Autotrophic production of PHB in R. eutropha has been studied previously, and the fermentation parameters to maximize culture density and mitigate explosion risk
have been studied in detail [14-16]. Also, stoichiometric formulae for autotrophic biomass and PHB production have been calculated, with the latter being [14]:

4CO2 + 33H2 + 12O2 ^ C4H6O2 + 30H2O (1)

Adapting this stoichiometry for IBT production, the overall mass balance on the gaseous inputs and liquid products would be:

4CO2 + 36H2 + 12O2 ^ C4H10O + 31H2O (2)

The standard free energy (AG°) for this overall reaction is -5.0 MJ/mol IBT, which demonstrates that IBT production is not in violation of thermodynamic laws. Assuming that 50% of the electrical energy used for H2O electrolysis (AG° = 0.237 MJ/ mol H2O) is lost as heat, approximately ((0.237 MJ/mol H2) x (36 mol H2)/ (0.5)) = 17.1 MJ of electrical energy are needed to produce 1 mol of IBT (C4H10O). As the approximate heat of combustion of IBT is 2.4 MJ/mol, the IBT production process involving a recombinant R. eutropha strain discussed here should be able to convert approximately 10% of the input electrical energy to transportation fuel energy. The required input H2:O2 ratio of 3:1 can easily be achieved via H2O elec­trolysis, which produces separate H2 and O2 product streams that can be fed to IBT — producing R. eutropha cultures in any desired ratio. However, integrating the fermentation with in situ generation of H2 and O2 presents a reactor design chal­lenge, which is discussed below (see Sects. 4.1 and 4.2).

When grown autotrophically, R. eutropha generates the energy and reducing equivalents required to drive carbon fixation by the oxidation of H2 gas, catalyzed by hydrogenase enzymes. There are three types of hydrogenases present in R. eutro­pha. Two of these are energy conserving hydrogenases (Fig. 2): a membrane bound hydrogenase (MBH, Fig. 2a) and a soluble hydrogenase (SH, Fig. 2b). The third is a regulatory hydrogenase (RH), which serves as a hydrogen sensor [17].

Electrons generated by the MBH are directed into the respiratory chain, provid­ing the reducing equivalents for reduction of O2 to H2O and the proton gradient for ATP synthesis [18] . The cytoplasmic SH directly reduces NAD+ to NADH [19], which is necessary to drive carbon fixation via the Calvin-Benson-Bassham (CBB) cycle [20]. Because all three hydrogenases in R. eutropha are resistant to inhibition by ambient oxygen concentrations, H2 oxidation can be coupled to the reduction of O2, a rare property among microorganisms [21]. Despite the presence of a hydrogen sensor, expression of both energy-conserving hydrogenases is linked to global energy level, not the amount of available hydrogen [22]. The combination of consti­tutive expression and oxygen resistance allows use of the hydrogenases in aerobic processes, such as the autotrophic production of IBT.

For design of an IBT production pathway in R. eutropha. carbon must first be diverted from PHB biosynthesis. A deletion of the PHA synthase gene, phaC, abol­ishes PHB production [23]. However, to maximize carbon flow to IBT, the b-keto — thiolase and acetoacetyl-CoA reductase genes (phaA and phaB, respectively) have also been deleted. Further optimization of carbon flow requires that many of the

genes and enzymes needed for the IBT biosynthesis pathway (Fig. 1), most which are native to R. eutropha, be expressed concomitantly and that the temporal expres­sion of the pathway is highest during “carbon storage” conditions.

The CBB cycle is used by a vast majority of autotrophic microorganisms for CO2 assimilation, and is often coupled with photosynthesis. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the key enzyme of the cycle. RuBisCO is a bifunctional enzyme that is involved both in photosynthesis and photorespiration in photosynthetic organisms. The enzyme catalyzes the initial step in the fixation of CO2. In this reaction, one molecule of CO2 is added to ribulose-1,5-bisphosphate, yielding two molecules of 3-phosphoglycerate (3-PGA) which can be metabolized to pyruvate or other central metabolites. When R. eutropha is growing autotrophi- cally, the reducing equivalents required for the CBB cycle are generated by the oxidation of hydrogen, rather than photosynthesis. Figure 3 shows a schematic of the R. eutropha CBB pathway.

To enhance CO2 fixation, organisms have developed efficient methods to acquire inorganic carbon. Carbonic anhydrase (carbonate dehydratase) enzymes play important

