ACS Sustainable Chemistry & Engineering
·
2025
·
IF 7.3
Chaoqun Huang,Xuerui Jin,Yeyi Kan,Can Cui,Longchen Wei,Huayi Liu,Dawen Niu,Yunzi Luo
Nonnatural amino acids hold the promise of enhancing enzyme activity and bolstering the efficacy of drugs. Enzymatic synthesis emerges as a valuable adjunct to traditional synthetic methodologies, providing an alternative pathway for the generation of nonnatural amino acids. This study introduces a one-pot enzymatic strategy for the synthesis of aryl and heteroaryl γ-hydroxy-α-amino acids. The process involved the utilization of a newly identified aldolase from Streptomyces to catalyze the formation of C–C bond from the cost-effective fluorinated pyridine aldehyde substrate, and an α-transaminase sourced from Saccharomyces cerevisiae to transfer an amino group. After optimization, a 94% conversion efficiency was achieved, along with a product diastereoselectivity ratio of up to 13:87. Furthermore, the one-pot reaction system demonstrated efficacy across a broad spectrum of substrates, including fluorinated pyridines, quinolines, azabiphenyls, thiazoles, and phenyl derivatives. This study significantly broadened the scope of nonnatural amino acids containing heteroaromatic rings and diversified the array of enzymes accessible for the production of functionally diverse bioactive fluorinated compounds.
研究领域:
生物化学
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合成生物学
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酶工程
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DOI
高影响因子
Journal of Pharmaceutical Analysis
·
2024
·
IF 6.1
Xin Yang,Guang-Yuan Ma,Xiao-Qiang Li,Na Tang,Yang Sun,Xiao-Wei Hao,Ke-Han Wu,Yu-Bo Wang,Wen Tian,Xin Fan,Zezhi Li,Caixia Feng,Xu Chao,Yu-Fan Wang,Yao Liu,Di Li,Wei Cao
Hepatocellular carcinoma (HCC) expresses abundant glycolytic enzymes and displays comprehensive glucose metabolism reprogramming. Aldolase A (ALDOA) plays a prominent role in glycolysis; however, little is known about its role in HCC development. In the present study, we aim to explore how ALDOA is involved in HCC proliferation. HCC proliferation was markedly suppressed both in vitro and in vivo following ALDOA knockout, which is consistent with ALDOA overexpression encouraging HCC proliferation. Mechanistically, ALDOA knockout partially limits the glycolytic flux in HCC cells. Meanwhile, ALDOA translocated to nuclei and directly interacted with c-Jun to facilitate its Thr93 phosphorylation by P21-activated protein kinase; ALDOA knockout markedly diminished c-Jun Thr93 phosphorylation and then dampened c-Jun transcription function. A crucial site Y364 mutation in ALDOA disrupted its interaction with c-Jun, and Y364S ALDOA expression failed to rescue cell proliferation in ALDOA deletion cells. In HCC patients, the expression level of ALDOA was correlated with the phosphorylation level of c-Jun (Thr93) and poor prognosis. Remarkably, hepatic ALDOA was significantly upregulated in the promotion and progression stages of diethylnitrosamine-induced HCC models, and the knockdown of ALDOA strikingly decreased HCC development in vivo . Our study demonstrated that ALDOA is a vital driver for HCC development by activating c-Jun-mediated oncogene transcription, opening additional avenues for anti-cancer therapies.
研究领域:
肿瘤学
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分子生物学
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癌症研究
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DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2025
·
IF 8.5
Qianqian Jiang,Qi Chu,Qi Zhang,Chaoqiang Wu,Tongqing Tang,Xin Wang,Feifei Chen,Alei Zhang,Kequan Chen
Adol condensation mediated by L-threonine aldolase (LTA) is a promising route to synthesize β-hydroxy-α-amino acids, such as the anti-Parkinson's disease drug L- threo -3,4-dihydroxyphenylserine (L- threo -DOPS; droxidopa). However, the biocatalytic approach generally suffered from poor stereoselectivity at the C β chiral center. Herein, several LTAs from diverse microorganisms were examined for the stereoselective synthesis of L- threo -DOPS, and an LTA from Firmicutes bacterium ( Fb LTA) was identified with relatively high stereoselectivity. A semi-rational design was then employed to further enhance the stereoselectivity, resulting in the identification of beneficial mutations and the generation of beneficial mutants with markedly elevated stereoselectivity (up to 80 % de) over the wild-type (23 % de). Molecular dynamics (MD) simulation revealed that the significant improvement of stereoselectivity could be attributed to the structural reconstitution of substrate tunnels. Finally, the best mutant was applied to a scaled-up reaction, affording 1.3 g/L yield of L- threo -DOPS with 80 % de. This study provides new insights into the stereoselectivity control of LTAs and potential engineered LTAs for the manufacture of L- threo -DOPS.
