INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2025
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IF 8.5
Kai Yang,Zhenjie Tang,Chong Zhang,Ruide Zhang,Sheng Hu,Changjiang Lv,Jun Huang,Jiaqi Mei,Weirui Zhao,Lehe Mei
Salvianic acid A (SAA) is a catechol compound known for its diverse physiochemical functions and has significant applications in the food and pharmaceutical industries. 4-Hydroxyphenylacetate-3-hydroxylase (4HPA3H) is a critical enzyme for SAA biosynthesis, and improving its activity towards p -hydroxyphenyllactate acid (4HPLA) is essential for highly efficient SAA production in stable biosynthetic pathways. To address this, the distal site and loops of the substrate pocket were modified to improve 4HPA3H catalytic activity towards 4HPLA using computer-aided molecular modification methods. As a result, we identified and mutated two critical sites in Ec HpaB, a 4HPA3H monooxygenase from Escherichia coli : T398, a substrate loop site, and M205, a distal site. The mutants M205F, T398S, and M205F/T398S enabled 2.51-, 2.07-, and 2.20-fold increases in catalytic efficiency ( k cat / K m towards 4HPLA), respectively. Molecular dynamics simulations showed that the overall decreased flexibility of the substrate pocket loop in Ec HpaB might help improve the enzyme's catalytic activity for 4HPLA. Owing to the T398 site in the substrate pocket coming from another monomer, molecular modification of Ec HpaB with multi-monomer interactions was also worthy of attention. These findings not only enhanced the biosynthetic efficiency of SAA but also provided insights into enzyme distal site modification and the engineering of multi-monomer enzymes.
研究领域:
生物化学
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酶工程
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计算生物学
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PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2024
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IF 5.7
Gege Ma,Xiangyu Zhu,Dongxin Zhang,Haoran Li,Jinping Lin,Dongzhi Wei
(R)-Citronellal is a key chiral precursor of high-value chemicals, such as the best-selling flavor compound (−)-menthol; however, the conventional synthesis suffers from low yield and unsatisfactory enantioselectivity. In this study, we developed a highly atom-efficient hydrogen-borrowing cascade for the synthesis of (R)-citronellal from geraniol using alcohol dehydrogenase from Escherichia coli K12 (AdhP) and ene-reductase from Saccharomyces cerevisiae YJM1341 (OYE2p). The key rate-limiting enzyme, AdhP, was subjected to structure-guided semirational engineering, and the triple mutant AdhP260T/284A/268P (M3) was obtained that demonstrated a 1.28-fold improvement in catalytic efficiency (kcat/Km) toward geraniol. After optimization of the reaction conditions, the hydrogen-borrowing cascade system achieved the conversion of 23.14 g/L geraniol into (R)-citronellal at a conversion rate of 98.23% with 96.7% ee. This work represents an alternative approach for the biosynthesis of (R)-citronellal without sacrificing a cosubstrate or additional enzymes.
研究领域:
生物化学
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合成生物学
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酶学
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DOI
PubMed
高影响因子
Biotechnology Journal
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2024
·Vol. 19
·期 11
·e202400489
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IF 3.2
Bo Zhang,Sifang Zhu,Yi Zhu,Xin Sui,Junping Zhou,Zhiqiang Liu,Yuguo Zheng
Boldenone (BD), a protein anabolic hormone, is commonly used to treat muscle damage, osteoporosis, and off-season muscle building in athletes. Traditional BD synthesis methods rely on chemical processes, which are costly and environmentally impactful. Therefore, developing a more sustainable and economical biosynthetic pathway is crucial for BD production. This study aimed to achieve efficient production of BD. Firstly, the catalytic performance of 17β-hydroxysteroid dehydrogenase and 3-ketosteroid-Δ 1 -dehydrogenase was improved through enzyme engineering, and their expression in the new strain of Mycobacterium neoaurum was enhanced using metabolic engineering. These improvements significantly increased BD production to 4.05 g/L, with a significant decrease in by-product generation. To further increase the yield, a multi-enzyme fusion expression system was constructed, and a key cell wall gene kasB was knocked out, resulting in a spatial-time yield of BD reaching 1.02 g/(L·d). Subsequent optimization of the transformation system further increased the BD production to 5.56 g/L, with a spatiotemporal yield of 1.39 g/(L·d). The green biosynthetic route of phytosterol one-step conversion to BD developed in this study lays the foundation for industrial production.
