Nature Communications
·
2025
·Vol. 16
·期 1
·1-13
·
IF 15.7
Li Fei,Yuan Zhenbo,Gao Yue,Deng Zhiwei,Zhang Yan,Luo Zhengshan,Rao Yijian
The application and drug development of plant-derived natural products are often limited by their low abundance in medicinal plants and the lack of structural complexity and diversity. Herein, we design a concise enzyme cascade to efficiently produce natural and unnatural protoberberine alkaloids from cost-effective, readily available substrates. Through enzyme discovery and engineering, along with systematic optimization of the berberine bridge enzyme to address remaining manufacturing challenges in protoberberine alkaloid biosynthesis, the high production of drug Rotundine is achieved at an impressive gram-scale titer, demonstrating its industrial potential. More importantly, this cascade also enables the efficient biosynthesis of various unnatural halogenated protoberberine alkaloids. Thus, this work not only unlocks the potential of enzyme cascades in overcoming longstanding challenges in the efficient biosynthesis of plant-derived alkaloids, but also opens avenues to introduce structural complexity and diversity into alkaloids through synthetic biology, offering significant potential for drug development.
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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2023
·
IF 4.6
Bin Wu,Xiangyun Wang,Bo Yang,Yunjian Ma,Frank Hollmann,Yonghua Wang
In the present work we propose a bienzymatic cascade for the oxyfunctionalisation of cycloalkanes to cyclic alcohols/cyclic ketones. By combining a H 2 O 2 -dependent peroxygenase with a O 2 -consuming and H 2 O 2 -producing alcohol oxidase an overall aerobic oxidation system was established. A convincing proof-of-concept is presented and some current limitations are outlined.
研究领域:
生物化学
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酶工程
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DOI
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2023
·Vol. 89
·期 4
·
IF 4.4
Xuling Jiang,Wanqing Wei,Yaozhong Cui,Wei Song,Yingying Li,Xiulai Chen,Cong Gao,Jia Liu,Liang Guo,Liming Liu,Jing Wu
Pyrrolidone is a high value-added monomer and an important active drug intermediate. However, the efficient enzymatic synthesis of pyrrolidone remains a challenge. Here, we developed and reconstructed a three-enzyme cascade pathway using Escherichia coli BL21(DE3) for the production of pyrrolidone from l-glutamate (l-Glu). The carnitine-CoA ligase from Escherichia coli (EcCaiC) at a low expression level and with a low activity is regarded as the rate-limiting enzyme. Here, we obtained the best EcCaiCF380M/N430D double mutant with a kcat/Km value 1.5 times higher than that of the wild type via mechanism-based protein engineering. For this, we (i) eliminated the steric hindrance of the loop ring to improve the precatalytic conformation of the adenylation intermediate and (ii) fixed the hinge region to stabilize the closed conformation of the enzyme. Furthermore, ribosome-binding site (RBS) optimization led to an increase in the expression level of EcCaiCF380M/N430D, which was then cloned into the plasmid pET-EcCaiCF380M/N430D-DegoPPK2. Finally, under optimal induction and transformation conditions, 16.62 g/L of pyrrolidone was generated from 30 g/L l-Glu (batch feeding) within 24 h with a molar conversion rate of 95.2% and the highest productivity ever obtained, to our knowledge (0.69 g/L/h). Our findings demonstrate a strategy that is potentially attractive for the industrial production of pyrrolidone.IMPORTANCE This study developed a three-enzyme cascade pathway for the production of pyrrolidone from l-Glu. The catalytic efficiency of carnitine CoA ligase from Escherichia coli (EcCaiC) was improved by mechanism-based protein engineering, and the titer of pyrrolidone was further increased by ribosome-binding site (RBS), induction conditions, and conversion conditions optimization. Finally, we efficiently produced pyrrolidone by one pot in vivo with 95.2% conversion and 0.69 g/L/h productivity. Our study provides a new possibility for the industrial production of enzymatic synthesis of pyrrolidone.
