title: A de novo NADPH generation pathway for improving lysine production of Corynebacterium glutamicum by rational design of the coenzyme specificity of glyceraldehyde 3-phosphate dehydrogenase.
abstract: Engineering the cofactor availability is a common strategy of metabolic engineering to improve the production of many industrially important compounds. In this work, a de novo NADPH generation pathway is proposed by altering the coenzyme specificity of a native NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to NADP, which consequently has the potential to produce additional NADPH in the glycolytic pathway. Specifically, the coenzyme specificity of GAPDH of Corynebacterium glutamicum is systematically manipulated by rational protein design and the effect of the manipulation for cellular metabolism and lysine production is evaluated. By a combinatorial modification of four key residues within the coenzyme binding sites, different GAPDH mutants with varied coenzyme specificity were constructed. While increasing the catalytic efficiency of GAPDH towards NADP enhanced lysine production in all of the tested mutants, the most significant improvement of lysine production (~60%) was achieved with the mutant showing similar preference towards both NAD and NADP. Metabolic flux analysis with (13)C isotope studies confirmed that there was no significant change of flux towards the pentose phosphate pathway and the increased lysine yield was mainly attributed to the NADPH generated by the mutated GAPDH. The present study highlights the importance of protein engineering as a key strategy in de novo pathway design and overproduction of desired products.
authors: Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickestrasse 15, D-21073 Hamburg, Germany.
authors: Rajesh Reddy
authors: RR
authors: Bommareddy
authors: Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickestrasse 15, D-21073 Hamburg, Germany.
authors: Zhen
authors: Z
authors: Chen
authors: Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickestrasse 15, D-21073 Hamburg, Germany.
authors: Sugima
authors: S
authors: Rappert
authors: Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickestrasse 15, D-21073 Hamburg, Germany. Electronic address: aze@tu-harburg.de.
authors: An-Ping
authors: AP
authors: Zeng
mesh_terms: D048230:Corynebacterium glutamicum / Q000502:physiology*; D024861:Genetic Enhancement / Q000379:methods*; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000235:genetics* / Q000378:metabolism; D008239:Lysine / Q000096:biosynthesis* / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods*; D009249:NADP / Q000096:biosynthesis* / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics*
chemical_list: D009249:NADP; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+); D008239:Lysine
keywords: Cofactor availability; Glyceraldehyde 3-phosphate dehydrogenase; Lysine; Rational protein design