COBIOT-1202; NO. OF PAGES 6 Please cite this article in press as: Wang Y, et al.: Cofactor engineering for advancing chemical biotechnology, Curr Opin Biotechnol (2013), http://dx.doi.org/10.1016/j.copbio.2013.03.022 Cofactor engineering for advancing chemical biotechnology Yipeng Wang 1 , Ka-Yiu San 2,3 and George N Bennett 1 Cofactors provide redox carriers for biosynthetic reactions, catabolic reactions and act as important agents in transfer of energy for the cell. Recent advances in manipulating cofactors include culture conditions or additive alterations, genetic modification of host pathways for increased availability of desired cofactor, changes in enzyme cofactor specificity, and introduction of novel redox partners to form effective circuits for biochemical processes and biocatalysts. Genetic strategies to employ ferredoxin, NADH and NADPH most effectively in natural or novel pathways have improved yield and efficiency of large-scale processes for fuels and chemicals and have been demonstrated with a variety of microbial organisms. Addresses 1 Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77005, USA 2 Department of Bioengineering, Rice University, Houston, TX 77005, USA 3 Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX 77251, USA Corresponding author: Bennett, George N (gbennett@rice.edu, gbennett@bioc.rice.edu) Current Opinion in Biotechnology 2013, 24:xx–yy This review comes from a themed issue on Chemical biotechnology Edited by Kristala LJ Prather 0958-1669X/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.copbio.2013.03.022 Introduction Cofactors provide redox carriers for biosynthetic reac- tions, catabolic reactions and act as important agents in transfer of energy for the cell. Some examples illustrating the requirement for cofactor balance and availability include: the conversion of biomass feedstocks containing xylose to ethanol where the formation of xylitol is a problem [1,2  ,3–7]; as a driving force for more effective production of reduced compounds such as biofuels [8  ]; in using cytochrome P450s in specific oxidation reactions where the recycling of active enzyme is required [9–11]; and the production of chiral pharmaceutical intermedi- ates where specific reductions require a certain cofactor [12,13]. Experimental studies along with more complete computational models have shown a global picture of the flow of reducing equivalents and its connection to cell physiology and allowed these insights to be considered for metabolic engineering purposes [14–17]. The synthetic biology revolution has allowed easier ways to construct combinations of redox complexes and connect them to give a focused controllable circuit that can be directed to a desired metabolite [18]. Advances in protein engineering also have helped direct cell redox metabolism toward desired ends [6,19  ,20–23]. These advances intertwine with the theme of generating effective bioprocesses for fuels and chemicals from biomass and more recently from H 2 /CO 2 or electrodes [24,25]. The brief review will cover some recent developments in analyzing redox flow in microbial cells and its physiological consequences [26], engineering proteins and pathways for optimal perform- ance and applications of cofactor engineering to com- mercial processes. Studies connecting cofactor balance to cell physiology The effect of extracellular additives on redox cofactor physiology has been observed in several reports. The addition of hydrogenase substrates/inhibitors can affect the availability of NADH and lead to higher butyrate formation in clostridium cultures [27]. Electron mediators such as methyl viologen or neutral red can also serve to increase butyrate productivity [28] and demonstrate poten- tial for electrically driven acidogenesis. The addition of pyridine nucleotide precursors to the medium improved NADPH pools in Escherichia coli and aided formation of a chiral pharmaceutical intermediate [29]. Experimental and modeling studies of the contribution of the pentose phos- phate pathway in forming NADPH have been published. As demand for NADPH was increased, cells increased flux through the pentose phosphate and acetate pathways and employed a cytosolic transhydrogenase [15]. The group also developed a constraint-based model to analyze the contribution of pathways producing NADPH [14]. The model supported the findings and they proposed that a glycerol-DHA futile cycle could provide additional NADPH. These studies have shown a variety of effects of cofactor manipulation on cellular metabolism. Recent studies of redox reactions in a variety of anaerobes have shown the importance of ferredoxins as electron carriers and the recent discovery of bifurcating systems coupling this cofactor to generating membrane gradients and regeneration of NADH/NADPH while catalyzing lower energy requiring reductions has brought new appreciation to their role as major carriers of electron flow and allowed initial efforts to engineer such systems in a useful fashion. Ferredoxin is an electron transfer carrier with a low midpoint potential so the reduced form is a potent reducing agent. The analysis of the specificity of the partner and coupling mechanism has been an active Available online at www.sciencedirect.com www.sciencedirect.com Current Opinion in Biotechnology 2013, 24:1–6