JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2025
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IF 6.2
Cunduo Tang,Taisong Shen,Xueyang Bai,Yao Wang,Jiashu Zhang,Yunchao Kan,Dandan Li,Lunguang Yao,Hongling Shi
Nicotinamide mononucleotide (NMN) is a very important bioactive nucleotide that is of great help to human health. However, its widespread application has been limited by its high production costs, especially the cost of the core substrates, coenzyme, and enzymes. In this study, the ADP/GDP-polyphosphate phosphotransferase RhPPK2 originating from Rhodobacter sphaeroides was successfully expressed in Escherichia coli with high-level solubility, and the enzyme activity in the lysate supernatant reached 21.9 ± 0.65 U/mL. And then, the temperature profiles, pH profiles, and kinetic parameters of purified reRhPPK2 were systematically characterized, which demonstrate its potential for application in enzymatic ATP regeneration systems. Furthermore, the introduction of reRhPPK2 for ATP regeneration significantly enhanced NMN production efficiency, achieving a 2.3-fold increase compared to the conventional ATP supplementation method. Finally, the production efficiency of NMN was further improved by a single-factor experiment and L9(34) orthogonal design, and the yield was up to 14.6 ± 0.51 g/L, about 5.4 times of the initial yield. This research substantially reduced NMN production costs and established a robust foundation for industrial-scale NMN production.
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
Biotechnology for Biofuels and Bioproducts
·
2023
·Vol. 16
·期 1
·1-13
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IF 0
Gao Hui,Li Mengxuan,Wang Qing,Liu Tingting,Zhang Xian,Yang Taowei,Xu Meijuan,Rao Zhiming
ATP, an important cofactor, is involved in many biocatalytic reactions that require energy. Polyphosphate kinases (PPK) can provide energy for ATP-consuming reactions due to their cheap and readily available substrate polyphosphate. We determined the catalytic properties of PPK from different sources and found that PPK from Cytophaga hutchinsonii ( Ch PPK) had the best catalytic activity for the substrates ADP and polyP 6 . An extracellular–intracellular dual system was constructed to high-throughput screen for better catalytic activity of Ch PPK mutants. Finally, the specific activity of Ch PPK D82N-K103E mutant was increased by 4.3 times. Therefore, we focused on the production of L-theanine catalyzed by GMAS as a model of ATP regeneration. Supplying 150 mM ATP, GMAS enzyme could produce 16.8 ± 1.3 g/L L-theanine from 100 mM glutamate. When 5 mM ATP and 5 U/mL Ch PPK D82N-K103E were added, the yield of L-theanine was 16.6 ± 0.79 g/L with the conversion rate of 95.6 ± 4.5% at 4 h. Subsequently, this system was scaled up to 200 mM and 400 mM glutamate, resulting in the yields of L-theanine for 32.3 ± 1.6 g/L and 62.7 ± 1.1 g/L, with the conversion rate of 92.8 ± 4.6% and 90.1 ± 1.6%, respectively. In addition, we also constructed an efficient ATP regeneration system from glutamate to glutamine, and 13.8 ± 0.2 g/L glutamine was obtained with the conversion rate of 94.4 ± 1.4% in 4 h after adding 6 U/ mL GS enzyme and 5 U/ mL Ch PPK D82N-K103E , which further laid the foundation from glutamine to L-theanine catalyzed by GGT enzyme. This proved that giving the reaction an efficient ATP supply driven by the mutant enzyme enhanced the conversion rate of substrate to product and maximized the substrate value. This is a positively combination of high yield, high conversion rate and high economic value of enzyme catalysis. The mutant enzyme will further power the ATP-consuming biocatalytic reaction platform sustainably.