研究领域:
生物化学
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合成生物学
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药物开发
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DOI
PubMed
高影响因子
Molecular Catalysis
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2022
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IF 5.089
Li-Chao Wang,Lian Xu,Bing-Mei Su,Xin-Qi Xu,Juan Lin
L- threo -4-methylsulfonylphenylserine ethyl ester [(2 S , 3 R )- 1d ], a key building block of florfenicol which synthesized by esterification using ethanol and L- threo -4-methylsulfonylphenylserine [(2 S , 3 R )- 1b ] as substrates. L-threonine aldolase (LTA) is a promising biocatalyst for producing (2 S , 3 R )- 1b through a one-step process taking 4-Methylsulphonyl benzaldehyde ( 1a ) and glycine as substrates under the mild condition. However, the moderate C β -stereoselective blocked the industrial application of LTA. To address this issue, rational design by combination with prereaction state molecular dynamics (MD) simulation and per-residue energy decomposition algorithm are employed to engineer LTA for enhancing C β -stereoselective. As a result, a triple mutant N16A/E98S/Y314R (Mu3) is screened out to produce (2 S , 3 R )- 1b with 93.7% de and 90.2% conversion under the 300 mM 1a substrate loading. Furthermore, esterification is applied to synthesize (2 S , 3 R )- 1d with over 99% chiral purity and 99% product purity. The success of this study provides new insights for the rational design of LTA with improved C β -stereoselectivity and proves a chemo-enzymatic route for green synthesis of (2 S , 3 R )- 1d .
研究领域:
生物催化
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酶工程
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绿色化学
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药物合成
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DOI
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2020
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IF 4.192
Zijie Li,Fen Li,Li Cai,Zhou Chen,Ling Qin,Xiao-Dong Gao
A facile approach is introduced here for the synthesis of rare ketoses from glycerol and d-/l-glyceraldehyde (d-/l-GA). The reactions were carried out in a one-pot multienzyme fashion in which the only carbon source is glycerol. In the enzymatic cascade, glycerol is phosphorylated and then oxidized at C2 to afford dihydroxyacetone phosphate (DHAP), the key donor for enzymatic aldol reaction. Meanwhile, the primary alcohol of glycerol is also oxidized to give the acceptor molecule GA in situ (d- or l-isomer could be formed stereospecifically with either alditol oxidase or horse liver alcohol dehydrogenase). Different DHAP-dependent aldolases were used to generate the aldol adducts (rare ketohexose phosphates) with various stereoconfigurations and diastereomeric ratios. It is worth noting that the enzyme that catalyzes the phosphorylation reaction in the first step could also help recycle the phosphate in the last step to provide free rare sugar molecules. This study provides a useful method for rare ketose synthesis on a 100 mg to g scale, starting from relatively inexpensive materials which solved the problem of supplying both glycerol 3-phosphate and GA in our previous work. It also demonstrates an example of green synthesis due to highly efficient carbon usage and recycling of cofactors.
研究领域:
生物催化
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合成生物学
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绿色化学
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DOI
PubMed
高影响因子
Catalysts
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2025
·Vol. 15
·期 12
·
IF 4
Yang Yang,Kangkang Shang,Xiaorui Gao,Xingmiao Zhu,Mengying Ling,Pu Zheng,Shichao Xu,Pengcheng Chen
Raspberry ketone (RK) is the primary aromatic compound in raspberry fruit, which is widely utilized in perfume, cosmetics, and food additive industries. Currently, RK is predominantly produced chemically. RK biosynthesis through enzyme or whole cell has garnered significant attention due to the mild reaction conditions and the process being regarded as ‘natural’. This study proposed a ‘dual-microorganism, two-phase’ stepwise cascade strategy to produce RK from an economical precursor, 4-hydroxybenzaldehyde (4-HBD). An acetone-tolerant deoxyribose-phosphate aldolase DERAEc(S238D) mutant was obtained through a site-specific rigidification strategy for converting 4-HBD to 4-hydroxybenzylaceton (4-HBA). Then, an engineeredE. colico-expressing isocitrate dehydrogenase and raspberry ketone synthaseRiRZS1 with a citrate-sodium citrate buffer to recycle nicotinamide adenine dinucleotide phosphate (NADPH) was constructed for the conversion of 4-HBA to RK. The final concentration of RK was 50.00 ± 1.92 mmol·L−1with a yield of 86.96%. This strategy provides a scalable coenzyme self-recycling and two-phase catalysis platform for high-value phenolic compounds.
