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1.
Engineering microbial consortia to express complex biosynthetic pathways efficiently for the production of valuable compounds is a promising approach for metabolic engineering and synthetic biology. Here, we report the design, optimization, and scale-up of an Escherichia coli-E. coli coculture that successfully overcomes fundamental microbial production limitations, such as high-level intermediate secretion and low-efficiency sugar mixture utilization. For the production of the important chemical cis,cis-muconic acid, we show that the coculture approach achieves a production yield of 0.35 g/g from a glucose/xylose mixture, which is significantly higher than reported in previous reports. By efficiently producing another compound, 4-hydroxybenzoic acid, we also demonstrate that the approach is generally applicable for biosynthesis of other important industrial products.Metabolic engineering and synthetic biology have made great strides in constructing and optimizing metabolic pathways for biochemical product synthesis in a pure culture (1, 2). There are, however, situations where this approach may be limited, as in the cases where (i) a single host cell cannot provide an optimal environment for the functioning of all pathway enzymes, (ii) biosynthetic efficiency is reduced due to overwhelming metabolic stress from the overexpression of long and complex pathways (3, 4), or (iii) pathway intermediates are secreted yielding undesired byproducts and reducing substrate utilization (5, 6). The above limitations can potentially be overcome through the use of rationally designed microbial coculture systems. Different from previous modular engineering approaches, such coculture-based systems completely modularize and segregate a biosynthetic pathway into two separate cells, each of which carries a portion of the pathway, and thus can be engineered independently to achieve optimal functioning of the combined pathway.The concept of microbial cocultures is not new. However, previous research on microbial consortia was primarily concerned with the study of mixed population stability and dynamic interactions (711), although a few recent studies have reported the engineering of microbial consortia for utilization of simple sugars to make small molecules of central carbon metabolism, such as ethanol and lactate (12, 13). Progress was made recently, when a full n-butanol pathway was expressed in two separate E. coli cells to achieve higher production (14) and when a bacterium-yeast coculture was used to address the difficulties of functional reconstitution of a pathway involving prokaryotic and eukaryotic enzymes in a consortium, and thus improved production of complex pharmaceutical molecules (15). Here, we expand the generality of the coculture engineering by demonstrating that microbial cocultures can also be engineered to overcome more universal challenges in metabolic engineering, including high-level intermediate secretion and low-efficiency sugar mixture utilization. In particular, we show that without the employment of additional microbial species, two cells of the same species can be engineered to form an integrated system for accomplishing biosynthesis that is difficult to achieve by a single cell. The use of one microbial species may increase culture stability compared with multispecies consortia, and represents a promising and relatively unexplored approach in the context of metabolic engineering and synthetic biology. As a proof of concept, we show that a stable coculture system can be established to accommodate the requirements of the complex pathway of the synthesis of commodity chemical cis,cis-muconic acid (MA) optimally and result in high-level production from sugar mixtures that can be derived from renewable lignocellulose at an industrially relevant yield.The cis,cis-MA is an important compound for making a variety of high-demand bulk chemicals, including nylon- and polyurethane-precursor adipic acid and terephthalic acid, a monomer of the plastic polymer polyethylene terephthalate (16). Current industrial production of adipic acid and terephthalic acid relies exclusively on petroleum and coal feedstocks. Production methods using renewable resources, such as lignocellulose, are potentially attractive, provided that they can be cost-effective. However, despite progress in microbial MA production, previous studies have suffered from low yields and titers and high accumulation of intermediate metabolites (1722).  相似文献   
2.
骨髓间充质干细胞旁分泌HGF体外调控肝星状细胞   总被引:2,自引:0,他引:2  
目的 探讨大鼠骨髓间充质干细胞(BMSCs)与肝星状细胞(HSCs)体外共培养体系中,BMSCs旁分泌肝细 胞生长因子(HGF)对 HSCs 增殖、凋亡、活化的影响。方法 全骨髓贴壁法分离、培养、纯化大鼠 BMSCs,另培养 HSCs。6 孔板半透膜建立上下两层细胞非直接接触共培养体系,实验设 H 组(HSCs 单独培养)、H-H 组(HSCs 与 HSCs共培养)、M-H组(BMSCs与HSCs共培养)、M-H-C组(BMSCs与HSCs共培养并加c-met抑制剂),各组细胞培 养48 h后,流式细胞仪鉴定BMSCs,检测HSCs凋亡率,MTT法检测HSCs的增殖,免疫荧光共聚焦定量检测HSCs中 α-肌动蛋白(α-SMA)的表达量,ELISA法检测共培养体系上清液中HGF的浓度。结果 MSCs高表达阳性表面分子 CD29、CD90,低表达造血细胞表面标记CD45;BMSCs能明显抑制HSCs的增殖、活化并促进其凋亡,且M-H组上清液 中HGF的浓度明显高于其他组。结论 BMSCs与HSCs共培养过程中,BMSCs通过旁分泌HGF促进HSCs的凋亡,抑 制HSCs的增殖、活化。  相似文献   
3.