研究领域:
生物化学
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微生物学
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合成生物学
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DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2025
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IF 8.5
Guosi Li,Shanyong Yi,Haijiao Wang,Hulin Qiu,Wei Wang,Leilei Gao,Qilin Xu,Bangxing Han,Xinjian Yin
Salidroside is a phenylpropanoid glycoside with wide applications in the food, pharmaceutical, and cosmetic industries; however, the plant genus Rhodiola , the natural source of salidroside, has slow growth and limited distribution. In this study, we designed a novel six-enzyme biocatalytic cascade for the efficient production of salidroside, utilizing cost-effective bio-based L-Tyrosine as the starting material. A preliminary analysis revealed that the poor thermostability of the Bacillus licheniformis UDP-glycosyltransferase (EC 2.4.1.384) Bl YjiC M6 is a bottleneck in the cascade. Therefore, a combined computational strategy was used to engineer it and finally obtained a mutant TSM6 (T304V/G307A/N309W/F123W/T344V/D271G) with a 134-fold longer half-life at 40 °C and a 13 °C higher T m app compared to M6. The integration of TSM6 into the cascade improved salidroside productivity significantly, while reducing residual intermediates. After further optimization, the whole-cell biocatalytic cascade achieved a high salidroside titer of 12.8 g·L −1 in a 5 L bioreactor, giving a productivity of 0.53 g·L −1 ·h −1 . This study provides a green and efficient biosynthetic process for salidroside production and highlights the potential of enzyme engineering to enhance the biocatalytic cascade.
研究领域:
生物化学
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酶学
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合成生物学
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DOI
PubMed
高影响因子
ACS Sustainable Chemistry & Engineering
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2025
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IF 7.3
Ran Wang,Wei Song,Hangyuan Xu,Jian Wen,Wanqing Wei,Zhaoyang Chen,Guipeng Hu,Cong Gao,Xiaomin Li,Jia Liu,Jing Wu
l-Asparagine (l-Asn) is an important amino acid with broad applications in food, medicine, fine chemicals, and environmental protection. However, its industrial production is limited by the high cost of raw materials and the low catalytic efficiency of enzymes. In this study, a three-enzyme cascade pathway (FDN) was designed to produce l-Asn, using fumaric acid as the raw material. Within this pathway, EcAsnA was identified as the rate-limiting enzyme and was subsequently engineered using a product rescue strategy to reduce product inhibition by opening the closed gate. The optimal mutant, L109 K/K58R, exhibited a 6.61-fold reduction in product inhibition and a 4.24-fold improvement in catalytic efficiency. This mutant was then integrated into Escherichia coli along with the other two enzymes to construct the optimal recombinant strain E. coli 17. Using diatomite-glutaraldehyde cross-linking immobilized E. coli 17 as a biocatalyst, 267.74 g of l-Asn (with 5.35 g·L–1·h–1 STY, > 99% ee) was produced across 50 batch feedings in 1 L reaction volume. The purity of l-Asn exceeded 99% after isolation and purification. This study demonstrates the effective integration of cascade reaction design, enzyme engineering, and cell immobilization, providing a promising approach for synthesizing high-value products from cost-effective substrates.
研究领域:
生物化学
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酶学
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合成生物学
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产品关联
DOI
高影响因子
BIORESOURCE TECHNOLOGY
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2023
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IF 11.4
Yufei Zhang,Junhua Yun,Guoyan Zhang,Amreesh Parvez,Lei Zhou,Hossain M. Zabed,Jia Li,Xianghui Qi
3-Hydroxypropionic acid (3-HP) is a top value-added chemical with multifaceted application in chemical, material, and food field. However, limited availability of robust strains and elevated fermentation costs currently impose constraints on sustainable biosynthesis of 3-HP. Herein, transporter engineering, metabolic dynamic modulation, and enzyme engineering were combined to address above limitations. First, a glucose-utilizing 3-HP biosynthetic pathway was constructed in Escherichia coli , followed by recruiting alternative glucose transport system to overcome center metabolism overflow. Next, the Cra (a transcription factor)-dependent switch was applied to autonomously fine-tune carbon flux, which alleviated growth retardation and improved the 3-HP production. Subsequently, inactivation of glycerol facilitator (GlpF) increased intracellular glycerol levels and boosted 3-HP biosynthesis, but caused toxic intermediate 3-hydroxypropionaldehyde (3-HPA) accumulation. Furthermore, semi-rational design of aldehyde dehydrogenase (YdcW) increased its activity and eliminated 3-HPA accumulation. Finally, fed-batch fermentation of the final strain resulted in 52.73 g/L 3-HP, with a yield of 0.59 mol/mol glucose.