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高影响因子
Catalysts
·
2022
·Vol. 12
·期 9
·997
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IF 4.501
Xingmiao Zhu,Pengcheng Chen,Pu Zheng
4-Hydroxy benzylideneacetone (4-HBA) is an organic synthesis intermediate and can be used as a precursor for the synthesis of raspberry ketone. Herein, 2-deoxy-D-ribose 5-phosphate aldolase (DERA) was overexpressed inE. coliBL21 (DE3) as an attractive catalyst for enzymatic aldol reactions. The aldol reaction between 4-hydroxybenzaldehyde (4-HBD) and acetone to biosynthesize 4-HBA was catalyzed by whole-cellE. coliBL21 (DE3) (pRSF-Deoc). The yield and 4-HBA concentration were 92.8% and 111.35 mM, respectively, when using 120 mM 4-HBD and acetone as substrates. When the concentration of 4-HBD was increased to 480 mM, 376.4 mM 4-HBA was obtained by a fed-batch strategy with a yield of 78.4%, which was about a 28% improvement compared to the one-time addition strategy.E. coliBL21 (DE3) (pRSF-Deoc) cells were further immobilized withK-carrageenan, and the immobilized cells still maintained a residual activity of above 90% after 10 repeated uses. Our study provides a promising method of biosynthesizing 4-HBA.Keywords:4-hydroxybenzylideneacetone;2-deoxy-D-ribose 5-phosphate aldolases;whole-cell catalysis;fed-batch strategy;immobilized cells
研究领域:
生物化学
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合成生物学
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产品关联
DOI
高影响因子
Frontiers in Microbiology
·
2023
·
IF 5.2
Liu Quanzhen,Wei Guoguang,Yang Pengfan,Wang Chengyong,Chen Kequan,Ouyang Pingkai,Zhang Alei
N-acetylneuraminic acid (Neu5Ac) possesses the ability to promote mental health and enhance immunity and is widely used in both medicine and food fields as a supplement. Enzymatic production of Neu5Ac using N-acetyl-D-glucosamine (GlcNAc) as substrate was significant. However, the high-cost GlcNAc limited its development. In this study, an in vitro multi-enzyme catalysis was built to produce Neu5Ac using affordable chitin as substrate. Firstly, exochitinase SmChiA from Serratia proteamaculans and N-acetylglucosaminosidase CmNAGase from Chitinolyticbacter meiyuanensis SYBC-H1 were screened and combined to produce GlcNAc, effectively. Then, the chitinase was cascaded with N-acetylglucosamine-2-epimerase (AGE) and N-neuraminic acid aldolase (NanA) to produce Neu5Ac; the optimal conditions of the multi-enzyme catalysis system were 37°C and pH 8.5, the ratio of AGE to NanA (1:4) and addition of pyruvate (70 mM), respectively. Finally, 9.2 g/L Neu5Ac could be obtained from 20 g/L chitin within 24 h along with two supplementations with pyruvate. This work will lay a good foundation for the production of Neu5Ac from cheap chitin resources.
研究领域:
生物技术
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酶学
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食品科学
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医学
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DOI
PubMed
高影响因子
ADVANCED SYNTHESIS & CATALYSIS
·
2022
·Vol. 364
·期 24
·4363-4370
·
IF 5.981
Sai Fang,Haoran Yu,Lanxin Xiao,Zhe Wang,Yixuan Lei,Gang Xu,Lirong Yang,Wenlong Zheng,Jianping Wu
l -threonine aldolase ( L TA) is a vital tool for the production of β -hydroxy- α -amino acids, important pharmaceutical intermediates with two chiral centres. However, the trade-off between activity and diastereoselectivity seriously hinders the application of LTA. Here, microenvironment of the proton transfer was regulated to improve the enzyme activity while avoiding the loss of diastereoselectivity. A combinatorial active-site saturation test (CAST) strategy was applied to engineer the microenvironment of the key histidines H86 and H128 involved in proton transfer. Except for the amino acid residues tunning diastereoselectivity, a total of 18 (9+9) residues lining around H86 and H128 were investigated. As a result, two variants, RS1-T92V and RS1-E123R, were obtained with specific activity from 9.61 U/mg to 11.24 U/mg and 14.41 U/mg, respectively. By combinatorial mutagenesis, a double-point mutant RS1-VR (T92V/E123R) was obtained with specific activity reaching 18.65 U/mg that was two-fold of the original strain (RS1). Notably, the mutant RS1-VR remained a high de value of 94.21%. Molecular dynamics (MD) simulations provided insights into the mechanism of activity-diastereoselectivity trade-off. The improvement of microenvironment contributes to reduce the swing amplitude of the side chain of H86, resulting in the proton transfer more efficient. This work provides a strategy of regulating the proton transfer microenvironment for counteracting the trade-off between activity and diastereoselectivity in protein engineering.
