Profiling of Microbial Colonies for High-Throughput Engineering of Multistep Enzymatic Reactions via Optically Guided Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.
Profiling of Microbial Colonies for High-Throughput Engineering of Multistep Enzymatic Reactions via Optically Guided Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.
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DOI：
10.1021/jacs.7b04641
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发表时间：
2017-09-13
影响因子：
15
通讯作者：
Sweedler JV
中科院分区：
文献类型：
--
作者：
Si T;Li B;Comi TJ;Wu Y;Hu P;Wu Y;Min Y;Mitchell DA;Zhao H;Sweedler JV
基质辅助激光解吸/电离飞行时间（MALDI-ToF）质谱（MS）成像已用于酶活性的快速表型分析，但主要限于单步转换。在此，我们报告了一种基于光学引导的MALDI-ToF MS细菌菌落分析的多步生化反应的高通量工程化的无标记方法。细菌细胞提供多种酶的容纳，并通过代谢获得底物和辅因子。从随机分布的菌落中自动采集MALDI-ToF MS简化了制备菌株文库的程序，无需液体处理。利用MALDI-ToF MS分析来筛选天然产物生物合成的底物和酶文库。开发了计算算法来处理和可视化所得到的质谱数据集。对于肽类抗生素plantazolicin的类似物，通过t-分布随机近邻嵌入的多变量分析被用于分组相似的光谱以快速鉴定非同量异位素变体。在MALDI-ToF MS筛选后，使用高分辨率MS和串联MS的后续分析很容易在相同的样品靶标上进行。单独地，评价具有各种脂质部分的鼠李糖脂同系物的相对离子强度以工程化酶特异性。每个菌落的糖脂谱与光学图像重叠，以促进所需突变体的回收。对于抗生素和鼠李糖脂的情况，在分子水平上迅速调查了大量的菌落，提供了用传统筛选测定法不易获得的信息丰富的见解。利用常规显微镜和MALDI-ToF MS仪器的标准微生物学技术，这种简单而有效的工作流程适用于针对多步酶促反应的广泛筛选活动。
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS) imaging has been used for rapid phenotyping of enzymatic activities, but is mainly limited to single-step conversions. Herein we report a label-free method for high-throughput engineering of multistep biochemical reactions based on optically guided MALDI-ToF MS analysis of bacterial colonies. The bacterial cells provide containment of multiple enzymes and access to substrates and cofactors via metabolism. Automated MALDI-ToF MS acquisition from randomly distributed colonies simplifies procedures to prepare strain libraries without liquid handling. MALDI-ToF MS profiling was utilized to screen both substrate and enzyme libraries for natural product biosynthesis. Computational algorithms were developed to process and visualize the resulting mass spectral data sets. For analogues of the peptidic antibiotic plantazolicin, multivariate analyses by t-distributed stochastic neighbor embedding were used to group similar spectra for rapid identification of nonisobaric variants. After MALDI-ToF MS screening, follow-up analyses using high-resolution MS and tandem MS were readily performed on the same sample target. Separately, relative ion intensities of rhamnolipid congeners with various lipid moieties were evaluated to engineer enzymatic specificity. The glycolipid profiles of each colony were overlaid with optical images to facilitate the recovery of desirable mutants. For both the antibiotic and rhamnolipid cases, large populations of colonies were rapidly surveyed at the molecular level, providing information-rich insights not easily obtained with traditional screening assays. Utilizing standard microbiological techniques with routine microscopy and MALDI-ToF MS instruments, this simple yet effective workflow is applicable for a wide range of screening campaigns targeting multistep enzymatic reactions.
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影响因子：
18.3
作者：
Dunham SJ;Ellis JF;Li B;Sweedler JV
通讯作者：
Sweedler JV
影响因子：
7.4
作者：
Do TD;Comi TJ;Dunham SJ;Rubakhin SS;Sweedler JV
通讯作者：
Sweedler JV
影响因子：
4
作者：
Deane, Caitlin D.;Melby, Joel O.;Molohon, Katie J.;Susarrey, Aziz R.;Mitchell, Douglas A.
通讯作者：
Mitchell, Douglas A.
影响因子：
4.2
作者：
Masyuko, Rachel N.;Lanni, Eric J.;Bohn, Paul W.
通讯作者：
Bohn, Paul W.
影响因子：
14.8
作者：
Kim E;Moore BS;Yoon YJ
通讯作者：
Yoon YJ