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
PubMed
高影响因子
ANALYTICAL CHEMISTRY
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2024
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IF 6.7
Qingzhi Peng,Huakun Wan,Zhenguo Yu,Shiyao Li,Hui Huang,Li Zhang,Yinli Guo,Dong Wang,Zhentan Lu
Bacteria can cause infectious diseases even at ultralow concentrations (<1 CFU/mL). It is important to rapidly identify bacterial contamination at ultralow concentrations. Herein, FITC-labeled gelatinase-sensitive nanoparticles (GNPs@FITCs) and NFM@GNP@FITCs are designed and fabricated as ultralow concentration bacteria detection platforms based on an enzymatic cascade reaction–amplifying strategy. Bacterial secretions could trigger the dissociation of GNPs@FITCs to release FITC, with gelatinase used as the model secretion. The detectable signal of ultralow concentration bacteria could be amplified effectively by the gelatinase-triggered cascade reaction. Bacterial concentration was evaluated by the change in fluorescence density. The results showed that the GNPs@FITCs and NFM@GNP@FITCs could be used for identifying bacterial contamination qualitatively, even when the bacterial contamination is lower than 1 CFU/mL. Moreover, the method has better timeliness and convenience, when compared with national standards. As solid films, NFM@GNP@FITCs have better long-term storage stability than GNPs@FITCs. The potential applications of GNPs@FITC and NFM@GNP@FITCs were proved by detecting pathogenic bacteria in food. All the results showed that the method has great potential for screening pathogenic bacterial contamination qualitatively.
研究领域:
微生物学
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生物医学工程
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食品安全
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DOI
PubMed
高影响因子
ACS Omega
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2025
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IF 4.3
Tianfu Wu,Wei Song,Jing Wu,Jian Wen,Wanqing Wei,Liming Liu
Chiral amino alcohols are vital in many biologically active molecules and natural products and are crucial building blocks for the pharmaceutical industry. However, the chemosynthesis of chiral amino alcohols, particularly bichiral amino alcohols, faces challenges related to chemoselectivity and enantioselectivity. Here, a dual-enzyme cascade pathway for the synthesis of bichiral amino alcohols through difunctionalization, exhibiting both high chemo- and stereoselectivity, was proposed for the first time. Among them, NADPH-dependent meso-diaminopyrimidate dehydrogenase from Proteus vulgaris (PvDAPDH) exhibited (R)-selective reductive amination activity, converting ketones into chiral amines; natural glucose dehydrogenase from Bacillus megaterium (BmGDH) not only facilitated NADPH regeneration but also catalyzed the reductive hydroxylation of ketones to chiral alcohols. Second, the rational design of the rate-limiting enzyme PvDAPDH generated the mutant M4 with increased specific activity and catalytic efficiency (kcat/Km) for 16a up to 3.2- and 10.3-fold, respectively, compared to the wild type. Additionally, the yield of mutant M4 for 1a–16a increased by 1.1- to 37-fold compared to the wild type. Finally, two high-value bichiral amino alcohols (14b and 16b) were asymmetrically synthesized from the corresponding bietone via a one-pot dual-enzyme cascade, exhibiting excellent stereoselectivity (dr ≥ 98:2). These findings provide a potential biosynthetic pathway for the green synthesis of bichiral amino alcohols and complement existing synthetic methods.
研究领域:
生物化学
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合成生物学
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制药工程
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DOI
PubMed
高影响因子
Molecular Catalysis
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2025
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IF 4.9
Shuo Wang,Binhao Wang,Guochao Xu,Ye Ni
Chiral lactones are valuable aroma compounds which have wide applications in food and fragrance industries. However, the synthesis of chiral lactones often faces challenges such as low yield, poor stereoselectivity, high production costs and harsh reaction conditions. To overcome these issues, a novel multi-enzyme cascade reaction was designed to achieve dynamic resolution of decalactones through stereoconvergent preparation of hydroxydecanoic acids. Utilizing lactonase Rpa 3624 for hydrolysis, Hb KR for stereoselective oxidation, and Sm CR V4 for stereoselective reduction, 50 mM racemic γ -decalactone was stereoselectively converted into ( R )- γ -decalactone with 96.5 % ee and 89.2 % yield in 1 h. By replacing Sm CR V4 with Sm CR M5 , 50 mM racemic δ -decalactone was also efficiently converted into ( R )- δ -decalactone with excellent stereoselectivity of 95.5 % and 84.9 % yield. The one-pot process could be operated under mild conditions, enhanced atom economy and reduced production costs, and provides a sustainable approach for the synthesis of chiral decalactones. This work demonstrates the potential of multi-enzyme systems in synthesis of chiral lactones for industrial application.