研究领域:
生物化学
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酶工程
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代谢工程
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DOI
PubMed
ENZYME AND MICROBIAL TECHNOLOGY
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2021
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IF 3.493
Caixia Cui,Mengyuan Kong,Yihan Wang,Chenyan Zhou,Hong Ming
Polyphosphate kinase (PPK) is important for industrial processes involving ATP regeneration. While a variety of methods have been reported for regenerating ATP from ADP, few have explored enzyme catalyzed ATP regeneration from cheaper and stable AMP. In this work, PPKs from different sources were expressed and their catalytic activity were tested at different reaction temperatures, reaction pH and with different polyphosphate (polyP n ) types. The ATP regeneration system for glutathione (GSH) synthesis was established using a single PPK capable of phosphorylating AMP to synthesize ATP from AMP and short chain polyP n . GSH yield was obtained using adenosine mono-, di- and triphosphates, which confirmed the flexibility of our constructed ATP regeneration system coupled with GSH synthesis via bifunctional GSH synthase. Finally, optimization of the GSH synthesis yielded conversion value above 80 %. Overall, these results illustrate that PPK is suitable for a broader range of substrates than previously expected, and has great untapped potential for applications involving ATP regeneration.
研究领域:
生物化学
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生物技术
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工业生物处理
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DOI
PubMed
高影响因子
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
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2017
·Vol. 185
·期 2
·385-395
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IF 1.751
Cao Hao,Li Chengcheng,Zhao Jing,Wang Fang,Tan Tianwei,Liu Luo
Glutathione (GSH) is an important reducing agent in the living cells. It is synthesized by a two-step reaction and requires two molecules of adenosine triphosphate (ATP) for one molecule GSH. The enzymatic cascade reaction in vitro is a promising approach to achieve a high titer and limit side reactions; although, a cost-effective phosphate donor for ATP regeneration is required. Triphosphate (PolyP (3) ), tetraphosphate (PolyP (4) ), and hexametaphosphate (PolyP (6) ) were investigated in this study. Triphosphate inhibited the bifunctional GSH synthetase (GshF) from Streptococcus agalactiae , while no significant inhibition was observed by adding hexametaphosphate. The polyphosphate kinase from Corynebacterium glutamicum was hence investigated to use hexametaphosphate for regeneration of ATP. Further, the orthogonal experiment, which includes seven factors (buffer concentration, pH value, ADP concentration, GshF dosage, polyphosphate kinase (PPK) dosage, reaction temperature, substrate ratio of amino acid, and reaction times), indicated that the capacity of buffer is the most significant factor of the reaction conditions for enzymatic production of glutathione coupling with a PPK-based ATP regeneration system. After optimizing the Mg 2+ concentration, the reaction was scaled up to 250 mL in a stirred reactor with pH feedback control to stabilize the pH value of reaction system and nitrogen protection to avoid the oxidation of product. A yield of 12.32 g/L was achieved. This work provided a potential GshF-based enzymatic way coupling the PPK-based ATP regeneration to product GSH in the optimal conditions towards cost-effectiveness at the industrial scale.
研究领域:
生物化学
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酶工程
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工业微生物学
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DOI
PubMed
高影响因子
Biotechnology Journal
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2025
·Vol. 20
·期 2
·e202400707
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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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PubMed
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
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2017
·Vol. 101
·期 13
·5325-5332
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IF 3.42
Cao Hao,Nie Kaili,Li Chengcheng,Xu Haijun,Wang Fang,Tan Tianwei,Liu Luo
Adenosine-5′-triphosphate (ATP) is the energy equivalent of the living system. Polyphosphate (polyP) is the ancient energy storage equivalent of organisms. Polyphosphate kinases (PPKs) catalyze the polyP formation or ATP formation, to store energy or to regenerate ATP, respectively. However, most PPKs are active only in the presence of long polyPs, which are more difficult and more expensive to generate than the short polyPs. We investigated the PPK preference towards polyPs by site-directed mutagenesis and computational simulation, to understand the mechanism and further design enzymes for effective ATP regeneration using short polyPs for in vitro cascade reactions, which are highly desired for research and applications. The results suggest that the short polyPs inhibit PPK by blocking the ADP-binding pocket. Structural comparison between PPK ( Corynebacterium glutamicum ) and PPK ( Sinorhizobium meliloti ) indicates that three amino acid residues, i.e., lysine, glutamate, and threonine, are involved in the activity towards short polyP by fixing the adenosine group of ADP in between the subunits of the dimer, while the terminal phosphate group of ADP still offers an active site, which presents a binding pocket for ADP. A proposed triple mutant PPK (SMc02148-KET) demonstrates significant activity towards short polyP to form ATP from ADP. The obtained high glutathione titer (38.79 mM) and glucose-6-phosphate titer (87.35 mM) in cascade reactions with ATP regeneration using the triple mutant PPK (SMc02148-KET) reveal that the tailored PPK establishes the effective ATP regeneration system for ATP-dependent reactions.