PurposeLimbal niche cells (LNCs) play a vital role in the maintenance of limbal epithelial stem/progenitor cells (LESCs). Four methods have been reported to isolate and expand LNCs: digestion by collagenase alone (C-LNC), collagenase following dispase removal of the limbal epithelium (DC-LNC), dissection of dispase-isolated limbal epithelial sheets (D-LNC), and explant cultures of limbal stromal tissues (Ex-LNC). This study aimed to isolate LNCs using those four methods and to compare their capacity to maintain LESCs.MethodsLNCs were isolated from the rat corneal limbus by the following methods: C-LNC, DC-LNC, D-LNC, and Ex-LNC. Quantitative real-time PCR and immunofluorescence staining were used to analyze the expression of embryonic stem cell (ESC) markers. The ability to maintain LESCs was assessed on the basis of colony-forming capacity and the expression of progenitor, proliferation, and differentiation markers in three-dimensional (3D) Matrigel and Transwell systems. Notch signaling of LESCs supported by different LNCs in Transwell inserts was analyzed by quantitative real-time PCR.ResultsDC-LNCs exhibited lower expression of CK12 during isolation and expansion. Among P4-expanded LNCs, DC-LNCs expressed significantly higher levels of Sox2, Oct4, Nanog, and N-cadherin than C-LNCs, D-LNCs, and Ex-LNCs. Compared with other LNCs, DC-LNCs were more effective in maintaining LESCs with higher holoclone-forming efficiency, greater expression of ΔNp63α and Ki67, and lower expression of CK12. DC-LNCs were also more capable of downregulating Notch signaling of LESCs.ConclusionsDC-LNCs were more effective in expressing ESC markers and maintaining LESCs compared to other LNCs. This study identifies an optimal method for the isolation of LNCs in tissue engineering and ocular surface reconstruction.  相似文献   
4.
目的探究高职护生科学素养与人文精神融合培养模式在教学中的应用效果。方法选择本校2011级高职护生为对照组,采用传统的教学方法;2012级高职护生为实验组,入学即采用科学素养与人文精神融合教学模式。实施1年后,通过问卷调查及考试成绩,以评价融合模式的实施效果。结果观察组对科学素养与人文精神的认知、态度、行为及教学效果和学习成绩明显优于对照组(P0.05);90%以上护生认为护理知识、批判能力、执业资格证等科学素养比较重要,89%以上的护生认为人际关系、沟通能力、心理素质等人文精神也比较重要,显示护生对科学与人文重要性认识基本相当;83%~91%的护生认为融合模式有利于培养批判思维能力、创新意识、提高学习兴趣及学习效果等科学素养;85%~93%的护生认为融合模式有利于培养吃苦精神、礼仪礼节和职业道德等人文精神。结论融合教学模式克服了传统教学中专业与人文脱节的弊端,符合现代护理教育观;使护生科学素养与人文精神得到提升;有助于提高护生综合素质。  相似文献   
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背景:软骨细胞通过自分泌及旁分泌的作用可以为滑膜间充质干细胞向软骨细胞分化提供所需的生长因子及微环境,三维条件下更有利于细胞的黏附增殖与分化。目的:观察滑膜间充质干细胞与软骨细胞混合培养于壳聚糖/Ⅰ型胶原复合支架材料中向成软骨细胞分化的能力。方法:取SD大鼠滑膜组织及软骨组织,用酶消化法获得滑膜间充质干细胞及软骨细胞分别进行培养。取第3代滑膜间充质干细胞及第2代软骨细胞,将二者以1∶2的比例混合培养负载于壳聚糖/Ⅰ型胶原复合支架材料21 d,进行激光共聚焦扫描及免疫组织化学检测。结果与结论:培养72 h后,扫描电镜观察细胞黏附于支架材料表面,并可见细胞分泌大量基质成分。培养21 d后,激光共聚焦扫描可见细胞在支架表面分布均匀,逐层扫描后细胞逐渐减少。免疫组织化学检测可见基质能被Ⅱ型胶原染色,细胞染色呈现棕黄色。结果表明壳聚糖/Ⅰ型胶原复合支架材料提供三维生长空间,利用软骨细胞分泌生长因子及细胞间的相互作用可以诱导滑膜间充质干细胞向软骨细胞分化。  相似文献   
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AIM: To determine the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells (hUCMSCs) on the expression of vascular endothelial growth factor A (VEGF-A) in human retinal vascular endothelial cells (HRECs). METHODS: Exosomes were isolated from hUCMSCs using cryogenic ultracentrifugation and characterized by transmission electron microscopy, Western blotting and nanoparticle tracking analysis. HRECs were randomly divided into a normal control group (group A), a high glucose model group (group B), a high glucose group with 25 μg/mL (group C), 50 μg/mL (group D), and 100 μg/mL exosomes (group E). Twenty-four hours after coculture, the cell proliferation rate was detected using flow cytometry, and the VEGF-A level was detected using immunofluorescence. After coculture 8, 16, and 24h, the expression levels of VEGF-A in each group were detected using PCR and Western blots. RESULTS: The characteristic morphology (membrane structured vesicles) and size (diameter between 50 and 200 nm) were observed under transmission electron microscopy. The average diameter of 122.7 nm was discovered by nanoparticle tracking analysis (NTA). The exosomal markers CD9, CD63, and HSP70 were strongly detected. The proliferation rate of the cells in group B increased after 24h of coculture. Immunofluorescence analyses revealed that the upregulation of VEGF-A expression in HRECs stimulated by high glucose could be downregulated by cocultured hUCMSC-derived exosomes (F=39.03, P<0.01). The upregulation of VEGF-A protein (group C: F=7.96; group D: F=17.29; group E: F=11.89; 8h: F=9.45; 16h: F=12.86; 24h: F=42.28, P<0.05) and mRNA (group C: F=4.137; group D: F=13.64; group E: F=22.19; 8h: F=7.253; 16h: F=16.98; 24h: F=22.62, P<0.05) in HRECs stimulated by high glucose was downregulated by cocultured hUCMSC-derived exosomes (P<0.05). CONCLUSION: hUCMSC-derived exosomes downregulate VEGF-A expression in HRECs stimulated by high glucose in time and concentration dependent manner.  相似文献   
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