研究领域:
生物化学
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合成生物学
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微生物学
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DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2023
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IF 6.1
Bin Wu,Shiyu Wang,Yunjian Ma,Shuguang Yuan,Frank Hollmann,Yonghua Wang
Alcohol oxidases (AOxs) catalyze the aerobic oxidation of alcohols to the corresponding carbonyl products (aldehydes or ketones), producing only H2O2 as the byproduct. The majority of known AOxs, however, have a strong preference for small, primary alcohols, limiting their broad applicability, e.g., in the food industry. To broaden the product scope of AOxs, we performed structure-guided enzyme engineering of a methanol oxidase from Phanerochaete chrysosporium (PcAOx). The substrate preference was extended from methanol to a broad range of benzylic alcohols by modifying the substrate binding pocket. A mutant (PcAOx-EFMH) with four substitutions exhibited improved catalytic activity toward benzyl alcohols with increased conversion and kcat toward the benzyl alcohol from 11.3 to 88.9% and from 0.5 to 2.6 s–1, respectively. The molecular basis for the change of substrate selectivity was analyzed by molecular simulation.
研究领域:
生物化学
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酶学
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食品科学
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DOI
PubMed
高影响因子
MOLECULES
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2024
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IF 4.2
Jiming Zhang,Hui Lin,Chaosong Zheng,Bin Yang,Miao Liang,Yi Lin,Liaoyuan Zhang
2,3-butanediol (2,3-BD) is a versatile bio-based platform chemical. An artificial four-enzyme synthetic biosystem composed of ethanol dehydrogenase, NADH oxidase, formolase and 2,3-butanediol dehydrogenase was designed for upgrading ethanol to 2,3-BD in our previous study. However, a key challenge in developing in vitro enzymatic systems for 2,3-BD synthesis is the relatively sluggish catalytic efficiency of formolase, which catalyzes the rate-limiting step in such systems. Herein, this study reports how engineering the tunnel and substrate binding pocket of FLS improved its catalytic performance. A series of single-point and combinatorial variants were successfully obtained which displayed both higher catalytic efficiency and better substrate tolerance than wild-type FLS. Subsequently, a cell-free biosystem based on the FLS:I28V/L482E enzyme was implemented for upgrading ethanol to 2,3-BD. Ultimately, this system achieved efficient production of 2,3-BD from ethanol by the fed-batch method, reaching a concentration of 1.39 M (124.83 g/L) of the product and providing both excellent productivity and yield values of 5.94 g/L/h and 92.7%, respectively. Taken together, this modified enzymatic catalysis system provides a highly promising alternative approach for sustainable and cost-competitive production of 2,3-BD.
研究领域:
生物化学
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合成生物学
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酶学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2025
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IF 6.2
Yu Du,Xiang Lv,Till Siebenmorgen,Grzegorz Popowicz,Chenhao Feng,Yunjian Ma,Yonghua Wang
Enzymatic synthesis of δ-lactones using Baeyer–Villiger monooxygenases (BVMOs) has potential in the fragrance and flavor industries but is constrained by poor activity toward ortho-alkyl-substituted cyclopentanones, key δ-lactone precursors. We determined the crystal structure of a BVMO derived from Oceanicola granulosus (OgBVMO), uncovering a unique loop adjacent to key catalytic residue R335. Site-saturation mutagenesis of loop residues A338 and A339 identified the A339E variant, obtaining a 2.4- to 3-fold increase in catalytic activity toward ortho-alkyl-substituted cyclopentanones (2-methyl-, 2-ethyl-, 2-hexyl-, and 2-heptylcyclopentanone). Further engineering the substrate-binding pocket yielded the Q442N variant, improving activity by 2.7–3.8-fold. Remarkably, the combinatorial mutant A339E/Q442N achieved 3.3–5.2-fold higher activity than the wild-type. Molecular dynamics simulations indicated that these improvements were driven by more favorable nucleophilic attack distances, underscoring the synergistic effects of distal and active-site mutations. These findings offer valuable insights into enhancing the catalytic activity of BVMOs, supporting the green manufacturing of high-value flavor compounds.
