Biomolecules
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2024
·Vol. 14
·期 12
·1553
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IF 4.8
Zifu Ni,Linshang Zhang,Azhen Nie,Huan Wang,Xiaoling Wu
As the only naturally occurring β-amino acid, β-alanine has important application prospects in many fields. Driven by the huge demand, biosynthesis is becoming more and more popular as a potential alternative to the chemical synthesis of β-alanine. Although the direct pathway from L-aspartic acid to β-alanine, catalyzed by L-aspartic acid-α-decarboxylase (PanD), is ideal for β-alanine synthesis, it is hindered by the high cost of the substrate and limited economic viability. In this work, a cell-free dual enzyme cascade system based on methylaspartate lyase (EcMAL) and panD was constructed to safely and efficiently synthesize β-alanine using fumarate as a substrate. Taking the previously engineered EcMAL as the target, CgPanD was finally screened as the best candidate through gene mining, sequence alignment, and enzyme property analysis. Finally, under the optimal conditions of 35 °C, pH 8.0, and EcMAL: CgPanD concentration ratio of 1:5, the yield of β-alanine reached 80% theoretical yield within 120 min. This study provides a potential strategy for the biosynthesis of β-alanine, paving the way for future industrial-scale production.
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
BIOMACROMOLECULES
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2025
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IF 5.4
Xinghan Wu,Wenxin Xu,Xuantao Huang,Nianhua Dan,Yining Chen,Zhengjun Li,Yunbing Wang
Emergency repair of complicated full-thickness abdominal wall defects remains one of the most common and challenging surgical emergencies globally. Here, an integrated polydopamine permeating-cross-linking strategy was innovatively proposed to convert porcine acellular dermal matrix (pADM) into versatile, degradation-resistant biopatches (PDA-pADM) for efficiently repairing full-thickness abdominal wall defects. The strategy significantly addresses the challenge that natural-property improvement and biocompatibility of biomaterials are difficult to balance. Molecularly, dopamine (DA) molecules could fully permeate into the collagen fibers of the acellular dermal matrix and then automatically trigger the interfacial in situ polymerization of dopamine monomers among collagen fibers to achieve the efficient cross-linking of pADM. Surprisingly, the enzymatic durability of the biopatch shows significant improvements, extending the original duration from 3 to 20 d. Comprehensive in vivo experiments have shown that PDA-pADM can effectively promote angiogenesis and inhibit inflammatory response, so as to achieve regeneration and repair of abdominal wall damage.
研究领域:
生物医学工程
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组织工程
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外科手术
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产品关联
DOI
PubMed
高影响因子
Frontiers in Bioengineering and Biotechnology
·
2021
·
IF 5.89
Yang Chen,Liu Yushi,Liu Wan-Qiu,Wu Changzhu,Li Jian
Cell-free systems have been used to synthesize chemicals by reconstitution of in vitro expressed enzymes. However, coexpression of multiple enzymes to reconstitute long enzymatic pathways is often problematic due to resource limitation/competition (e.g., energy) in the one-pot cell-free reactions. To address this limitation, here we aim to design a modular, cell-free platform to construct long biosynthetic pathways for tunable synthesis of value-added aromatic compounds, using (S)-1-phenyl-1,2-ethanediol ((S)-PED) and 2-phenylethanol (2-PE) as models. Initially, all enzymes involved in the biosynthetic pathways were individually expressed by an E. coli-based cell-free protein synthesis (CFPS) system and their catalytic activities were confirmed. Then, three sets of enzymes were coexpressed in three cell-free modules and each with the ability to complete a partial pathway. Finally, the full biosynthetic pathways were reconstituted by mixing two related modules to synthesize (S)-PED and 2-PE, respectively. After optimization, the final conversion rates for (S)-PED and 2-PE reached 100 and 82.5%, respectively, based on the starting substrate of l-phenylalanine. We anticipate that the modular cell-free approach will make a possible efficient and high-yielding biosynthesis of value-added chemicals.
