Quantification of Biocatalytic Transformations by Single Microbial Cells Enabled by Tailored Integration of Droplet Microfluidics and Mass Spectrometry

Abstract

Improving the performance of chemical transformations catalysed by microbial biocatalysts requires a deep understanding of cellular processes. While the cellular heterogeneity of cellular characteristics, such as the concentration of high abundant cellular content, is well studied, little is known about the reactivity of individual cells and its impact on the chemical identity, quantity, and purity of excreted products. Biocatalytic transformations were monitored chemically specific and quantifiable at the single-cell level by integrating droplet microfluidics, cell imaging, and mass spectrometry. Product formation rates for individual Saccharomyces cerevisiae cells were obtained by i) incubating nanolitresized droplets for product accumulation in microfluidic devices, ii) an imaging setup to determine the number of cells in the droplets, and iii) electrospray ionisation mass spectrometry for reading the chemical contents of individual droplets. These findings now enable the study of whole-cell biocatalysis at single-cell resolution.

Toward nanofluidics‐based mass spectrometry for exploring the unknown complex and heterogeneous subcellular worlds

“…Recently, Belder et al. combined the droplet‐based microfluidic mass spectrometry with a capillary method (Figure 3A) to quantify biocatalytic transformations at the single‐cell level 67 …”

Screening megasynthetase mutants at high throughput using droplet microfluidics

“…Droplet-based microfluidics is a rapidly developing ultrahigh-throughput screening platform, allowing compartmentalization of molecules, cells, or library members into picolitre-volume reaction chambers, linking products to producers. [11][12][13] Diverse molecular properties have been screened for in microfluidics at ultrahigh throughput, [14][15][16][17][18][19] but application on megasynthetases such as NRPSs remains elusive. Fluorescent reporter strains growing in microfluidic droplets together with potential producers of antibiotics have been employed to identify antibiotics in environmental samples [20] or libraries of ribosomally produced and posttranslationally-modified peptides.…”

Mass Spectrometry-Based High-Throughput Quantification of Bioproducts in Liquid Culture

“…We recently reported the development of MALDI-time-of-flight (TOF)-based approaches for screening microbial products directly from large numbers of colonies in a high-throughput manner (∼2 s per colony). − As is typical for MALDI approaches, the analytes remain on the surface, and the analyte vaporization and ionization processes depend on both the sample and matrix crystallization, making absolute quantitation problematic even when using standards. Other approaches involve microfluidics coupled to ESI–MS or MALDI–MS, − the integration of self-assembled monolayers with matrix-assisted laser desorption ionization (SAMDI) , and the use of desorption ESI (DESI) …”