Department of Chemistry, Vanderbilt University, Nashville, TN 37235.
Center for Innovative Technology, Vanderbilt University, Nashville, TN 37235.
Proc Natl Acad Sci U S A. 2021 Dec 7;118(49). doi: 10.1073/pnas.2109633118.
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 This has been replaced by the search for the optimal target sequences to produce systems with desired properties.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 10 genetic variants. At a rate of 10 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 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.
在合成生物学工作流程中，阅读和编写 DNA 曾经是限速步骤。现在，人们已经将重点转移到寻找产生具有所需特性的系统的最佳目标序列上。定向进化和筛选突变文库是一种经过验证的技术，可以在有专门的检测方法来快速检测感兴趣的表型时，分离出性能增强的菌株。有了 CRISPR-Cas9 等技术，这些实验能够生成多达 10 个遗传变体的文库。对于每天 10 个样本的速度，评估代谢表型的标准分析方法代表了现代合成生物学工作流程的主要瓶颈。为了解决这个问题，我们开发了一种解吸电喷雾电离-成像质谱筛选测定法，可直接对微生物进行采样。该技术通过减少样品制备和以多路复用的方式分析生物体，提高了代谢测量的通量。为了进一步加速合成生物学工作流程，我们利用无目标采集和无监督分析在单个采集过程中评估多个目标，以确定未来的工程策略。我们使用经过工程改造以过量生产游离脂肪酸的 菌株来证明所开发方法的实用性。我们确定了与每个菌株相关的离散代谢表型，其中包括主要脂肪酸产物、次要产物以及工程产物途径之外的其他代谢产物。此外，我们还测量了氨基酸水平和膜脂组成的变化，这些变化会影响细胞活力。总之，我们提出了一种分析方法，通过快速、无目标和多路复用的代谢组学分析来加速合成生物学工作流程。
