Optimal cofactor swapping can increase the theoretical yield for chemical production in Escherichia coli and Saccharomyces cerevisiae.

Maintaining cofactor balance is a critical function in microorganisms, but often the native cofactor balance does not match the needs of an engineered metabolic flux state. Here, an optimization procedure is utilized to identify optimal cofactor-specificity "swaps" for oxidoreductase enzymes utilizing NAD(H) or NADP(H) in the genome-scale metabolic models of Escherichia coli and Saccharomyces cerevisiae. The theoretical yields of all native carbon-containing molecules are considered, as well as theoretical yields of twelve heterologous production pathways in E. coli. Swapping the cofactor specificity of central metabolic enzymes (especially GAPD and ALCD2x) is shown to increase NADPH production and increase theoretical yields for native products in E. coli and yeast--including L-aspartate, L-lysine, L-isoleucine, L-proline, L-serine, and putrescine--and non-native products in E. coli-including 1,3-propanediol, 3-hydroxybutyrate, 3-hydroxypropanoate, 3-hydroxyvalerate, and styrene.

[1]  Adam M. Feist,et al.  Model-driven evaluation of the production potential for growth-coupled products of Escherichia coli. , 2010, Metabolic engineering.

[2]  G. Bennett,et al.  The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures. , 2002, Metabolic engineering.

[3]  R. Cabrera,et al.  The cofactor preference of glucose‐6‐phosphate dehydrogenase from Escherichia coli– modeling the physiological production of reduced cofactors , 2012, The FEBS journal.

[4]  Y. Jang,et al.  Bio‐based production of C2–C6 platform chemicals , 2012, Biotechnology and bioengineering.

[5]  S. M. Raj,et al.  Production of 3-hydroxypropionic acid via malonyl-CoA pathway using recombinant Escherichia coli strains. , 2012, Journal of biotechnology.

[6]  M. Penttilä,et al.  Engineering Redox Cofactor Regeneration for Improved Pentose Fermentation in Saccharomyces cerevisiae , 2003, Applied and Environmental Microbiology.

[7]  Stephen P. Miller,et al.  The Biochemical Architecture of an Ancient Adaptive Landscape , 2005, Science.

[8]  J. Liao,et al.  Driving Forces Enable High-Titer Anaerobic 1-Butanol Synthesis in Escherichia coli , 2011, Applied and Environmental Microbiology.

[9]  N. Price,et al.  Genome-Scale Consequences of Cofactor Balancing in Engineered Pentose Utilization Pathways in Saccharomyces cerevisiae , 2011, PloS one.

[10]  Ka-Yiu San,et al.  Metabolic engineering of aerobic succinate production systems in Escherichia coli to improve process productivity and achieve the maximum theoretical succinate yield. , 2005, Metabolic engineering.

[11]  Bernhard O. Palsson,et al.  Connecting Extracellular Metabolomic Measurements to Intracellular Flux States in Yeast , 2022 .

[12]  B. Hahn-Hägerdal,et al.  Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains , 2008, Biotechnology for biofuels.

[13]  Kristala L. J. Prather,et al.  Biosynthesis of chiral 3-hydroxyvalerate from single propionate-unrelated carbon sources in metabolically engineered E. coli , 2010, Microbial cell factories.

[14]  Kenneth J. Kauffman,et al.  Advances in flux balance analysis. , 2003, Current opinion in biotechnology.

[15]  S. Haynie,et al.  Functional expression of prokaryotic and eukaryotic genes in Escherichia coli for conversion of glucose to p-hydroxystyrene. , 2007, Metabolic engineering.

[16]  K. Zhao,et al.  Microbial Conversion of Glycerol to 1,3-Propanediol by an Engineered Strain of Escherichia coli , 2009, Applied and Environmental Microbiology.

[17]  L. Stols,et al.  Production of succinic acid through overexpression of NAD(+)-dependent malic enzyme in an Escherichia coli mutant , 1997, Applied and environmental microbiology.

[18]  G. Bennett,et al.  Effect of different levels of NADH availability on metabolite distribution in Escherichia coli fermentation in minimal and complex media , 2004, Applied Microbiology and Biotechnology.

[19]  A. Burgard,et al.  Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol. , 2011, Nature chemical biology.

[20]  K. Prather,et al.  Metabolic Engineering of Escherichia coli for Enhanced Production of (R)- and (S)-3-Hydroxybutyrate , 2009, Applied and Environmental Microbiology.

[21]  Adam M. Feist,et al.  A comprehensive genome-scale reconstruction of Escherichia coli metabolism—2011 , 2011, Molecular systems biology.

[22]  Christoph Wittmann,et al.  Systems and synthetic metabolic engineering for amino acid production - the heartbeat of industrial strain development. , 2012, Current opinion in biotechnology.

[23]  J. Russell,et al.  Energetics of bacterial growth: balance of anabolic and catabolic reactions. , 1995, Microbiological reviews.

[24]  U. Sauer,et al.  The Soluble and Membrane-bound Transhydrogenases UdhA and PntAB Have Divergent Functions in NADPH Metabolism of Escherichia coli* , 2004, Journal of Biological Chemistry.

[25]  G. Bennett,et al.  The effect of NAPRTase overexpression on the total levels of NAD, the NADH/NAD+ ratio, and the distribution of metabolites in Escherichia coli. , 2002, Metabolic engineering.

