ABSTRACT
Metabolically engineered production strains are living microorganisms that convert a substrate to a structurally dissimilar value-added product through a pathway of biochemical reactions, catalyzed by native and recombinant enzymes. The substrates are usually derived from renewable feedstock and simultaneously serve for growth and product formation. 2.3 Bioreaction and Pathway Engineering
Single recombinant enzyme activities and whole biosynthetic pathways catalyze product formation in whole-cell biocatalysts and production strains, respectively. Various protein engineering strategies have been developed to make the involved enzymes more stable, active, or specific (Romero and Arnold, 2009; Bornscheuer et al., 2012). Here, we present strategies to engineer enzymes and pathways in order to make catalysis as a whole more effective when implemented into the reaction network of the host organism. In simple terms, this means to find the optimal balance between product formation and the availability of cofactors or metabolites provided by the host metabolism. 2.3.1 Cofactor Dependency
Many enzymes require cofactors, small organic molecules or metal ions, for their catalytic activity. Many oxidoreductases use the redox cofactors nicotinamide adenine dinucleotide NAD(H) or its phosphorylated derivative NADP(H) for electron transfer in stoichiometric amounts (Gamenara et al., 2013). The ability of the host metabolism to regenerate cofactors is therefore an important aspect when using intact cells for both whole-cell biocatalysis and metabolic engineering and influences the choice of host organism (see also Section 2.4.2).For most whole-cell biotransformations, the global redox regeneration capacity of the host metabolism does not limit pro-duction rate. However, NADPH consumption can compete with biomass formation and NAD consumption can compete with the uptake of substrates such as glycerol. If the enzyme activity used for biotransformation is well characterized, its native cofactor preference can be adapted in order to fit the metabolic prereq-uisites of the host organism. Interchanging the preference of
oxidoreductases for NAD(H) or NAD(P)H can thus minimize competition with the host metabolism and broaden the applicability of these enzymes as cofactor-regeneration system (Serov et al., 2002; Andreadeli et al., 2008; Hoelsch et al., 2013). A change of cofactor preference can also be attractive in cell-fee biotransfor-mations because NADH is more stable and cheaper than NADPH (Rosell et al., 2003; Weckbecker et al., 2010). In addition, some popular host strains such as E. coli posses the ability to transfer reducing power between NAD(H) and NADP(H) catalyzed by tran-shydrogenases, kinases, and phosphatases (Jackson, 2003; Sauer et al., 2004; Fuhrer and Sauer, 2009).
