Engineering of a glycerol utilization pathway for amino acid production by Corynebacterium glutamicum

Abstract

The amino acid-producing organism Corynebacterium glutamicum cannot utilize glycerol, a stoichiometric by-product of biodiesel production. By heterologous expression of Escherichia coli glycerol utilization genes, C. glutamicum was engineered to grow on glycerol. While expression of the E. coli genes for glycerol kinase (glpK) and glycerol 3-phosphate dehydrogenase (glpD) was sufficient for growth on glycerol as the sole carbon and energy source, additional expression of the aquaglyceroporin gene glpF from E. coli increased growth rate and biomass formation. Glutamate production from glycerol was enabled by plasmid-borne expression of E. coli glpF, glpK, and glpD in C. glutamicum wild type. In addition, a lysine-producing C. glutamicum strain expressing E. coli glpF, glpK, and glpD was able to produce lysine from glycerol as the sole carbon substrate as well as from glycerol-glucose mixtures.

FIG. 1.

Growth of C. glutamicum WT(pVWEx1) (open symbols) and WT(pVWEx1-glpFKD) (closed symbols) on mineral medium containing 110 mM glycerol (A) or 110 mM glycerol and 55 mM glucose (B) as carbon sources. Growth was monitored as OD₆₀₀ (circles); the concentrations of glycerol (squares) and glucose (triangles) are indicated. The data are averages and standard deviations of three replicates.

FIG. 2.

Biomass yields (squares) and growth rates (triangles) of C. glutamicum WT(pVWEx1-glpFKD) for growth on minimal medium containing different concentrations of glycerol as the sole carbon source.

FIG. 3.

Growth rates and specific glycerol kinase activities of various recombinant C. glutamicum strains during growth on glycerol-glucose mixtures with different IPTG concentrations. Growth rates of C. glutamicum WT(pVWEx1) (squares), WT(pVWEx1-glpF) (triangles), and WT(pVWEx1-glpFK) (solid circles) and specific glycerol kinase activities of WT(pVWEx1-glpFK) (open circles) are plotted against the IPTG concentration used for inducible gene expression from plasmid pVWEx1. Crude extracts of WT(pVWEx1) and WT(pVWEx1-glpF) contained specific glycerol kinase activities of 0.01 to 0.02 U mg⁻¹.

FIG. 4.

Intracellular glycerol 3-phosphate concentrations of various recombinant C. glutamicum strains during growth on 110 mM glycerol and 55 mM glucose. Concentrations were measured 4 h after induction with 0.1 (white bars) or 0.5 mM (black bars) IPTG at an OD₆₀₀ of 4 and are given as arithmetic means and absolute errors of two independent cultivations.