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[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.
[学术文献 ] Directed evolution of aminoacyl-tRNA synthetases through in vivo hypermutation 进入全文
Nature Communications
Genetic code expansion (GCE) is a critical approach to the site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Central to GCE is the development of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs wherein engineered aaRSs recognize chosen ncAAs and charge them onto tRNAs that decode blank codons (e.g., the amber stop codon). However, evolving new aaRS/tRNA pairs traditionally relies on a labor-intensive process that often yields aaRSs with suboptimal ncAA incorporation efficiencies. Here, we present an OrthoRep-mediated strategy for aaRS evolution, which we demonstrate in 8 independent aaRS evolution campaigns, yielding multiple aaRSs that incorporate an overall range of 13 ncAAs tested. Some evolved systems enable ncAA-dependent translation at single amber codons with similar efficiency as natural translation at sense codons. Additionally, we discover an aaRS that regulated its own expression to enhance ncAA dependency. These findings demonstrate the potential of OrthoRep-driven aaRS evolution platforms to advance the field of GCE.
[学术文献 ] Mechanistic rules for de novo design of enzymes 进入全文
Chem Catalysis
While the last two decades have witnessed the development of a number of different strategies to build synthetic nanomotors that deliver mechanical work, making systems that possess engineered catalytic functionality has not so far been demonstrated either theoretically or experimentally in the context of (wet) molecular nanotechnology. We describe a fundamentally new paradigm in the bottom-up design of systems that give direction to chemistry, which will enable future technologies to control how catalytic activity is organized. Our work is inspired by the key observation that the non-equilibrium dynamics of an enzyme during catalysis simultaneously involve energy transduction and conformational changes, i.e., displacements. This suggests that mechanical considerations should play a key role in the stochastic dynamics of an enzyme, and consequently, in its optimal design with the aim of achieving the desired catalytic cycle. Our proposed dynamical paradigm, built on appropriate implementation of the relevant physical constraints on the minimal reaction coordinates, allows us to identify the following three golden rules for the optimal function of a fueled enzyme driven by mechanochemical coupling: (1) the enzyme and the molecule should be attached at the smaller end of each (i.e., friction matching); (2) the conformational change of the enzyme must be comparable to or larger than the conformational change required of the molecule; and (3) the conformational change of the enzyme must be fast enough so that the molecule actually stretches, rather than just following the enzyme without stretching. The mechanistic rules can provide useful input to the complementary perspectives of de novo enzyme design based on machine learning, as they can be used for training the algorithm, as well as fine-tuning the force fields and phenomenological parameters in all-atom simulations.
[学术文献 ] An mRNA-display derived cyclic peptide scaffold reveals the substrate binding interactions of an N-terminal cysteine oxidase 进入全文
Nature Communications
N-terminal cysteine oxidases (NCOs) act as enzymatic oxygen (O2) sensors, coordinating cellular changes to hypoxia in animals and plants. They regulate the O2-dependent stability of proteins bearing an N-terminal cysteine residue through the N-degron pathway. Despite their important role in hypoxic adaptation, which renders them potential therapeutic and agrichemical targets, structural information on NCO substrate binding remains elusive. To overcome this challenge, we employed a unique strategy by which a cyclic peptide inhibitor of the mammalian NCO, 2-aminoethanethiol dioxygenase (ADO), was identified by mRNA display and used as a scaffold to graft substrate moieties. This allowed the determination of two substrate analogue-bound crystal structures of ADO. Key binding interactions were revealed, including bidentate coordination of the N-terminal residue at the metal cofactor. Subsequent structure guided mutagenesis identified aspartate-206 as an essential catalytic residue, playing a role in reactive oxygen intermediate orientation or stabilisation. These findings provide fundamental information on ADO substrate interactions, which can elucidate enzyme mechanism and act as a platform for chemical discovery.
[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。
