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[学术文献 ] Combinatorial High-Throughput Screening of Complex Polymeric Enzyme Immobilization Supports 进入全文
Journal of the American Chemical Society
Recent advances have demonstrated the promise of complex multicomponent polymeric supports to enable supra-biological enzyme performance. However, the discovery of such supports has been limited by time-consuming, low-throughput synthesis and screening. Here, we describe a novel combinatorial and high-throughput platform that enables rapid screening of complex and heterogeneous copolymer brushes as enzyme immobilization supports, named combinatorial high-throughput enzyme support screening (CHESS). Using a 384-well plate format, we synthesized arrays of three-component polymer brushes in the microwells using photoactivated surface-initiated polymerization and immobilized enzymes in situ. The utility of CHESS to identify optimal immobilization supports under thermally and chemically denaturing conditions was demonstrated usingBacillus subtilisLipase A (LipA). The identification of supports with optimal compositions was validated by immobilizing LipA on polymer-brush-modified biocatalyst particles. We further demonstrated that CHESS could be used to predict the optimal composition of polymer brushes a priori for the previously unexplored enzyme, alkaline phosphatase (AlkP). Our findings demonstrate that CHESS represents a predictable and reliable platform for dramatically accelerating the search of chemical compositions for immobilization supports and further facilitates the discovery of biocompatible and stabilizing materials.
[学术文献 ] Design of a Genetically Encoded Biosensor for High-Throughput Screening and Engineering 5-Aminolevulinic Acid Hyper-Producing Escherichia coli 进入全文
ACS Sustainable Chemistry & Engineering
5-Aminolevulinic acid (ALA) is a multifunctional nonprotein amino acid used in agriculture, medicine, and other fields. As current environmental problems become increasingly serious, it is of great significance to establish a cheap, environmentally friendly, and sustainable way to synthesize ALA. Therefore, the biosynthesis of ALA is receiving increasing attention. However, the lack of high-throughput screening methods for ALA has become a limiting factor in improving microbial production of ALA. In this study, a high-throughput screening method was developed based on the relationship between reactive oxygen species (ROS) caused by ALA and cyclic adenosine monophosphate (cAMP). First, the relationship between the ROS accumulation and changes in cAMP levels was verified. Subsequently, the selected promoter was optimized by adding cAMP receptor protein (CRP) binding sites at its upstream, and a high-throughput screening method for ALA was established. HemA mutant and ALA-producing Escherichia coli strain mutant were obtained and combined with a series of metabolic engineering strategies to improve ALA production. Finally, the ALA fed-batch fermentation in a 5 L fermenter achieved the highest ALA titer of 58.54 g/L with a productivity of 1.58 g/L/h. This laid the foundation for the industrialization of ALA through biosynthesis.
[学术文献 ] Machine learning-aided design and screening of an emergent protein function in synthetic cells 进入全文
Nature Communications
Recently, utilization of Machine Learning (ML) has led to astonishing progress in computational protein design, bringing into reach the targeted engineering of proteins for industrial and biomedical applications. However, the design of proteins for emergent functions of core relevance to cells, such as the ability to spatiotemporally self-organize and thereby structure the cellular space, is still extremely challenging. While on the generative side conditional generative models and multi-state design are on the rise, for emergent functions there is a lack of tailored screening methods as typically needed in a protein design project, both computational and experimental. Here we describe a proof-of-principle of how such screening, in silico and in vitro, can be achieved for ML-generated variants of a protein that forms intracellular spatiotemporal patterns. For computational screening we use a structure-based divide-and-conquer approach to find the most promising candidates, while for the subsequent in vitro screening we use synthetic cell-mimics as established by Bottom-Up Synthetic Biology. We then show that the best screened candidate can indeed completely substitute the wildtype gene in Escherichia coli. These results raise great hopes for the next level of synthetic biology, where ML-designed synthetic proteins will be used to engineer cellular functions.