研究领域:
生物化学
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酶工程
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合成生物学
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PubMed
高影响因子
Molecular Catalysis
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2020
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IF 3.687
Caixia Cui,Hong Ming,Linjing Li,Mingjie Li,Jian Gao,Tao Han,Yunyun Wang
We report here adopting a glucose template for synthesizing mesoporous silica for enzyme in-situ immobilization. Bifunctional GSH synthetase (GSHF) and polyphosphate kinase (PPK) were selected as model system for synthesizing GSH with ATP regeneration. Results indicated that a pore size that was slightly smaller than that of enzyme was responsible for the high performance of immobilized enzyme inside mesoporous without protein leaking. Then, the cationic dopamine was used for PPK immobilization by self-polymerization. The co-immobilized enzyme activity was much higher than that of the free enzyme. The catalytic efficiency kcat/Km of co-immobilized enzyme was higher than that of the free enzyme (8.5 and 1.5 times higher for GSHF and PPK, respectively). After optimizing the reaction parameters, the co-immobilized enzymes exhibited excellent operational and storage stability, and could be re-used seven times and retained the original activity after 30 days. It is hoped that these findings will pave the way for applying enzyme co-immobilization to biocatalytic system.
研究领域:
生物化学
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生物技术
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材料科学
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DOI
高影响因子
Journal of the American Chemical Society
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2025
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IF 15.6
Yiming Liu,Mingrui Zhang,Jingjing Zhao,Yongshuo Ren,Shubin Li,Weichen Wang,Wei Mu,Xiaojun Han
The reconstitution of the de novo nucleotide synthesis pathway from inorganic compounds for RNA transcription is essential for an autonomous artificial cell. Here, a de novo uridine triphosphate (UTP) synthesis pathway is successfully constructed starting from NH4HCO3, involving eight enzymes of carbamoyl phosphate synthetase (CPS), aspartate transcarbamoylase (ATC), dihydroorotase (DHO), dihydroorotate dehydrogenase (DHODH), ribose-phosphate pyrophosphokinase (RPPK), uridine 5′-monophosphate synthase (UMPs), uridylate kinase (UK), and nucleoside diphosphate kinase (NDK). ATP regeneration system composed of creatine kinase (CK) and creatine phosphate is incorporated to drive this pathway. 0.85 mM UTP is produced within 180 min under the optimized conditions, which is further used for RNA transcription along with preadded CTP, GTP, regenerated ATP, and T7 RNA polymerase. The de novo UTP synthesis pathway and RNA transcription components are encapsulated into giant unilamellar vesicles to build artificial cells capable of nucleotide de novo production for subsequent RNA transcription. The successful RNA transcription inside artificial cells is visualized using pyronin Y. The successful reconstitution of the de novo UTP synthesis pathway for RNA transcription inside artificial cells lays a solid foundation for the reproduction of an autonomous artificial cell.
研究领域:
合成生物学
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生物化学
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分子生物学
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人工细胞
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系统生物学
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产品关联
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PubMed
高影响因子
危险品化学品经营许可证（带存储）