182 / 1096-7176/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved. Metabolic Engineering 4, 182–192 (2002) doi:10.1006/mben.2001.0220, available online at http://www.idealibrary.comon Metabolic Engineering through Cofactor Manipulation and Its Effects on Metabolic Flux Redistribution in Escherichia coli Ka-Yiu San,* ,‡ George N. Bennett, † Susana J. Berríos-Rivera, ‡ Ravi V. Vadali,* Yea-Tyng Yang, † Emily Horton, † Fred B. Rudolph, † Berna Sariyar,* and Kimathi Blackwood † *Department of Bioengineering, ‡ Department of Chemical Engineering, and † Department of Biochemistry and Cell Biology, Institute of Biosciences and Bioengineering, Rice University, Houston, Texas 77005 Received October 19, 2001; accepted November 26, 2001; published online February 27, 2002 Applications of genetic engineering or metabolic engineering have increased in both academic and industrial institutions. Most current metabolic engineering studies have focused on enzyme levels and on the effect of the amplification, addition, or deletion of a particular pathway. Although it is generally known that cofactors play a major role in the production of different fermentation products, their role has not been thoroughly and systematically studied. It is conceivable that in cofactor-dependent production systems, cofactor availability and the proportion of cofactor in the active form may play an impor- tant role in dictating the overall process yield. Hence, the manipula- tion of these cofactor levels may be crucial in order to further increase production. We have demonstrated that manipulation of cofactors can be achieved by external and genetic means and these manipulations have the potential to be used as an additional tool to achieve desired metabolic goals. We have shown experimentally that the NADH/NAD + ratio can be altered by using carbon sources with different oxidation states. We have shown further that the metabolite distribution can be influenced by a change in the NADH/NAD + ratio as mediated by the oxidation state of the carbon source used. We have also demonstrated that the total NAD(H/ + ) levels can be increased by the overexpression of the pncB gene. The increase in the total NAD(H/ + ) levels can be achieved even in a complex medium, which is commonly used by most industrial processes. Finally, we have shown that manipulation of the CoA pool/flux can be used to increase the productivity of a model product, isoamyl acetate. © 2002 Elsevier Science (USA) INTRODUCTION The production of chemicals by biocatalysts has attained considerable attention. Technological advances for making genetically engineered strains, improvements in experimen- tal measurement, and theoretical analysis of metabolic fluxes have contributed to the production of high value added products ( for example, recombinant proteins for diagnostic and therapeutic uses) and the commercial intro- duction of new processes for the manufacture of certain novel chemicals (examples using engineered Escherichia coli are the production of indigo dye by Genencor and 1,3-pro- panediol by DuPont). The production of lactate, biopoly- mer PHB, and ethanol using metabolic engineering tech- niques is currently being pursued with commercial interest. Metabolic engineering has the potential to considerably improve process productivity by manipulating the through- put of certain pathways. However, despite its powerful and precise nature, the current status of metabolic engineering is still hindered by the lack of our full understanding of cellular metabolism. The aspects of integrated dynamics and overall control structure are the common obstacles for the optimal design of pathways to achieve a desired goal. Applications of genetic engineering or metabolic engineering have increased in both academic and industrial institutions and the area has been reviewed (reviews: Stephanopoulos and Vallino, 1990; Bailey, 1990; Carmeron and Chaplen, 1997; Stephanopoulos et al., 1998; Lee and Papousakis, 1999). Most current metabolic engineering studies have mainly focused on manipulating enzyme levels through the amplification, addition, or deletion of a par- ticular pathway. However, cofactors play an essential role in a large number of biochemical reactions and their mani- pulation has the potential to be used as an additional tool to achieve desired metabolic engineering goals. Furthermore, it will also provide an additional means to study cellular metabolism, in particular the interplay between cofactor levels/fluxes and metabolic fluxes. In this project, we focus on two common yet very impor- tant cofactors, nicotinamide adenine dinucleotide (NAD) and acetyl-CoA. NAD functions as a cofactor in over 300 oxidation–reduction reactions. The NADH/NAD + cofac- tor pair plays a major role in microbial catabolism, in which a carbon source, such as glucose, is oxidized using NAD + and producing reducing equivalents in the form of NADH. It is critically important for continued cell growth and product formation that this reduced NADH be oxidized to NAD + and a redox balance be achieved. Under anaerobic growth, and in the absence of an alternate oxidizing agent,