研究领域:
生物催化
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酶工程
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计算化学
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蛋白质工程
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产品关联
DOI
高影响因子
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
·
2021
·
IF 3.575
Yongxin Li,Hua Huang,Xinshuai Zhang
1-Deoxy-D-sorbitol, the 1-deoxy analogue of D-sorbitol, has been detected in human urine as well as in natural herbs and spices. Although there are sporadic reports about 1-deoxy-D-sorbitol dehydrogenase, the complete catabolic pathway of 1-deoxy-D-sorbitol remains unsolved. Informed by the promiscuous activities of fructose-6-phosphate aldolase (FSA) which is involved in the sorbitol (glucitol) utilization ( gut ) operon and guided by the large scale bioinformatics analysis, we predicted and then experimentally verified the gut operon encoded by Bacillus licheniformis ATCC14580 is responsible for the catabolism of both D-sorbitol and 1-deoxy-D-sorbitol by in vitro activity assays of pathway enzymes , in vivo growth phenotypes, and transcriptomic studies. Moreover, the phylogenetic distribution analysis suggests that the D-sorbitol and 1-deoxy-D-sorbitol catabolic gene cluster is mostly conserved in members of Firmicutes phylum.
研究领域:
微生物学
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生物化学
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分子生物学
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基因组学
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DOI
PubMed
高影响因子
ACS Sustainable Chemistry & Engineering
·
2024
·
IF 7.1
Xuerui Jin,Chaoqun Huang,Can Cui,Huayi Liu,Zhenyu Liu,Dawen Niu,Yunzi Luo
Fluorinated amino acids play a crucial role as fundamental components in drug synthesis owing to their favorable attributes that enhance the pharmacokinetic properties of drugs. Nevertheless, the conventional chemical synthesis of fluorinated amino acids faces challenges in achieving stereoselectivity control. Additionally, the high cost of substrates and the intricate cofactor recycling process in enzymatic synthesis have limited their industrial production and applications. In this study, we developed a three-step chemoenzymatic cascade for the asymmetric synthesis of chiral fluorinated amino acids. The methodology involves the utilization of a variety of cost-effective aldehydes as starting materials, enabling economical aldol reactions catalyzed by aldolase. Subsequent chemical decarboxylation produces substrates for reductive amination through phenylalanine ammonia lyase, resulting in a diverse library of fluorinated aromatic l-α-amino acid products with a relative high yield and high enantiomeric purity (up to 99%). Furthermore, we synthesized three pharmaceutically relevant fluorinated l-α-amino acids on a preparative scale using whole-cell biocatalysts, demonstrating the versatility of the approach. This study has introduced a simplified, efficient, and cost-effective route for synthesizing fluorinated l-α-amino acids under mild conditions, offering insights into the production of valuable fluorine-containing pharmaceuticals and other biologically active compounds.
研究领域:
有机化学
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生物化学
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药学
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DOI
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2016
·
IF 7.476
Xiaonan Wang,Meiwen Wang,Yuanyuan Zhang,Xiaocao Miao,Yuanyuan Huang,Juan Zhang,Lizhou Sun
A new strategy to fabricate electrochemical biosensor is reported based on the linkage of enzyme substrate , thereby an electrochemical method to detect aldolase activity is established using pectin-thionine complex (PTC) as recognization element and signal probe . The linkage effect of fructose-1,6-bisphosphate (FBP), the substrate of aldolase, can be achieved via its strong binding to magnetic nanoparticles (MNPs)/aminophenylboronic acid (APBA) and the formation of phosphoramidate bond derived from its reaction with p -phenylenediamine (PDA) on the surface of electrode. Aldolase can reversibly catalyze the substrates into the products which have no binding capacity with MNPs/APBA, resulting in the exposure of the corresponding binding sites and its subsequent recognization on signal probe. Meanwhile, signal amplification can be accomplished by using the firstly prepared PTC which can bind with MNPs/APBA, and accuracy can be strengthened through magnetic separation. With good precision and accuracy, the established sensor may be extended to other proteins with reversible catalyzed ability.
研究领域:
分析化学
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生物化学
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生物传感器
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电化学
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纳米技术
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DOI
PubMed
高影响因子
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