Fructose-6-Р

Fig-3 Schematic diagram of the Calvin-Benson-Bassham (CBB) cycle in R. eutropha. Ribulose- 5-phosphate is phosphorylated by the enzyme phosphoribulose kinase (CbbP). The resulting com­pound, ribulose-1,5-bisphosphate is then carboxylated by ribulose-1,5-bisphosphate carboxylase/ oxygenase (RuBisCO) (CbbL and CbbS) The outcome of this carboxylation are two molecules of 3-phosphoglycerate (3-PGA). 3-PGA is phosphorylated by phosphoglycerate kinase (CbbK) to yield 1,3-bisphosphoglycerate (1,3-BP). 1,3-Bisphosphoglycerate is reduced by NADPH to yield NADP+ and glyceraldehyde-3-phosphate (GAP) by glyceraldehyde-3-phosphate dehydrogenase (CbbG). GAP is then converted fructose-6-phosphate (F6P) by aldolase (CbbA) and fructose bis — phosphatase (CbbF). The reversible reactions of the reductive pentose phosphate cycle involving erythrose-4-phosphate, fructose-6P, sedoheptulose-7P, xylulose-5P, and ribose-5-P are catalyzed by the enzymes: Transketolase (CbbT), fructose-bisphosphate aldolase (CbbA), fructose/sedohep — tulose bisphosphatase (CbbF), ribulose-5-epimerase (CbbE), and triosephosphate isomerase (TpiA). Ribose-5P is isomerized by ribose-5-phosphate isomerase (RpiA) to yield ribulose-5P, which can then be put back into the cycle [27, 96] roles in this process [24]. Carbonic anhydrases (CAs) are zinc-containing enzymes that catalyze the reversible formation of bicarbonate (HCO3-) from water and carbon diox­ide [25]. These enzymes are important to many physiological processes as well, such as respiration, photosynthesis, transport, and autotrophic fixation of CO2 as well as HCO3- or H+ coupled ion transport, pH regulation, or carboxylation reactions. Recent work has shown that CAs are present in a wide range of metabolically diverse species from both Archaea and Bacteria, indicating that the enzyme has a more extensive and fundamental role in prokaryotic biology than previously recognized [24, 26]. Maintenance of the optimal CO2 concentration in the R. eutropha cell during auto­trophic fermentation avoids CO2 limitation during carbon fixation by the CBB cycle and, in consequence, ensures optimal IBT production. The action of CAs could play a central role in this process.

The 3-PGA produced in the CBB cycle is converted into pyruvate [27] , which can be utilized by the branched-chain amino acid (BCAA) production pathway to produce the intermediate ketoisovalerate (KIV) (Fig. 1). The BCAA valine, leucine, and isoleucine are synthesized by plants, algae, fungi, Bacteria, and Archaea through

Fig. 4 Schematic diagram of BCAA metabolism in R. eutropha. Pyruvate is the common precur­sor, which is reacted by acetohydroxyacid synthase (AHAS). AHAS can also incorporate 2-keto — butyrate, allowing a branch point to isoleucine biosynthesis. Ketoacid reductoisomerase (KARI) and dihydroxyacid dehydratase (DHAD) then produce the key intermediate 2-ketoisovalerate (KIV). A transaminase (TA) produces valine from 2-KIV. The IBT production pathway competes with TA for 2-KIV. Other enzymes: IPMS isopropylmalate synthase; IPMD isopropylmalate syn­thase; IPMDH isopropylmalate dehydrogenase

a common pathway [28] . The common enzymes in BCAA biosynthesis pathways (Fig. 4) are acetohydroxyacid synthase (AHAS), ketoacid reductoisomerase (KARI), dihydroxyacid dehydratase (DHAD), and transaminase (TA). These enzymes are involved in synthesis of all three BCAAs, and their expression and activity are tightly regulated through tRNABCAA repression, substrate specificity, and feedback inhibition. R. eutropha BCAA biosynthesis enzymes have not been studied previ­ously. However, sequence alignment with other characterized BCAA biosynthesis enzymes from E. coli, Corynebacterium glutamicum, Bacillus subtilis, and Streptomyces avermitilis revealed that the R. eutropha enzymes are most similar to the ones from E. coli on the level of primary sequence.

The KIV produced in the BCAA pathway is a key intermediate in IBT production (Fig. 4). To decarboxylate KIV to isobutyraldehyde, the precursor of IBT, a heter­ologous enzyme must be expressed in R. eutropha. To accomplish this, the ketois — ovalerate decarboxylase (kivd) gene from Lactococcus lactis is expressed, either on a plasmid or inserted into the R. eutropha genome, to allow production of isobutyr — aldehyde from 2-KIV (data not shown). Subsequently, an alcohol dehydrogenase (Adh) converts isobutyraldehyde to IBT. Initial assays for Adh activity in cell extracts of R. eutropha using isobutyraldehyde as the substrate yielded no detectable activity. Multifunctional Adh activity is repressed in R. eutropha during growth under ambient O2 concentrations [29-31]. Thus, a native, constitutively expressed Adh or a heterologous Adh with substrate specificity for isobutyraldehyde is needed for the final step of IBT production. A “short-chain alcohol dehydrogenase” has
been described in R. eutropha, but in wild-type cells is only expressed under anaerobic conditions [31]. Thus, the enzyme must be expressed under the right conditions for use in IBT production. Additionally, heterologous Adh enzymes, such as YqhD, have shown promise for converting isobutyraldehyde to IBT. The yqhD gene from

E. coli [32] has been used in previous iterations of IBT production pathways in heterotrophic organisms [4, 33].