Cometabolism of a nongrowth substrate: L-serine utilization by Corynebacterium glutamicum

Abstract

Despite its key position in central metabolism, L-serine does not support the growth of Corynebacterium glutamicum. Nevertheless, during growth on glucose, L-serine is consumed at rates up to 19.4 +/- 4.0 nmol min(-1) (mg [dry weight])(-1), resulting in the complete consumption of 100 mM L-serine in the presence of 100 mM glucose and an increased growth yield of about 20%. Use of 13C-labeled L-serine and analysis of cellularly derived metabolites by nuclear magnetic resonance spectroscopy revealed that the carbon skeleton of L-serine is mainly converted to pyruvate-derived metabolites such as L-alanine. The sdaA gene was identified in the genome of C. glutamicum, and overexpression of sdaA resulted in (i) functional L-serine dehydratase (L-SerDH) activity, and therefore conversion of L-serine to pyruvate, and (ii) growth of the recombinant strain on L-serine as the single substrate. In contrast, deletion of sdaA decreased the L-serine cometabolism rate with glucose by 47% but still resulted in degradation of L-serine to pyruvate. Cystathionine beta-lyase was additionally found to convert L-serine to pyruvate, and the respective metC gene was induced 2.4-fold under high internal L-serine concentrations. Upon sdaA overexpression, the growth rate on glucose is reduced 36% from that of the wild type, illustrating that even with glucose as a single substrate, intracellular L-serine conversion to pyruvate might occur, although probably the weak affinity of L-SerDH (apparent Km, 11 mM) prevents substantial L-serine degradation.

FIG. 1.

Growth (A, B) and substrate consumption (C) of wild-type C. glutamicum in minimal medium containing different glucose concentrations (+, 0 mM; ▪, 20 mM; •, 40 mM; ▴, 60 mM; ▾, 80 mM; ♦, 100 mM) in the absence (A) or presence (B) of l-serine. (C) Consumption of glucose (open symbols) and serine (closed symbols) as a function of OD₆₀₀.

FIG. 2.

¹³C NMR spectra of the C-2 of alanine and illustration of the signal fine structure composition. s, singlet peak of [2-¹³C]alanine (no neighboring labels); d₋₁, ¹³C in the preceding position ([1,2-¹³C₂]alanine) produces a doublet peak, split by scalar coupling; d₊₁, ¹³C in the following position ([2,3-¹³C₂]alanine) yields another doublet split with a different coupling constant; dd, “doublet of doublet” signal of [¹³C₃]alanine.

FIG. 3.

Specific activity (v) of l-SerDH in crude extracts of C. glutamicum 13032(pXMJ19sdaA) as a function of the l-serine concentration (S). Inset represents the respective Eadie-Hofstee plot.

FIG. 4.

Growth of different C. glutamicum strains in minimal medium containing 100 mM glucose (open symbols) or 100 mM l-serine (closed symbols). Triangles, 13032(pXMJ19sdaA); circles, 13032(pXMJ19); squares, 13032ΔsdaA(pXMJ19).

FIG. 5.

Growth (open symbols) and l-serine consumption (closed symbols) of different C. glutamicum strains in minimal medium containing 100 mM glucose plus 100 mM l-serine. Triangles, 13032ΔsdaA(pSL173); circles, 13032ΔsdaA(pZ1); squares, 13032ΔsdaAΔmetC.