Increased NADPH concentration obtained by metabolic engineering of the pentose phosphate pathway in Aspergillus niger.
Many biosynthetic reactions and bioconversions are limited by low availability of NADPH. With the purpose of increasing the NADPH concentration and/or the flux through the pentose phosphate pathway in Aspergillus niger, the genes encoding glucose 6-phosphate dehydrogenase (gsdA), 6-phosphogluconate dehydrogenase (gndA) and transketolase (tktA) were cloned and overexpressed in separate strains. Intracellular NADPH concentration was increased two- to ninefold as a result of 13-fold overproduction of 6-phosphogluconate dehydrogenase. Although overproduction of glucose 6-phosphate dehydrogenase and transketolase changed the concentration of several metabolites it did not result in increased NADPH concentration. To establish the effects of overexpression of the three genes, wild-type and overexpressing strains were characterized in detail in exponential and stationary phase of bioreactor cultures containing minimal media, with glucose as the carbon source and ammonium or nitrate as the nitrogen source and final cell density limiting substrate. Enzymes, intermediary metabolites, polyol pools (intra- and extracellular), organic acids, growth rates and rate constant of induction of acid production in postexponential phase were measured. None of the modified strains had a changed growth rate. Partial least square regressions showed the correlations between NADPH and up to 40 other variables (concentration of enzymes and metabolites) and it was possible to predict the intracellular NADPH concentration from relatively easily obtainable data (the concentration of enzymes, polyols and oxalate). This prediction might be used in screening for high NADPH levels in engineered strains or mutants of other organisms.
R Poulsen
Bjarne
B
Molecular Genetics of Industrial Microorganisms, Wageningen University, The Netherlands.
Nøhr
Jane
J
Douthwaite
Stephen
S
Hansen
Line V
LV
Iversen
Jens J L
JJ
Visser
Jaap
J
Ruijter
George J G
GJ
D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics; D016018:Least-Squares Analysis; D008954:Models, Biological; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism*; D010427:Pentose Phosphate Pathway / Q000502:physiology*; D010734:Phosphogluconate Dehydrogenase / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D012044:Regression Analysis; D014174:Transketolase / Q000235:genetics
D017931:DNA Primers; D011994:Recombinant Proteins; D009249:NADP; D010734:Phosphogluconate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; D014174:Transketolase