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[前沿资讯 ] 天津工业生物技术研究所通过电击诱变和代谢工程改造创制高产虾青素的裂殖壶菌细胞工厂 进入全文
中国科学院天津工业生物技术研究所
虾青素(astaxanthin)是一种强效抗氧化剂,在医药、保健品、化妆品等领域具有广泛的应用。在虾青素单酯的各种分子构型中,DHA与虾青素形成的单酯具有最高的生物利用度和稳定性。裂殖壶菌(Schizochytrium sp)因其高油脂含量及产量,并含有EPA、DHA等多不饱和脂肪酸,以及少量角鲨烯、虾青素等类胡萝卜素而受到广泛关注。然而天然的裂殖壶菌虾青素产量极低,无法满足工业化需求。 近日,中国科学院天津工业生物技术研究所李德茂研究员带领的工业生物系统工程研究团队成功构建出一株高产虾青素的裂殖壶菌细胞工厂。研究团队通过电穿孔诱变技术强化MVA途径前体供应,成功构建Schizochytrium sp. AST32底盘菌株,使虾青素含量从7.56μg/g显著提升至55.17μg/g。基于该突变株,通过理性设计策略进一步优化虾青素生物合成途径,在Schizochytrium sp. AST32中同时表达五个关键基因(idi、crtE、crtIBY、crtZ、crtW),使虾青素含量跃升至374.85μg/g。进一步,应用PLIN融合蛋白技术将虾青素合成模块定向锚定于细胞脂滴,使虾青素产量提升至400.38μg/g。经发酵工艺优化后,最终获得虾青素产量峰值29.53mg/L(1913.07μg/g,较初始突变株提升169.72倍),同时维持DHA产量3.83g/L(占总脂质含量的45%)。该研究为推进裂殖壶菌虾青素生物合成的商业化应用奠定了关键技术基础。
[前沿资讯 ] 一种用于新月柄杆菌高效快速基因组编辑的CRISPR/SpCas9M报告系统 进入全文
中国科学院深圳先进技术研究院
近日,中国科学院深圳先进技术研究院合成生物学研究所赵国屏院士,赵维研究员团队联合上海交通大学在Nucleic Acids Research上发表题为“A CRISPR/SpCas9M-reporting system for efficient and rapid genome editing in Caulobacter crescentus”的方法论文,针对上述问题,开发了一套基于CRISPR/SpCas9M的基因编辑报告系统,实现了对新月柄杆菌高效、快速且无痕的基因编辑。该项工作不仅为新月柄杆菌的遗传操作提供了强有力的工具,也为解决其它难以进行遗传操作的非模式菌提供了新的技术思路和方法学参考。 研究团队最初构建了一个基于同源重组(HR)的CRISPR/Cas系统。将Streptococcus pyogenes来源的SpCas9、sgRNA和同源臂(H-arms)克隆至携带pBBR1复制子的复制型质粒pBXMCS2中。为了实现无痕编辑,在同源臂之间未设计任何抗生素抗性基因。然而,将该编辑质粒电转化至新月柄杆菌后,发现获得极少甚至无菌落,并且存活菌株的靶位点均未发生预期编辑。这表明CRISPR/SpCas9的切割具有致死性,而该菌细胞内的同源重组(HR)效率又相对较低。 为实现有效的靶向基因编辑,研究团队对编辑质粒进行了系统性分析,并围绕三个关键方向进行了优化。首先,研究人员筛选了来自不同物种的Cas蛋白,包括Streptococcus pyogenes Cas9 (SpCas9)、Francisella novicida Cas12a (FnCas12a), Streptococcus thermophilus CRISPR1-Cas9 (Sth1Cas9)和Streptococcus thermophilus CRISPR3-Cas9 (Sth3Cas9),以评估它们在新月柄杆菌中的编辑效率,结果发现上述所有Cas蛋白均未检测到编辑克隆。然而,当根据新月柄杆菌的密码子偏好性优化SpCas9编码序列(命名为SpCas9M)后,基因编辑效率可达到15%左右。其次,研究团队通过调控SpCas9M的表达水平来提升编辑效率。研究人员测试了两种诱导型启动子,香草酸诱导型启动子(Pvan)和木糖诱导型启动子(Pxyl),检测不同诱导剂浓度下的基因编辑效率。结果发现相对较低的SpCas9M表达水平更有利于新月柄杆菌的基因组编辑,编辑效率最高可达到40%左右。最后,通过分析未发生基因组编辑的克隆中的编辑质粒,研究人员惊奇的发现这些克隆的SpCas9M编码序列均存在缺失。这一现象证实了CRISPR/SpCas9系统的致死性,并表明新月柄杆菌中存在着强烈的选择性压力。基于此,研究人员提出假设:通过预先识别并排除这些SpCas9M突变体,可有效提升在新月柄杆菌中的表观编辑效率。为此,研究人员在SpCas9M的C端融合了超折叠绿色荧光蛋白(sfGFP)作为报告系统,通过荧光信号指示,在菌落PCR前筛选并排除SpCas9M异常表达的克隆。最后,总体实现编辑效率达到80%左右,研究团队将其命名为CRISPR/SpCas9M-报告系统。
[学术文献 ] De novo design of porphyrin-containing proteins as efficient and stereoselective catalysts 进入全文
Science
De novo design of protein catalysts with high efficiency and stereoselectivity provides an attractive approach toward the design of environmentally benign catalysts. Here, we design proteins that incorporate histidine-ligated synthetic porphyrin and heme ligands. Four of 10 designed proteins catalyzed cyclopropanation with an enantiomeric ratio greater than 99:1. A second class of proteins were designed to catalyze a silicon-hydrogen insertion and were optimized by directed evolution in whole cells. The evolved proteins incorporated features unlikely to be generated by computational design alone, including a proline in an α helix. Molecular dynamics simulations showed that as the proteins evolved toward higher activity, their conformational ensembles narrowed to favor more productive conformations. Our work demonstrates that efficient de novo protein catalysts are designable and should be useful for manifold chemical processes.