研究领域:
生物化学
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合成生物学
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酶学
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产品关联
DOI
PubMed
高影响因子
METABOLIC ENGINEERING
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2017
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IF 8.142
Lu Dai,Fei Tao,Hongzhi Tang,Yali Guo,Yaling Shen,Ping Xu
Primordial enzymes are proposed to possess broad specificities. Through divergence and evolution, enzymes have been refined to exhibit specificity towards one reaction or substrate, and are thus commonly assumed as “specialists”. However, some enzymes are “generalists” that catalyze a range of substrates and reactions. This property has been defined as enzyme promiscuity and is of great importance for the evolution of new functions. The promiscuities of two enzymes, namely glycerol dehydratase and diol dehydratase, were herein exploited for catalyzing long-chain polyols , including 1,2-butanediol, 1,2,4-butanetriol, erythritol , 1,2-pentanediol, 1,2,5-pentanetriol, and 1,2,6-hexanetriol. The specific activities required for catalyzing these six long-chain polyols were studied via in vitro enzyme assays , and the catalytic efficiencies were increased through protein engineering. The promiscuous functions were subsequently applied in vivo to establish 1,4-butanediol pathways from lignocellulose derived compounds, including xylose and erythritol. In addition, a pathway for 1-pentanol production from 1,2-pentanediol was also constructed. The results suggest that exploiting enzyme promiscuity is promising for exploring new catalysts, which would expand the repertoire of genetic elements available to synthetic biology and may provide a starting point for designing and engineering novel pathways for valuable chemicals.
研究领域:
生物化学
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合成生物学
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DOI
PubMed
高影响因子
PROTEIN SCIENCE
·
2024
·Vol. 33
·期 10
·e5160
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IF 4.5
Shuai Xu,Zong-Lin Li,Zhi-Min Li,Hong-Lai Liu
L-cysteine is an essential component in pharmaceutical and agricultural industries, and synthetic biology has made strides in developing new metabolic pathways for its production, particularly in archaea with unique O-phosphoserine sulfhydrylases (OPSS) as key enzymes. In this study, we employed database mining to identify a highly catalytic activity OPSS from Acetobacterium sp. (AsOPSS). However, it was observed that the enzymatic activity of AsOPSS suffered significant feedback inhibition from the product L-cysteine, exhibiting an IC 50 value of merely 1.2 mM. A semi-rational design combined with tunnel analysis strategy was conducted to engineer AsOPSS. The best variant, AsOPSS A218R was achieved, totally eliminating product inhibition without sacrificing catalytic efficiency. Molecular docking and molecular dynamic simulations indicated that the binding conformation of AsOPSS A218R with L-cys was altered, leading to a reduced affinity between L-cysteine and the active pocket. Tunnel analysis revealed that the AsOPSS A218R variant reshaped the landscape of the tunnel, resulting in the construction of a new tunnel. Furthermore, random acceleration molecular dynamics simulation and umbrella sampling simulation demonstrated that the novel tunnel improved the suitability for product release and effectively separated the interference between the product release and substrate binding processes. Finally, more than 45 mM of L-cysteine was produced in vitro within 2 h using the AsOPSS A218R variant. Our findings emphasize the potential for relieving feedback inhibition by artificially generating new product release channels, while also laying an enzymatic foundation for efficient L-cysteine production.
研究领域:
生物化学
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酶学
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合成生物学
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DOI
PubMed
高影响因子
Nanoscale Advances
·
2023
·Vol. 5
·期 23
·6606-6616
·
IF 4.7
Taotao Feng,Jiaxu Liu,Xiaoyan Zhang,Daidi Fan,Yunpeng Bai
In the past decade, virus-like particles (VLPs) that can encapsulate single or multiple enzymes have been studied extensively as typical nanoreactors for biocatalysis in vitro, yet their catalytic efficiencies are usually inadequate for real applications. These biocatalytic nanoreactors should be engineered like their free-enzyme counterparts to improve their catalytic performance for potential applications. Herein we engineer biocatalytic VLPs for the enhanced synthesis of chiral alcohols. Different methods including directed evolution were applied to the entire bacteriophage P22 VLPs (except the coat protein), which encapsulated a carbonyl reductase from Scheffersomyces stipitis (SsCR) and a glucose dehydrogenase from Bacillus megaterium (BmGDH) in their capsids. The best variant, namely M5, showed an enhanced turnover frequency (TOF, min−1) up to 15-fold toward the majority of tested aromatic prochiral ketones, and gave up to 99% enantiomeric excess in the synthesis of chiral alcohol pharmaceutical intermediates. A comparison with the mutations of the free-enzyme counterparts showed that the same amino acid mutations led to different changes in the catalytic efficiencies of free and confined enzymes. Finally, the engineered M5 nanoreactor showed improved efficiency in the scale-up synthesis of chiral alcohols. The conversions of three substrates catalyzed by M5 were all higher than those catalyzed by the wild-type nanoreactor, demonstrating that enzyme-encapsulating VLPs can evolve to enhance their catalytic performance for potential applications.
研究领域:
生物化学
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合成生物学
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酶学
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DOI
PubMed
高影响因子
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