireoide , Humanos , Metástase Linfática/patologia , Neoplasias da Glândula Tireoide/cirurgia , Neoplasias da Glândula Tireoide/patologia , Carcinoma Papilar/cirurgia , Carcinoma Papilar/patologia , Linfonodos/patologia , Estudos Retrospectivos , Fatores de Risco , TireoidectomiaRESUMO
Introduction: The Camellia oleifera (C. oleifera) cultivars 'Huashuo' (HS) and 'Huaxin' (HX) are new high-yielding and economically valuable cultivars that frequently encounter prolonged cold weather during the flowering period, resulting in decreased yields and quality. The flower buds of HS sometimes fail to open or open incompletely under cold stress, whereas the flower buds of HX exhibit delayed opening but the flowers and fruits rarely drop. Methods: In this study, flower buds at the same development stage of two C. oleifera cultivars were used as test materials for a combination of physiological, transcriptomic and metabolomic analyses, to unravel the different cold regulatory mechanisms between two cultivars of C. oleifera. Results and discussion: Key differentially expressed genes (DEGs) and differentially expressed metabolites (DEMs) involved in sugar metabolism, phenylpropanoid biosynthesis, and hormone signal transduction were significantly higher in HX than in HS, which is consistent with phenotypic observations from a previous study. The results indicate that the flower buds of HX are less affected by long-term cold stress than those of HS, and that cold resistance in C. oleifera cultivars varies among tissues or organs.This study will provide a basis for molecular markers and molecular breeding of C. oleifera.
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The current study was performed to examine the acute toxicity of mercuric chloride (HgCl2) on the silver carp (Hypophthalmichthys molitrix) larvae. Probit analysis was used to determine the median lethal concentration (LC50). The LC50 values of Hg2+ for the fish larvae at 24, 48, 72, and 96 h were 267.72, 252.97, 225.57, and 97.80 µg/L, respectively. The safe concentration of Hg was 9.78 µg/L for fish larvae. Based on the 96 h LC50, fish were exposed to four different groups including 0, 6.11, 12.23, and 24.45 µg/L for 96 h to assess the effects of different concentrations of Hg2+ on antioxidant capacity, energy metabolism parameters, and related gene expression. The findings revealed that there were no significant differences in the activities of superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) in fish larvae among all the groups (P < 0.05). In the 12.23 µg/L group, fish larvae had a maximum in catalase (CAT) activity. The creatine kinase (CK) activities of fish larvae in control and 6.11 µg/L groups were significantly lower than those groups (P < 0.05). A high concentration of Hg2+ significantly upregulated the mRNA levels of heat shock protein 70 (HSP70) and metallothionein (MT) genes in fish larvae. Furthermore, the IBR index value showed the highest value in the 24.45 µg/L group. Overall, this study provides an increased understanding of the effects of Hg-acute toxicity on silver carp larvae.
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Carpas , Mercúrio , Animais , Antioxidantes/metabolismo , Carpas/metabolismo , Larva , Superóxido Dismutase/metabolismo , Mercúrio/metabolismoRESUMO
Carbonyl reductase (CR)-catalyzed bioreduction in the organic phase and the neat substrate reaction system is a lasting challenge, placing higher requirements on the performance of enzymes. Protein engineering is an effective method to enhance the properties of enzymes for industrial applications. In the present work, a single point mutation E145A on our previously constructed CR mutant LsCRM3 , coevolved thermostability, and activity. Compared with LsCRM3 , the catalytic efficiency kcat /KM of LsCRM3 -E145A (LsCRM4 ) was increased from 6.6 to 21.9 s-1 mM-1 . Moreover, E145A prolonged the half-life t1/2 at 40°C from 4.1 to 117 h, T m ${T}_{m}$ was increased by 5°C, T 50 30 ${T}_{50}^{30}$ was increased by 14.6°C, and Topt was increased by 15°C. Only 1 g/L of lyophilized Escherichia coli cells expressing LsCRM4 completely reduced up to 600 g/L 2-chloro-1-(3,4-difluorophenyl)ethanone (CFPO) within 13 h at 45°C, yielding the corresponding (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol ((S)-CFPL) in 99.5% eeP , with a space-time yield of 1.0 kg/L d, the substrate to catalyst ratios (S/C) of 600 g/g. Compared with LsCRM3 , the substrate loading was increased by 50%, with the S/C increased by 14 times. Compared with LsCRWT , the substrate loading was increased by 6.5 times. In contrast, LsCRM4 completely converted 600 g/L CFPO within 12 h in the neat substrate bioreaction system.