title: In silico model-driven cofactor engineering strategies for improving the overall NADP(H) turnover in microbial cell factories.
abstract: Optimizing the overall NADPH turnover is one of the key challenges in various value-added biochemical syntheses. In this work, we first analyzed the NADPH regeneration potentials of common cell factories, including Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, and Pichia pastoris across multiple environmental conditions and determined E. coli and glycerol as the best microbial chassis and most suitable carbon source, respectively. In addition, we identified optimal cofactor specificity engineering (CSE) enzyme targets, whose cofactors when switched from NAD(H) to NADP(H) improve the overall NADP(H) turnover. Among several enzyme targets, glyceraldehyde-3-phosphate dehydrogenase was recognized as a global candidate since its CSE improved the NADP(H) regeneration under most of the conditions examined. Finally, by analyzing the protein structures of all CSE enzyme targets via homology modeling, we established that the replacement of conserved glutamate or aspartate with serine in the loop region could change the cofactor dependence from NAD(H) to NADP(H).
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01, Centros, Singapore, 138668, Singapore.
authors.forename: Meiyappan
authors.initials: M
authors.lastname: Lakshmanan
authors.affiliation: Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117585, Singapore.|NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 28 Medical Drive, Singapore, 117456, Singapore.
authors.forename: Kai
authors.initials: K
authors.lastname: Yu
authors.affiliation: Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117585, Singapore.
authors.forename: Lokanand
authors.initials: L
authors.lastname: Koduru
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01, Centros, Singapore, 138668, Singapore. cheld@nus.edu.sg.|Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117585, Singapore. cheld@nus.edu.sg.|NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 28 Medical Drive, Singapore, 117456, Singapore. cheld@nus.edu.sg.
authors.forename: Dong-Yup
authors.initials: DY
authors.lastname: Lee
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology / Q000378:metabolism*; D019149:Bioreactors / Q000382:microbiology*; D003198:Computer Simulation; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010843:Pichia / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*; D012694:Serine / Q000378:metabolism
chemical_list: D004798:Enzymes; D009243:NAD; D012694:Serine; D009249:NADP; D005990:Glycerol
keywords: Cofactor modification analysis (CMA); Cofactor specificity engineering (CSE); Flux balance analysis (FBA); Metabolic engineering; NADPH