研究领域:
生物化学
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工业生物技术
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香料化学
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产品关联
DOI
高影响因子
ACS Sustainable Chemistry & Engineering
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2025
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IF 7.3
Yixin Cai,Louqian Zhang,Weixia Cheng,Wenxuan Xu,Kangrui Yuan,Ranhua Xiong,Chaobo Huang
Various active substances are encapsulated within microspheres’ independent compartments, forming an orderly system that can effectively improve chemical reaction efficiency and system stability. Herein, chitosan-based three-compartment microspheres (TCMs) were reported that encapsulate three enzymes in an orderly manner through a gas-shearing strategy. The TCMs can effectively enhance the efficiency of multienzyme cascade reactions, simultaneously controlling blood sugar levels and effectively scavenging ROS. The three compartments of TCMs independently encapsulated glucose oxidase (GOD)/insulin, superoxide dismutase (SOD), and horseradish peroxidase (HRP), corresponding to the insulin releasing unit, superoxide radical (•O2–) scavenging unit, and hydrogen peroxide (H2O2) scavenging unit. TCMs confine the enzyme cascade reaction within the same microsphere, significantly reducing the transmission distance of chemical signals and increasing the concentration of the reaction substrates. TCMs demonstrate a higher cascade catalytic efficiency than one-compartment microspheres (OCMs). In vitro experiments show that TCMs significantly reverse the increased oxidative stress. This high-efficiency multienzyme cascade reaction enables TCMs to promote insulin absorption and effectively control blood sugar levels in insulin-resistant cell model experiments. The multicompartmental structure of TCMs is capable of independently encapsulating multiple active substances, making them attractive carrier materials for biomedicine, food, and cosmetics applications.
研究领域:
生物医学工程
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材料科学
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药物递送
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产品关联
DOI
高影响因子
MACROMOLECULAR RAPID COMMUNICATIONS
·
2021
·Vol. 43
·期 1
·2100559
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IF 5.734
Bolei Yuan,Tingting Huang,Xinghuo Wang,Yi Ding,Lin Jiang,Yunhe Zhang,Jun Tang
An enzyme cascade system including glucose oxidase (GOx) and iron porphyrin (DhHP-6) is encapsulated in a metal–organic framework called zeolitic imidazolate framework-8 (ZIF-8) through one-step facile synthesis. The composite (GOx&DhHP-6@ZIF-8) is then used to initiate oxygen-tolerant reversible addition-fragmentation chain-transfer polymerization for different methacrylate monomers, such as 2-diethylaminoethyl methacrylate, 2-hydroxyethyl methacrylate, and poly(ethylene glycol) methyl ether methacrylate ( M n = 500 g mol −1 ). The composite shows the robustness toward solvent and temperatures, all polymerizations using above monomers and catalyzing by GOx&DhHP-6@ZIF-8 exhibits high monomer conversion (>85%) and narrow molar mass dispersity (<1.3). Besides, acrylic and acrylamide monomers such as 2-hydroxyethyl acrylate and N,N-dimethylacrylamide are also carried to demonstrate the broad applicability. Proton nuclear magnetic resonance characterization and chain extension experiments confirm the retaining end groups of the resultant polymers, which is a significant feature of living polymerization. More importantly, the process of recycling the composite through a centrifuge is simplistic, and the composite still maintains similar activity compared to the original composites after five times. This low-cost and easily separated composite catalyst represents a versatile strategy to synthesize well-defined functional polymers suitable for industrial-scale production.
研究领域:
材料科学
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生物化学
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DOI
高影响因子
Small
·
2023
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IF 13.3
Rui Pei,Jing Liu,Chuanyong Jing,Min Zhang
The reduction of carbon dioxide to valuable chemicals through enzymatic processes is regarded as a promising approach for the reduction of carbon dioxide emissions. In this study, an in vitro multi-enzyme cascade pathway is constructed for the conversion of CO 2 into dihydroxyacetone (DHA). This pathway, known as FFFP, comprises formate dehydrogenase (FDH), formaldehyde dehydrogenase (FaldDH), formolase (FLS), and phosphite dehydrogenase (PTDH), with PTDH serving as the critical catalyst for regenerating the coenzyme NADH. Subsequently, the immobilization of the FFFP pathway within the hydrogen-bonded organic framework (HOF-101) is accomplished in situ. A 1.8-fold increase in DHA yield is observed in FFFP@HOF-101 compared to the free FFFP pathway. This enhancement can be explained by the fact that within FFFP@HOF-101, enzymes are positioned sufficiently close to one another, leading to the elevation of the local concentration of intermediates and an improvement in mass transfer efficiency. Moreover, FFFP@HOF-101 displays a high degree of stability. In addition to the establishment of an effective DHA production method, innovative concepts for the tailored synthesis of fine compounds from CO 2 through the utilization of various multi-enzyme cascade developments are generated by this work.