研究领域:
合成生物学
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代谢工程
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生物技术
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DOI
PubMed
高影响因子
BMC BIOTECHNOLOGY
·
2023
·Vol. 23
·期 1
·1-9
·
IF 3.5
Jiang Huiming,Lu Jun,Li Jiawei,Liu Zizheng,Chen Fufei,Wu Rui,Xu Xingquan,Liu Yuan,Jiang Yiqiu,Shi Dongquan
Background Cartilage defects are common sports injuries without significant treatment. Articular cartilage with inferior regenerative potential resulted in the poor formation of hyaline cartilage in defects. Acellular matrix scaffolds provide a microenvironment and biochemical properties similar to those of native tissues and are widely used for tissue regeneration. Therefore, we aimed to design a novel acellular cartilage matrix scaffold (ACS) for cartilage regeneration and hyaline-like cartilage formation. Methods Four types of cartilage injury models, including full-thickness cartilage defects (6.5 and 8.5 mm in diameter and 2.5 mm in depth) and osteochondral defects (6.5 and 8.5 mm in diameter and 5 mm in depth), were constructed in the trochlear groove of the right femurs of pigs (n = 32, female, 25–40 kg). The pigs were divided into 8 groups (4 in each group) based on post-surgery treatment differences. was assessed by macroscopic appearance, magnetic resonance imaging (MRI), micro–computed tomography (micro-CT), and histologic and immunohistochemistry tests. Results At 6 months, the ACS-implanted group exhibited better defect filling and a greater number of chondrocyte-like cells in the defect area than the blank groups. MRI and micro-CT imaging evaluations revealed that ACS implantation was an effective treatment for cartilage regeneration. The immunohistochemistry results suggested that more hyaline-like cartilage was generated in the defects of the ACS-implanted group. Conclusions ACS implantation promoted cartilage repair in full-thickness cartilage defects and osteochondral defects with increased hyaline-like cartilage formation at the 6-month follow-up.
研究领域:
骨科
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组织工程
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再生医学
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DOI
PubMed
高影响因子
Metabolic Engineering Communications
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2022
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IF 0
Yushi Liu,Wan-Qiu Liu,Shuhui Huang,Huiling Xu,Haofan Lu,Changzhu Wu,Jian Li
Fatty acid-derived products such as alkanes, fatty aldehydes, and fatty alcohols have many applications in the chemical industry. These products are predominately produced from fossil resources, but their production processes are often not environmentally friendly. While microbes like Escherichia coli have been engineered to convert fatty acids to corresponding products, the design and optimization of metabolic pathways in cells for high productivity is challenging due to low mass transfer, heavy metabolic burden, and intermediate/product toxicity. Here, we describe an E. coli -based cell-free protein synthesis (CFPS) platform for in vitro conversion of long-chain fatty acids to value-added chemicals with product selectivity, which can also avoid the above issues when using microbial production systems. We achieve the selective biotransformation by cell-free expression of different enzymes and the use of different conditions (e.g., light and heating) to drive the biocatalysis toward different final products. Specifically, in response to blue light, cell-free expressed fatty acid photodecarboxylase (CvFAP, a photoenzyme) was able to convert fatty acids to alkanes with approximately 90% conversion. When the expressed enzyme was switched to carboxylic acid reductase (CAR), fatty acids were reduced to corresponding fatty aldehydes, which, however, could be further reduced to fatty alcohols by endogenous reductases in the cell-free system. By using a thermostable CAR and a heating treatment, the endogenous reductases were deactivated and fatty aldehydes could be selectively accumulated (>97% in the product mixture) without over-reduction to alcohols. Overall, our cell-free platform provides a new strategy to convert fatty acids to valuable chemicals with notable properties of operation flexibility, reaction controllability, and product selectivity.
研究领域:
代谢工程
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生物技术
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化学工程
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生物化学
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DOI
PubMed
ANALYTICAL BIOCHEMISTRY
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2025
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IF 2.5
Jiaxu Wang,Zhengrong Lu,Zhanmin Liu,Qiming Chen
Copy number of cell-free mitochondrial DNA (cf-mtDNA) has garnered significant attention as a biomarker for studying and diagnosing various diseases. However, Quantitative Real-time PCR (qPCR) and Droplet Digital PCR (ddPCR) assays for cf-mtDNA copy number detection require expensive equipment and high experiment conditions. In this study, a colorimetric and fluorescent dual-modality assay was developed for quantitative detection of cf-mtDNA copy number. With G-quadruplex (G4) sequence modified primers, the assay could quantitatively detect cf-mtDNA with spectrophotometry, RGB (Red, Green, Blue) visual method and fluorescence method, which made the application scenarios more diverse. The specificity of dual-mode method was better, and the detection limits of spectrophotometry, RGB visual method and fluorescence method were as low as 1.45 × 10 −1 copies/μL, 1.65 copies/μL and 1.58 × 10 −1 copies/μL, respectively. Compared with qPCR and ddPCR assays developed in previous studies, the dual-modality assay in this study had a lower detection limit. It was also independent of expensive qPCR and ddPCR equipment and the detection cost was low. Therefore, the colorimetric and fluorescent dual-modality assay represent a label-free and sensitive approach for assessing cf-mtDNA levels, offering promising implications for biomedical research and clinical diagnostics.
