Profiling of Microbial Colonies for High-Throughput Engineering of Multistep Enzymatic Reactions via Optically Guided Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

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.

A plug-and-play pathway refactoring workflow for natural product research in Escherichia coli and Saccharomyces cerevisiae.

Pathway refactoring serves as an invaluable synthetic biology tool for natural product discovery, characterization, and engineering. However, the complicated and laborious molecular biology techniques largely hinder its application in natural product research, especially in a high-throughput manner. Here we report a plug-and-play pathway refactoring workflow for high-throughput, flexible pathway construction, and expression in both Escherichia coli and Saccharomyces cerevisiae. Biosynthetic genes were firstly cloned into pre-assembled helper plasmids with promoters and terminators, resulting in a series of expression cassettes. These expression cassettes were further assembled using Golden Gate reaction to generate fully refactored pathways. The inclusion of spacer plasmids in this system would not only increase the flexibility for refactoring pathways with different number of genes, but also facilitate gene deletion and replacement. As proof of concept, a total of 96 pathways for combinatorial carotenoid biosynthesis were built successfully. This workflow should be generally applicable to different classes of natural products produced by various organisms. Biotechnol. Bioeng. 2017;114: 1847-1854. © 2017 Wiley Periodicals, Inc.