[26]  Ronan M. T. Fleming,et al.  Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0 , 2007, Nature Protocols.

[27]  John de la Mothe,et al.  Driving forces , 1991, Nature.

[28]  G. Bennett,et al.  Effect of Overexpression of a Soluble Pyridine Nucleotide Transhydrogenase (UdhA) on the Production of Poly(3‐hydroxybutyrate) in Escherichia coli , 2006, Biotechnology progress.

[29]  Meiyappan Lakshmanan,et al.  Genome-scale in silico modeling and analysis for designing synthetic terpenoid-producing microbial cell factories , 2013 .

[30]  Cong T. Trinh.  Elucidating and reprogramming Escherichia coli metabolisms for obligate anaerobic n-butanol and isobutanol production , 2012, Applied Microbiology and Biotechnology.

[31]  Costas D Maranas,et al.  Analysis of NADPH supply during xylitol production by engineered Escherichia coli , 2009, Biotechnology and bioengineering.

[32]  R. Gonzalez,et al.  Fermentative Utilization of Glycerol by Escherichia coli and Its Implications for the Production of Fuels and Chemicals , 2007, Applied and Environmental Microbiology.

[33]  N. Yakandawala,et al.  Metabolic engineering of Escherichia coli to enhance phenylalanine production , 2008, Applied Microbiology and Biotechnology.

[34]  G. Bennett,et al.  Metabolic engineering and transhydrogenase effects on NADPH availability in escherichia coli , 2013, Biotechnology progress (Print).

[35]  D. Nielsen,et al.  Styrene biosynthesis from glucose by engineered E. coli. , 2011, Metabolic engineering.

[36]  T. Ohtsuki,et al.  Production of l‐2,3‐butanediol by a new pathway constructed in Escherichia coli , 2004, Letters in applied microbiology.

[37]  Markus J. Herrgård,et al.  Integrating high-throughput and computational data elucidates bacterial networks , 2004, Nature.

[38]  Tom M. Conrad,et al.  Omic data from evolved E. coli are consistent with computed optimal growth from genome-scale models , 2010, Molecular systems biology.

[39]  Yajun Yan,et al.  Enantioselective synthesis of pure (R,R)-2,3-butanediol in Escherichia coli with stereospecific secondary alcohol dehydrogenases. , 2009, Organic & biomolecular chemistry.

[40]  A. Dean,et al.  The Selective Cause of an Ancient Adaptation , 2005, Science.

[41]  Xueli Zhang,et al.  Production of l-alanine by metabolically engineered Escherichia coli , 2007, Applied Microbiology and Biotechnology.

[42]  J. Knappe,et al.  Routes of flavodoxin and ferredoxin reduction in Escherichia coli. CoA-acylating pyruvate: flavodoxin and NADPH: flavodoxin oxidoreductases participating in the activation of pyruvate formate-lyase. , 2005, European journal of biochemistry.

[43]  Ka-Yiu San,et al.  Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase. , 2002, Metabolic engineering.

[44]  J. Nielsen,et al.  Chemicals from Metabolic Pathways , 2011 .

[45]  Adam M. Feist,et al.  A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information , 2007, Molecular systems biology.

[46]  B. Palsson,et al.  Genome-scale models of microbial cells: evaluating the consequences of constraints , 2004, Nature Reviews Microbiology.

[47]  Sang Woo Seo,et al.  Model-driven rebalancing of the intracellular redox state for optimization of a heterologous n-butanol pathway in Escherichia coli. , 2013, Metabolic engineering.

[48]  S. Lee,et al.  Metabolic engineering of Escherichia coli for the production of putrescine: a four carbon diamine. , 2009, Biotechnology and bioengineering.

[49]  Ronan M. T. Fleming,et al.  Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0 , 2007, Nature Protocols.

[50]  Chengcheng Liu,et al.  Cofactor Modification Analysis: a Computational Framework to Identify cofactor specificity Engineering targets for strain Improvement , 2013, J. Bioinform. Comput. Biol..

[51]  B. Hahn-Hägerdal,et al.  Xylose reductase from Pichia stipitis with altered coenzyme preference improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae , 2009, Biotechnology for biofuels.

[52]  J. Nielsen,et al.  Expression of a cytoplasmic transhydrogenase in Saccharomyces cerevisiae results in formation of 2‐oxoglutarate due to depletion of the NADPH pool , 2001, Yeast.

[53]  Peng Wang,et al.  Biochemical properties and physiological roles of NADP-dependent malic enzyme in Escherichia coli , 2011, The Journal of Microbiology.

[54]  Adam M. Feist,et al.  Optimizing Cofactor Specificity of Oxidoreductase Enzymes for the Generation of Microbial Production Strains—OptSwap , 2013 .

[55]  D. Nielsen,et al.  Engineering microbial chemical factories to produce renewable “biomonomers” , 2012, Front. Microbio..

[56]  Chelladurai Rathnasingh,et al.  Development and evaluation of efficient recombinant Escherichia coli strains for the production of 3‐hydroxypropionic acid from glycerol , 2009, Biotechnology and bioengineering.

[57]  Yong-Su Jin,et al.  Engineering of NADPH regenerators in Escherichia coli for enhanced biotransformation , 2013, Applied Microbiology and Biotechnology.

[58]  Ka-Yiu San,et al.  Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways. , 2008, Metabolic engineering.