研究领域:
医学诊断
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分子生物学
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DOI
PubMed
高影响因子
ACS Applied Materials & Interfaces
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2021
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IF 9.229
Yuling Sun,Jie Liu,Zhibo Li,Jianjun Wang,Yanbin Huang
Water-soluble, biodegradable, nonionic, and biocompatible polymers with multiple functional groups are highly desired for biomedical applications. Here, we report that water-soluble nonionic poly(d/l-serine) is chirality-controllable, biodegradation-controllable, and non-cytotoxic. Hence, it can be a highly sought-after alternative to the widely used poly(ethylene glycol), with an additional advantage of having multiple hydroxyl groups for further functionalization. As one example of its biomedical applications, poly(d/l-serine) demonstrated an obvious cryoprotective effect on the red blood cells. The usage of poly(d/l-serine) in the cryopreservation field would be of great promise to resolve the difficulties in separating cryoprotectants due to toxicity.
研究领域:
生物材料学
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医学工程
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细胞生物学
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2024
·Vol. 91
·期 1
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IF 3.9
Ji Zeng,Hao Wang,Yuxi Xu,Jianying Han,Yannan Li,Shu'an Wen,Changbu Wu,Dani Li,Zheng Liu,Xiaokang Zhang,Guo-Bao Tian,Min Dong
Clostridioides difficile is an obligate anaerobic, Gram-positive bacterium that produces toxins. Despite technological progress, conducting gene expression analysis of C. difficile under different conditions continues to be labor-intensive. Therefore, there is a demand for simplified tools to investigate the transcriptional and translational regulation of C. difficile. The cell-free gene expression (CFE) system has demonstrated utility in various applications, including prototyping, protein production, and in vitro screening. In this study, we developed a C. difficile CFE system capable of in vitro transcription and translation (TX-TL) in the presence of oxygen. Through optimization of cell extract preparation and reaction systems, we increased the protein yield significantly. Furthermore, our observations indicated that this system exhibited higher protein yield using linear DNA templates than circular plasmids for in vitro expression. The prototyping capability of the C. difficile CFE system was assessed using a series of synthetic Clostridium promoters, demonstrating a good correlation between in vivo and in vitro expression. Additionally, we tested the expression of tcdB and tcdR from clinically relevant C. difficile strains using the CFE system, confirming higher toxin expression of the hypervirulent strain R20291. We believe that the CFE system can not only serve as a platform for in vitro protein synthesis and genetic part prototyping but also has the potential to be a simplified model for studying metabolic regulations in Clostridioides difficile.
研究领域:
微生物学
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分子生物学
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生物技术
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DOI
高影响因子
ACS Sustainable Chemistry & Engineering
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2024
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IF 7.1
Yicong Lu,Xiao Zheng,Wan-Qiu Liu,Shengjie Ling,Jian Li
Cell-free protein synthesis (CFPS) has proven invaluable for expressing a wide array of proteins and enzymes, boasting significant advantages, such as facile manipulation, rapid mass transfer, and high productivity. However, traditional cell-free expression systems typically operate in batch format reactions, posing challenges for enzyme reuse in biocatalysis and rendering supplemented gene templates nonrecyclable. To overcome this limitation, we introduce a chitin-functionalized CFPS system designed to immobilize in vitro-expressed enzymes and gene templates for sustainable biocatalysis and protein synthesis. At its core, this system capitalizes on the strong binding affinity between a chitin-binding domain (ChBD) and crystalline chitin nanofibers (ChNFs). Specifically, we engineer ChBD fusion with target proteins and enzymes, allowing for their cell-free expression and in situ immobilization on ChNFs. This facilitates the effortless recycling of enzymes for multiple biocatalytic reactions, while ChNFs-immobilized enzymes can also be deployed in continuous flow biocatalysis setups. Leveraging the ChBD-ChNF pairing, gene templates can likewise be immobilized and recycled for sustained gene expression. Our results demonstrate that the chitin-functionalized cell-free system significantly enhances CFPS performance through immobilization with ChNF materials. This work underscores the flexibility and robustness of cell-free systems, which can seamlessly integrate with advanced techniques from fields such as chemistry and materials science for impactful applications.
