In R. eutropha cells grown in the presence of O2 under heterotrophic conditions, no detectable Adh activity is seen (Sinskey laboratory, data not shown). Adh has been assayed in R. eutropha grown under anaerobic conditions [31]. To ensure IBT production under aerobic autotrophic conditions, the use of a constitutively expressed, broad substrate specificity Adh enzyme is required. A broad substrate specificity Adh has been characterized in R. eutropha, encoded by the adh gene (locus tag H16_A0757). This enzyme has been shown to exhibit activity on ethanol and 2,3-propanediol [29-31]. Constitutively expressed adh mutants have been iso­lated and characterized. These mutant strains contain alterations in the promoter region of adh, and in some cases have been shown to utilize short-chain alcohols for growth [29]. The Adh enzyme has recently been demonstrated to use isobutyralde- hyde as a substrate to produce IBT (Sinskey laboratory, data not shown). The R. eutropha strains constitutively expressing adh can be used as parental strains to produce the IBT production strain.

E. coli alcohol dehydrogenase YqhD catalyzes the reaction between many alco­hols and their corresponding aldehydes. YqhD belongs to the NADPH-dependent Adh superfamily and is subclassified as zinc-dependent long chain Adh [32] . The crystal structure of YqhD reveals Zn(II) and NADPH as cofactors for catalysis [76]. The active site contains a relative large substrate-binding pocket, which explains its ability to catalyze reactions involving both normal and branched-chain aliphatic and aromatic alcohols and their corresponding aldehydes and ketones. Using YqhD for IBT production could affect the cofactor balance. Because many native metabolic pathways require NADPH, an IBT biosynthetic pathway constructed with an NADPH-dependent enzyme could disturb the native metabolism by competing for this cofactor necessary for growth and maintenance, thus potentially resulting in slower growth or a decreased production yield. Clostridium acetobutylicum has two NADH-dependent Adhs, AdhEl, and AdhE2. AdhEl is expressed under anaerobic conditions and is active towards ethanol, acetaldehyde, butanol, and butyraldehyde [77]. AdhE2 has the same substrate specificity as AdhEl, but is only expressed in high NADH/NAD+ ratio alcohologenic cultures. AdhE2 has a conserved iron-bind­ing motif and is hypothesized to require Fe2+ as a cofactor for catalytic activity [78]. The activities of AdhEl and AdhE2 towards the reduction of isobutyraldehyde have not been explored. AdhEl and AdhE2 are potentially advantageous for IBT produc­tion because they are both NADH-dependent and can use NADH produced directly from the oxidation of hydrogen by the soluble hydrogenase.

The UdhA reaction occurs in an energy (ATP)-independent manner. In a heterolo­gous E. coli system, the reversibility of UdhA was exploited via overexpression of the enzyme to increase NADPH availability and PHB production [82]. The increased intracellular NADPH concentration allowed a greater cofactor availability for the acetoacetyl-CoA dehydrogenase reaction [82]. A similar strategy was used in Pseudomonas fluorescens to increase production ofhydromorphone [83]. This strat­egy for altering redox cofactor pool size can be used in the IBT synthesizing strain of

R. eutropha to ensure efficient activities of NADPH-requiring enzymes in the biosyn­thetic pathway.

In most organisms, the redox cofactor NADPH is generated via the pentose — phosphate pathway by glucose-6-phosphate dehydrogenase and 6-phosphoglucon — ate dehydrogenase. Proteomic studies of R. eutropha revealed no active

6- phosphogluconate dehydrogenase under heterotrophic growth conditions [ 27], suggesting that R. eutropha synthesizes its cofactor NADPH from other pathway(s) during organoheterotrophic growth. One potential pathway involves the maeA or maeB genes, encoding malic enzyme. The malic enzyme catalyzes the conversion of malate to pyruvate and is part of a metabolic cycle that also includes pyruvate carboxylase and malate dehydrogenase (Fig. 5b). An increase in transcription of the malic enzyme gene also upregulates the transcription of both pyruvate carboxylase and malate dehydrogenase [84]. Another enzyme, nonphosphorylating glyceralde — hyde 3-phosphate dehydrogenase (GapN) bypasses 1,3-bisphospho-D-glycerate in glycolysis and generates an additional NADPH from NADH, at the expense of one ATP (Fig. 5c). In order to increase the intracellular NADPH pool, udhA and gapN can be heterologously expressed in R. eutropha or maeA could be overexpressed to increase IBT production.