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.

Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicum

“…C. 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 [167]. The engineered strains were able to produce glutamate efficiently as well as lysine.…”

Use of Glycerol in Biotechnological Applications

“…Pyruvate carboxylase has been shown to be indispensable under detergent triggered production conditions (Peters-Wendisch et al, 2001) and vice versa under biotin limiting conditions PEPcarboxylase is responsible for anaplerosis (Sato et al, 2008, Delaunay et al, 2004, Lapujade et al, 1999. C. glutamicum was engineered for glycerol utilization by expression of the genes for glycerol facilitator, glycerol kinase, and glycerol-3-phosphate dehydrogenase from E. coli (Rittmann et al, 2008). Under ethambutol triggered L-glutamate production conditions recombinant C. glutamicum showed reduced Lglutamate yields from glycerol compared to glucose, 0.11 g g -1 compared to 0.20 g g -1 , respectively (Rittmann et al, 2008).…”

“…C. glutamicum was engineered for glycerol utilization by expression of the genes for glycerol facilitator, glycerol kinase, and glycerol-3-phosphate dehydrogenase from E. coli (Rittmann et al, 2008). Under ethambutol triggered L-glutamate production conditions recombinant C. glutamicum showed reduced Lglutamate yields from glycerol compared to glucose, 0.11 g g -1 compared to 0.20 g g -1 , respectively (Rittmann et al, 2008). Production of L-lysine, which is used as a feed additive, is also carried out with C. glutamicum , Eggeling & Bott, 2005.…”

“…Besides products that can be derived from glycerol in one or two reactions further products requiring more complex conversions have been investigated, e.g. succinic acid production with Anaerobiospirillum succiniciproducens (Lee et al 2001) or Escherichia coli (Blankschien et al, 2010, citrate production with Yarrowia lipolytica , ethanol production with Escherichia coli, Saccharomyces cerevisiae, or Hansenula polymorpha , Shams Yazdani & Gonzalez, 2008, production of amino acids with Corynebacterium glutamicum (Rittmann et al, 2008), or propionate production with Propionibacteria (Himmi et al, 2000). Crude glycerol preparations from biodiesel factories differ considerably from the pure chemical glycerol, e.g.…”

Corynebacterium glutamicum harbours a molybdenum cofactor-dependent formate dehydrogenase which alleviates growth inhibition in the presence of formate

“…FDHs can be found in prokaryotic micro-organisms such as aerobic methylotrophs and chemoautotrophs, anaerobic or facultative anaerobic bacteria, as well as methanogenic archaea (Friedebold & Bowien, 1993;Jormakka et al, 2002;Karzanov et al, 1991;Schauer et al, 1986). Depending on the type of FDH, the electrons are transferred to an acceptor such as NAD Wendisch et al, 2006a, b) and has also been engineered for production of amino-acid-derived products, alcohols and organic acids from glucose and other carbon sources (Blombach et al, 2011;Buschke et al, 2011;Gopinath et al, 2011;Litsanov et al, 2012a, b;Niimi et al, 2011;Rittmann et al, 2008;Sasaki et al, 2009;Schneider et al, 2011Schneider et al, , 2012Stäbler et al, 2011). In initial experiments on the use of different carbon sources by this organism, we observed that C. glutamicum ATCC 13032 could consume formate.…”