Quantifying a Biocatalytic Product from a Few Living Microbial Cells Using Microfluidic Cultivation Coupled to FT-ICR-MS

Abstract

The in vivo quantification of metabolic products from microbial single cells is one of the last grand challenges in (bio)analytical chemistry. To date, no label-free analytical concept exists that is powerful enough to detect or even quantify the minute amounts of secreted low molecular weight compounds produced by living and isolated single bacteria or yeast cells. Coupling microfluidic cultivation systems with ultrahigh resolution electrospray-ionization mass spectrometry with its exquisite sensitivity and specificity offers the prospect of single-cell product analysis and quantification, but has not been successfully implemented yet. We report an analytical framework that interfaces noninvasive microfluidic trapping and cultivation of a few bacterial single cells with the analysis of their catalytic products by Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Cell trapping was performed with the microfluidic Envirostat platform for cultivating bacterial cells under continuous perfusion via negative dielectrophoresis (nDEP). A total of 1.5 μL of product-containing cell supernatant was sampled into microcapillaries using a dead volume-reduced world-to-chip interface. The samples were analyzed with a nanoESI ion source coupled to a FT-ICR-MS (limit of detection for lysine: 0.5 pg). As a biocatalytic model system, we analyzed few Corynebacterium glutamicum DM 1919 pSenLys cells that synthesized L-lysine from D-glucose. Secreted lysine was quantified from a few cells (down to 19). Single-cell specific lysine productivities were 2 and 10 fmol/cell/h. This demonstrates that coupling microfluidics and mass spectrometry (SIC-MS) now enables the quantification of catalytic products and extracellular metabolites from only a few living microbial cells.

Conversion Efficiencies of a Few Living Microbial Cells Detected at a High Throughput by Droplet-Based ESI-MS

“…Microbial cells are used as biocatalysts in various fields of industry and academia as they enable complex multistep chemistry at mild reaction conditions. − Their application as living and thus self-regenerating catalysts is often combined with high stereo-, chemo-, and regioselectivity. , In organic chemistry, whole-cell biocatalysts enable the design of elegant multistep one-pot reactions for the synthesis of fine chemicals or synthons. − Optimizing biocatalytic syntheses toward certain objectives requires a fundamental understanding of the underlying cell-specific mechanisms. Currently, biocatalyst analysis is advancing from population-based values to single cells for understanding catalytic processes in detail, including cellular heterogeneity in terms of reactivity and turnover numbers. − The determination of cell-specific reaction rates and yields is, however, highly challenging due to the low amount of catalytic products.…”

Single-Cell Technologies to Understand the Mechanisms of Cellular Adaptation in Chemostats

“…However, it is hard to control the environment in the droplets (Schmitz et al, 2019). Recent examples show how microfluidic cultivation systems can be coupled to mass spectrometry (MS) for label-free analysis of extracellular proteins or metabolites (Dusny et al, 2019;Haidas et al, 2020;Schirmer et al, 2020). These setups are promising as they expand the window of molecules, which can be analyzed in microfluidic cultivation systems.…”

Faster, better, and cheaper: harnessing microfluidics and mass spectrometry for biotechnology

“…Dusney et al reported an analytical framework that interfaces microfluidic trapping and cultivation of a few bacterial cells (using their previously developed negative dielectrophoresis (nDEP)-based device) with the analysis of their catalytic products by Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). 170 Using the biocatalytic model system of Corynebacterium glutamicum DM 1919 pSenLys cells, they analyzed cells that synthesized l -lysine from d -glucose. Cell trapping (with as few as 19 cells per experiment) was performed on chip for cultivating bacterial cells under continuous perfusion via negative dielectrophoresis ( Fig.…”