[前沿资讯 ] 深圳先进院合作构建“微生物特种兵”,可同时降解5种污染物 进入全文
中国科学院深圳先进技术研究院
近年来,合成生物学技术飞速发展为降解菌株的构建提供了可能。科学家们能够通过合成生物技术给微生物设计“智能工具箱”——不仅能给细菌安装多种污染物分解能力,还能让这些功能像“乐高积木”一样精准搭配。基于此,研究团队通过底盘菌株筛选与耐盐机制解析,精准锁定了具有最快繁殖速率、高盐耐受和易基因编辑等特性的理想底盘细胞——耐盐菌株“需钠弧菌(Vmax)”,并基于弧菌类细菌能吸收整合外源DNA的自然转化能力,通过调控基因精准构建可调控的具有高效自然转化能力的菌株VCOD-2。研究人员通过测试发现,这一菌株可高效整合外源DNA片段到细菌基因组,相较于自然界中微生物,转化效率可提升数倍。进一步研究中,研究团队将来自不同物种的降解基因模块进行适配优化,创新开发了迭代自然转化法,利用同源替换策略,将5个功能基因簇迭代整合到细菌基因组中,在单一菌株中构建了覆盖单环到多环化合物的五条人工代谢通路,得到的“微生物特种兵”VCOD-15,可实现五种典型芳香类有机污染物——联苯、苯酚、萘、二苯并呋喃和甲苯的同时降解,涵盖了从单环到多环化合物的广泛底物范围。 研究团队通过实际工业废水样本系统验证了“微生物特种兵”工程菌株VCOD-15从实验室到实际污染场地的全场景降解效果:例如,在污染物降解能力方面,这种“微生物特种兵”展现出多靶点同步处理优势——在48小时内对5种目标污染物的去除率均超60%,其中对联苯实现完全降解(100%),甲苯、二苯并呋喃等复杂污染物降解率近90%,较天然菌株提升2至3倍效能。面对极端工业环境挑战,VCOD-15在盐度高达102.5克每升的氯碱废水中仍保持活性,成功克服了传统菌株“遇盐即失活”的瓶颈;在活性污泥反应器中,12小时内可完全去除高浓度污染物;多平行生物反应器测试显示,48小时内工业废水中污染物残留量均低于检测范围的2%,且菌株在复杂微生物群落中占比稳定(40%以上),体现其强大的环境竞争力。
[学术文献 ] An Artificial Metal-Free Peroxidase Designed Using a Ferritin Cage for Bioinspired Catalysis 进入全文
Angewandte Chemie International Edition
Developing artificial enzymes is challenging because it requires precise design of active sites with well-arranged amino acid residues. Histidine-rich oligopeptides have been recently shown to exhibit peroxidase-mimetic activities, but their catalytic function relies on maintaining unique supramolecular structures. This work demonstrates the design of a specific array of histidine residues on the internal surface of the ferritin cage to function as an active center for catalysis. The crystal structures of the ferritin mutants revealed histidine–histidine interactions, forming well-defined histidine clusters (His-clusters). These mutants exhibit peroxidase-mimetic activities by oxidizing 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide. Molecular dynamics simulations further highlight the co-localization of TMB and hydrogen peroxide at the histidine-rich clusters, indicating that the confined environment of the ferritin cage enhances their interactions. This study presents a simple yet effective approach to design metal-free artificial enzymes, paving the way for innovations in bioinspired catalysis.
[学术文献 ] Glyceollin biosynthesis in a plant chassis engineered for isoflavone production 进入全文
Nature Chemical Biology
Glyceollins are structurally complex potent antimicrobial isoflavonoid phytoalexins produced by the crop soybean (Glycine max), yet their biosynthesis remains elusive, making it impossible to carry out synthetic biology-based production and engineering for further development. Here, via assembling synergistic engineering strategies, we successfully rewired the metabolic fluxes in Nicotiana benthamiana leaves for high-yield production of isoflavonoid precursor daidzein (7.04 g kg−1 dry weight (dw)), allowing for efficient screening and identification of six cytochrome P450 monooxygenases, namely glyceollin synthases, that furnish the pyrano/furano E ring and complete the 15-step biosynthetic pathways of diverse glyceollins. We establish that purified glyceollins are important for plant defense as they can effectively suppress the growth of Phytophthora sojae in vitro. Our engineered plant chassis can provide facile access to bioactive isoflavonoids, as manifested by the de novo total biosynthesis of glyceollins (for example, I, II, III and VII at up to 5.9 g kg−1, dw) and medicarpin (0.72 g kg−1, dw) for enhanced pathogen resistance and medicinal value.