Genome-Wide Identification of Genes Involved in NADPH Supply

Ye, L., Technical University of Denmark
Cardinale, S., Technical University of Denmark
Siedler, S., Technical University of Denmark
Ellabaan, M., Technical University of Denmark
Kim, S. H., Danmarks Tekniske Universitet

NADPH is a crucial cofactor in reductive enzymatic reactions to synthesize valuable bioactive compounds. Previous designs to increase intracellular NADPH availability mainly rely on regeneration of NADPH by overexpressing NADP+-dependent dehydrogenases, or strengthening of the oxidative branch of pentose phosphate pathway by abolishing the function of certain enzymes. However, modification in central metabolic pathway may cause unexpected growth defect, such as the deletion of phosphoglucose isomerase (pgi) gene would slow down more than 50% of the growth rate compares to wildtype. In this study, we prove the possibility to enrich the NADPH pool via engineering the E. coli K-12 strain based on genome wide screens. To identify mutants with both higher NADPH level and improved fitness, we combined the fast fitness quantification method by sequencing random barcoded transposon library (RB-TnSeq) and fluorescence-activated cell sorting (FACS) based on a NADPH biosensor. The mutant library (approximately 6×105 mutants each with unique barcode distributed in 3,550 genes) was constructed and characterized, and then transformed with plasmids containing the NADPH biosensor and different NADPH regenerating enzymes under an inducible promoter.  Libraries were selected in NADPH consuming and regenerating scenarios, fitness/fluorescence oscillation between the two opposite redox conditions allows us to profile the intracellular NADPH level of each mutant. Thirty mutants were selected for better performance in NADPH consuming condition. Fifteen of these mutants were confirmed to have increased NADPH/NADP+ ratio from 12% to 62% compared to the wildtype. These mutants have transposon insertion particularly in oxidoreductases, flagella assembly components, transcriptional regulators, or secretion proteins.