研究领域:
生物化学
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环境科学
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材料科学
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DOI
PubMed
高影响因子
Biotechnology Journal
·
2025
·Vol. 20
·期 2
·e202400707
·
IF 3.1
Fengrui Yu,Hongwen Li,Xianglong Li,Jianping Shi,Yanbin Feng,Song Xue
Nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide (NAD), provides a direct method for maintaining NAD levels, which may alleviate aging and metabolic disorders. However, the enzymatic conversion of NMN in cascade reactions is limited by intermediate product inhibition, and quantitative insights into these limitations remain scarce. Here, an efficient multienzyme cascade system was developed by quantifying intermediate inhibition, which synthesizes NMN from D-ribose in three tandem reactions with an Adenosine Triphosphate (ATP) regeneration system and pyrophosphatase (PPase). A critical Adenosine Diphosphate (ADP) concentration of 0.5 mM was determined, which inhibits phosphoribosyl pyrophosphate synthetase (Prs) at 0.08 µM. The incorporation of an ATP regeneration system and PPase markedly increased the NMN yield to 81.3%. The intermediate phosphoribosyl pyrophosphate (PRPP) hydrolysis rate was measured at 3 µM/min. The highly active nicotinamide phosphoribosyltransferase (Nampt) could compete with PRPP hydrolysis, thereby increasing the yield of NMN. This research facilitates large-scale, efficient NMN manufacturing.
研究领域:
生物化学
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代谢工程
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合成生物学
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产品关联
DOI
PubMed
高影响因子
BIOTECHNOLOGY LETTERS
·
2023
·Vol. 45
·期 10
·1355-1364
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IF 2.7
Gao Yahui,Li Fen,Wang Yulu,Chen Zhou,Li Zijie
Purpose In our previous study, we constructed a one-pot multi-enzyme system for rare ketoses synthesis based on L-rhamnulose-1-phosphate aldolase (RhaD) from accessible glycerol in vitro. To eliminate tedious purification of enzymes, a facile Escherichia coli whole-cell cascade platform was established in this study. Methods To enhance the conversion rate, the reaction conditions, substrate concentrations and expressions of related enzymes were extensively optimized. Results The biosynthetic route for the cascade synthesis of rare ketoses in whole cells was successfully constructed and three rare ketoses including D-allulose, D-sorbose and L-fructose were produced using glycerol and D/L-glyceraldehyde (GA). Under optimized conditions, the conversion rates of rare ketoses were 85.0% and 93.0% using D-GA and L-GA as the receptor, respectively. Furthermore, alditol oxidase (AldO) was introduced to the whole-cell system to generate D-GA from glycerol, and the total production yield of D-sorbose and D-allulose was 8.2 g l −1 only from the sole carbon source glycerol. Conclusion This study demonstrates a feasible and cost-efficient method for rare sugars synthesis and can also be applied to the green synthesis of other value-added chemicals from glycerol.
研究领域:
生物化学
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合成生物学
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代谢工程
✓ 已验证
DOI
高影响因子
Molecular Catalysis
·
2022
·
IF 5.089
Xuanru Zhong,Yunjian Ma,Xizhen Zhang,Jiahui Zhang,Bin Wu,Frank Hollmann,Yonghua Wang
Hydrocarbon synthesis from (waste)oils enabled by a cascade of lipase-catalysed hydrolysis and decarboxylase-catalysed decarboxylation has become an active area of research en route to alternative, biobased fuels. However, Poor substrate transport efficiency is a major issue causing low reaction rates. This study focused on a protein self-assembly strategy based on SpyTag/SpyCatcher to overcome diffusion limitations. For this, two fusion proteins, TLL-Linker-SpyCatcher based on the lipase from Thermomyces lanuginosus and Cv FAP-Linker-SpyTag based on the fatty acid photodecarboxylase from Chlorella variabilis were designed. A covalent multi-enzyme complex (TLL- Cv FAP) was formed spontaneously by self-assembly of each enzyme. The effects of temperature, pH and molar ratio of self-assembled components on assembly efficiency were investigated. The results showed that the multi-enzyme complex TLL- Cv FAP reached about 60% after 12 h of assembly, and the enzyme activity of the multienzyme complex was increased by about 50% compared to that of the corresponding non-assembled enzymes. Under optimized conditions 10 mM soybean oil were converted into 25 mM of the corresponding hydrocarbons, suggesting a good potential of biofuel synthesis.
研究领域:
生物技术
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酶工程
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可持续化学
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DOI
高影响因子
危险品化学品经营许可证（带存储）