研究领域:
生物化学
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合成生物学
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材料科学
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产品关联
DOI
高影响因子
CHEMICAL ENGINEERING JOURNAL
·
2020
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IF 10.652
Huachu Wu,Liang Shen,Zhu Zhu,Xusong Luo,Yishu Zhai,Xiaobin Hua,Shicheng Zhao,Lian Cen,Zhibing Zhang
This study was to co-encapsulate a chemokine (stromal cell-derived factor-1, SDF-1) and a chondroinductive molecule (kartogenin, KGN) within microspheres via microfluidics, and to incorporate them into a hyaluronic acid (HA) injectable scaffold for articular cartilage defect repair. HA injectable scaffold, as a cartilage-friendly microenvironment, was prepared by crosslinking HA with 1,4-butanediol diglycidyl ether. A microfluidic device was set up to prepare monodisperse PLGA microspheres (49 μm) to load SDF-1 and KGN. An in vivo model of full-thickness articular cartilage defects in rabbits was applied to evaluate the reparative capacity of the current package. The SDF-1 and KGN were co-encapsulated simultaneously within the core and shell area of the microsphere with high loading efficiency and sustained release profiles of more than 2 months. The release profiles of them were highly matched and well fitted to a first-order mathematical model. These microspheres when incorporated into HA injectable scaffold were demonstrated to heal the full-thickness articular cartilage defects in rabbits. The regenerated tissue had the typical cartilage histological characters and integrated well with the surrounding tissue at 12w. This developed cell-free system could serve as an efficient therapy for articular cartilage defects treatment, serving as a supplementary way to cell based therapies.
研究领域:
生物医学工程
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组织工程
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骨科
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DOI
高影响因子
Materials
·
2023
·Vol. 16
·期 5
·1937
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IF 3.4
Zhilin Jia,Hailin Ma,Jiaqi Liu,Xinyu Yan,Tianqing Liu,Yuen Yee Cheng,Xiangqin Li,Shuo Wu,Jingying Zhang,Kedong Song
Bone tissue engineering is a novel and efficient repair method for bone tissue defects, and the key step of the bone tissue engineering repair strategy is to prepare non-toxic, metabolizable, biocompatible, bone-induced tissue engineering scaffolds of suitable mechanical strength. Human acellular amniotic membrane (HAAM) is mainly composed of collagen and mucopolysaccharide; it has a natural three-dimensional structure and no immunogenicity. In this study, a polylactic acid (PLA)/Hydroxyapatite (nHAp)/Human acellular amniotic membrane (HAAM) composite scaffold was prepared and the porosity, water absorption and elastic modulus of the composite scaffold were characterized. After that, the cell–scaffold composite was constructed using newborn Sprague Dawley (SD) rat osteoblasts to characterize the biological properties of the composite. In conclusion, the scaffolds have a composite structure of large and small holes with a large pore diameter of 200 μm and a small pore diameter of 30 μm. After adding HAAM, the contact angle of the composite decreases to 38.7°, and the water absorption reaches 249.7%. The addition of nHAp can improve the scaffold’s mechanical strength. The degradation rate of the PLA+nHAp+HAAM group was the highest, reaching 39.48% after 12 weeks. Fluorescence staining showed that the cells were evenly distributed and had good activity on the composite scaffold; the PLA+nHAp+HAAM scaffold has the highest cell viability. The adhesion rate to HAAM was the highest, and the addition of nHAp and HAAM could promote the rapid adhesion of cells to scaffolds. The addition of HAAM and nHAp can significantly promote the secretion of ALP. Therefore, the PLA/nHAp/HAAM composite scaffold can support the adhesion, proliferation and differentiation of osteoblasts in vitro which provide sufficient space for cell proliferation, and is suitable for the formation and development of solid bone tissue.Keywords:human acellular amniotic membrane;acellular process;osteoblasts;polylactic acid;hydroxyapatite
研究领域:
生物医学工程
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组织工程
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材料科学
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产品关联
DOI
PubMed
高影响因子
Food Bioscience
·
2024
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IF 4.8
Qiang Wang,Feng Zhang,Yuhong Zhang,Yu Zhang,Hongwei Wang,Jiajia Song,Huayi Suo
This study investigated the antifungal effect, mechanism, and potential application in yogurt of Lactiplantibacillus plantarum ( L. plantarum ) LPP95 isolated from Chinese pickles. The L. plantarum LPP95 showed a high inhibitory effect on the growth of five molds , with the strongest inhibitory effect observed on Penicillium sp. The cell-free supernatants (CFS) of L. plantarum LPP95 resulted in the shrinking of Penicillium sp. spores , and destroyed the integrity of the cell wall and membrane of spores, causing the leakage of nucleic acid from cells. The antifungal activity of CFS was not decreased after heat or proteolytic enzyme treatment but was affected by pH change. The inhibitory activity of CFS was associated with organic acids, and 66 organic acids were identified by LC-MS analysis. In addition, the validation experiments showed that trans -cinnamic acid, citric acid , trans -3-indoleacrylic acid, and DL-4-hydroxyphenyllactic acid all exhibited antifungal activity. Moreover, L. plantarum LPP95 effectively inhibited the growth of Penicillium sp. in yogurt. Overall, L. plantarum LPP95 and its CFS have the potential as biological preservatives.
研究领域:
微生物学
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食品科学
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生物技术
✓ 已验证
产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）