[前沿资讯 ] 中国科学院微生物所在真菌混源萜生物合成机制研究中取得进展 进入全文
中国科学院微生物研究所
中国科学院微生物研究所刘钢研究团队与中国医学科学院药用植物研究所马国需团队合作在Journal of the American Chemical Society上发表论文，题为Two Cytochrome P450 Enzymes Form the Tricyclic Nested Skeleton of Meroterpenoids by Sequential Oxidative Reactions。该研究解析了大型真菌来源的混源萜clavilactone A的生物合成途径以及环化机制，有助于实现其高效精准制备。 Clavilactones是分离自大型真菌棒柄杯伞（Clitocybe clavipes）的一类莽草酸途径来源的混源萜，表现出良好的酪氨酸激酶抑制活性，具有开发成为抗肿瘤药物的潜力，但其生物合成途径和机制尚未被解析。 在前期工作基础上（ACS Catal. 2023, 13, 20, 13717–13728），该研究进一步阐明了clavilactones生物合成基因簇中的两个P450酶ClaR和ClaT的功能，完整解析了clavilactone A的生物合成途径和环化机制。其中P450酶ClaR通过催化底物geranylhydroquinone分子内对苯二酚及烯丙基的双自由基结合反应形成新颖的苯并十元碳环wigandol，接着多功能的P450酶ClaT催化十电子氧化反应，立体选择性的合成α，β-环氧-γ-内酯结构单元，完成clavilactone A中10/5/3三环嵌套结构的合成。该研究首次报道了P450酶在混源萜生物合成初期参与核心骨架的环化合成，扩展了P450酶的催化功能，为真菌来源的含有苯并大环的混源萜化合物的挖掘和生物合成解析奠定基础。
[前沿资讯 ] An e-nose could be a powerful tool in the feed chain for quality and safety control and mycotoxin monitoring 进入全文
FEED NAVIGATOR
Dr Matteo Ottoboni, from the Department of Veterinary Medicine and Animal Sciences, at the University of Milan, presented on the application of electronic noses for feed safety and animal nutrition at the World Mycotoxin Forum in Antwerp in Belgium last month.
[前沿资讯 ] 南京农业大学消化道微生物团队发现生命早期瘤胃微生物衍生物对反刍动物瘤胃发育影响 进入全文
南京农业大学
日粮在塑造胃肠道微生物群落方面扮演不可或缺的角色，微生物的代谢产物可作为信号或底物，影响宿主器官的发育和代谢健康。日粮、微生物和宿主之间的相互作用构建了一个复杂的共生体系，其如何共同促进人类和动物的健康已经引起了广泛关注，但它们之间的直接关联在很大程度上仍然是一个未解之谜。在瘤胃中，微生物发挥着多重作用，它们不仅能有效地将膳食纤维转化为重要的代谢前体，例如挥发性脂肪酸（VFA）、微生物蛋白质和维生素，还能显著影响瘤胃复层上皮和肌层的发育。瘤胃微生物的初始定植及其在出生后逐渐稳定的过程会受到不同日粮（如液态奶、谷物固体饲料、干草或这些日粮的组合）的显著影响。然而，目前仍然难以明确在出生后不同日粮营养条件下，瘤胃微生物群究竟如何影响瘤胃壁的发育以及动物的生长过程。 研究以哺乳羔羊为研究对象，利用极端化早期营养干预策略构建了瘤胃上皮/肌层的差异化发育表型；基于天然植物成分的微生物产物标准物质质谱库和极性/非极性物质的色谱选择策略，首次鉴定出与瘤胃上皮和肌层发育密切相关的代谢物吲哚-3-甲醛（IAld）和前列腺素D2（PGD2）；利用宏基因组技术厘清了IAld和PGD2在瘤胃内的微生物合成路径并筛选出候选代谢微生物菌株，进一步通过单菌体外纯培养试验，发现假长双歧杆菌可代谢色氨酸生成IAld，白色念珠球菌具有生成PGD2的能力；利用转录组技术锚定瘤胃差异化发育的信号调控通路，体内外试验揭示了IAld通过激活Wnt/β-catenin信号通路促进瘤胃上皮发育，PGD2依赖Ca2+信号通路调控瘤胃肌层发育。研究结果加深了对生命早期日粮-微生物-宿主互作的理解，首次明确了瘤胃特定菌株及其下游代谢物与瘤胃生理的直接联系，为幼龄反刍动物开食料添加剂的开发应用提供新思路。
