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Accelerating strain phenotyping with desorption electrospray ionization-imaging mass spectrometry and untargeted analysis of intact microbial colonies [Chemistry]
Proceedings of the National Academy of Sciences of the United States of America ( IF 9.4 ) Pub Date : 2021-12-07 , DOI: 10.1073/pnas.2109633118
Berkley M Ellis 1, 2, 3, 4, 5 , Piyoosh K Babele 6 , Jody C May 1, 2, 3, 4, 5 , Carl H Johnson 7, 8 , Brian F Pfleger 9 , Jamey D Young 6, 7 , John A McLean 2, 3, 4, 5, 10
Affiliation  

Reading and writing DNA were once the rate-limiting step in synthetic biology workflows. This has been replaced by the search for the optimal target sequences to produce systems with desired properties. Directed evolution and screening mutant libraries are proven technologies for isolating strains with enhanced performance whenever specialized assays are available for rapidly detecting a phenotype of interest. Armed with technologies such as CRISPR-Cas9, these experiments are capable of generating libraries of up to 1010 genetic variants. At a rate of 102 samples per day, standard analytical methods for assessing metabolic phenotypes represent a major bottleneck to modern synthetic biology workflows. To address this issue, we have developed a desorption electrospray ionization–imaging mass spectrometry screening assay that directly samples microorganisms. This technology increases the throughput of metabolic measurements by reducing sample preparation and analyzing organisms in a multiplexed fashion. To further accelerate synthetic biology workflows, we utilized untargeted acquisitions and unsupervised analytics to assess multiple targets for future engineering strategies within a single acquisition. We demonstrate the utility of the developed method using Escherichia coli strains engineered to overproduce free fatty acids. We determined discrete metabolic phenotypes associated with each strain, which include the primary fatty acid product, secondary products, and additional metabolites outside the engineered product pathway. Furthermore, we measured changes in amino acid levels and membrane lipid composition, which affect cell viability. In sum, we present an analytical method to accelerate synthetic biology workflows through rapid, untargeted, and multiplexed metabolomic analyses.



中文翻译:

用解吸电喷雾电离成像质谱法和完整微生物菌落的非靶向分析加速菌株表型分析[化学]

读取和写入 DNA 曾经是合成生物学工作流程中的限速步骤。这已被寻找最佳目标序列以产生具有所需特性的系统所取代。定向进化和筛选突变文库是经过验证的技术,可用于分离具有增强性能的菌株,只要有专门的检测方法可用于快速检测感兴趣的表型。借助 CRISPR-Cas9 等技术,这些实验能够生成多达 10 10 个基因变异的文库。以 10 2的速率每天的样本,用于评估代谢表型的标准分析方法是现代合成生物学工作流程的主要瓶颈。为了解决这个问题,我们开发了一种直接对微生物进行采样的解吸电喷雾电离成像质谱筛选试验。该技术通过减少样品制备和以多路复用方式分析生物体来提高代谢测量的吞吐量。为了进一步加快合成生物学工作流程,我们利用非目标收购和无监督分析来评估单个收购中未来工程战略的多个目标。我们展示了使用大肠杆菌开发的方法的实用性被设计成过量产生游离脂肪酸的菌株。我们确定了与每个菌株相关的离散代谢表型,包括初级脂肪酸产物、次级产物和工程产物途径之外的其他代谢物。此外,我们测量了影响细胞活力的氨基酸水平和膜脂组成的变化。总之,我们提出了一种分析方法,通过快速、非靶向和多重代谢组学分析加速合成生物学工作流程。

更新日期:2021-12-03
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