Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves l-lysine formation

Abstract

A critical factor in the biotechnological production of L-lysine with Corynebacterium glutamicum is the sufficient supply of NADPH. The membrane-integral nicotinamide nucleotide transhydrogenase PntAB of Escherichia coli can use the electrochemical proton gradient across the cytoplasmic membrane to drive the reduction of NADP + via the oxidation of NADH. As C. glutamicum does not possess such an enzyme, we expressed the E. coli pntAB genes in the genetically defined C. glutamicum lysineproducing strain DM1730, resulting in membrane-associated transhydrogenase activity of 0.7 U/mg protein. When cultivated in minimal medium with 10% (w/v) carbon source, the presence of transhydrogenase slightly reduced glucose consumption, whereas the consumption of fructose, glucose plus fructose, and, in particular, sucrose was stimulated. Biomass was increased by pntAB expression between 10 and 30% on all carbon sources tested. Most importantly, the lysine concentration was increased in the presence of transhydrogenase by ∼10% on glucose, ∼70% on fructose, ∼50% on glucose plus fructose, and even by ∼300% on sucrose. Thus, the presence of a proton-coupled transhydrogenase was shown to be an efficient way to improve lysine production by C. glutamicum. In contrast, pntAB expression had a negative effect on growth and glutamate production of C. glutamicum wild type.

Enhanced acetic acid and succinic acid production under microaerobic conditions by Corynebacterium glutamicum harboring Escherichia coli transhydrogenase gene pntAB

“…Similar results were obtained in the case of the budding yeast Saccharomyces cerevisiae (Anderlund et al, 1999). However, in the case of lysineproducing C. glutamicum mutants, introduction of pntAB increased lysine productivity, probably by increasing NADPH levels (Kabus et al, 2007). A similar effect was observed in valine production and isobutanol production by C. glutamicum recombinant strains (Bartek et al, 2011;Blombach et al, 2011).…”

“…However, C. glutamicum, which is used for production of amino acids, organic acids and alcohols, does not harbor transhydrogenase genes. Recently, the introduction of transhydrogenase genes into C. glutamicum cells has been examined in an attempt to improve production of amino acids and alcohol (Bartek et al, 2011;Blombach et al, 2011;Kabus et al, 2007). However, the effect of transhydrogenases on cellular metabolism in a C. glutamicum wild-type strain has not been analyzed.…”

“…In one approach, the pgi gene encoding phosphoglucose isomerase, was disrupted, and the zwf gene coding for glucose-6-phosphate dehydrogenase were overexpressed (Becker et al, 2007;Marx et al, 2003). In another approach, expression of E. coli pntAB in lysine-or valine-producing strains of C. glutamicum was also attempted (Bartek et al, 2011;Kabus et al, 2007). Blombach et al achieved isobutanol production by C. glutamicum using metabolic reactions for valine biosynthesis, and overexpression of E. coli pntAB in this system improved the productivity by allowing the cell to meet the NADPH requirements .…”

l -Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene

“…however, the PPP has been suggested to still contribute to the sufficient NADPH supply (Kabus et al, 2007).…”

Review and Hypothesis. New insights into the reaction mechanism of transhydrogenase: Swivelling the dIII component may gate the proton channel

“…In Escherichia coli, for example, about one third of the NADPH needed for biomass production can be supplied by proton-translocating transhydrogenase [1,2]. In some bacterial species over-expression of inserted transhydrogenase genes leads to commercially significant increases in the formation of fermentation products, such as lysine for animal feed, by increasing the rate of NADP + reduction [3].…”