Proteomics of FACS-sorted heterogeneous Corynebacterium glutamicum populations

Abstract

The metabolic status of individual cells in microbial cultures can differ, being relevant for biotechnology, environmental and medical microbiology. However, it is hardly understood in molecular detail due to limitations of current analytical tools. Here, we demonstrate that FACS in combination with proteomics can be used to sort and analyze cell populations based on their metabolic state. A previously established GFP reporter system was used to detect and sort single Corynebacterium glutamicum cells based on the concentration of branched chain amino acids (BCAA) using FACS. A proteomics workflow optimized for small cell numbers was used to quantitatively compare proteomes of a ΔaceE mutant, lacking functional pyruvate dehydrogenase (PD), and the wild type. About 800 proteins could be quantified from 1,000,000 cells. In the ΔaceE mutant BCAA production was coordinated with upregulation of the glyoxylate cycle and TCA cycle to counter the lack of acetyl CoA resulting from the deletion of aceE.

Biological significance: Metabolic pathways in C. glutamicum WT and ΔaceE, devoid of functional pyruvate dehydrogenase, were compared to understand proteome changes that contribute to the high production of branched chain amino acids (BCAA) in the ΔaceE strain. The data complements previous metabolome studies and corroborates the role of malate provided by the glyoxylate cycle and increased activity of glycolysis and pyruvate carboxylase reaction to replenish the TCA cycle. A slight increase in acetohydroxyacid synthase (ILV subunit B) substantiates the previously reported increased pyruvate pool in C. glutamicumΔaceE, and the benefit of additional ilv gene cluster overexpression for BCAA production.

Keywords: BCAA producer; Cellular heterogeneity; Corynebacterium glutamicum; FACS; Label-free quantification; Proteome analysis.