3.1 Carbon Sinks

As mentioned in the pathway overview, removing alternative sinks of pathway intermediates would improve the IBT yield. However, when the removal of carbon sink creates auxotrophy, supplementation of the essential nutrient will drive up the production cost. Promoter regions of genes exhibiting low-level constitutive expres­sion during conditions permissive for IBT production have been identified based on microarray analysis of R. eutropha during nitrogen limitation (Brigham et al., man­uscript in preparation). These promoters will be used to maintain low expression levels of genes encoding essential metabolic reactions, thus preventing auxotrophy while limiting carbon flux to alternative sinks.

The 3-phosphoglycerate (3-PGA) generated in the CBB-cycle is converted to pyruvate via glycerate-2-phosphate and PEP. Since pyruvate is the only substrate of valine synthesis, control of its concentration is essential for high IBT yield. As a key intermediate in central carbon metabolism, pyruvate concentration is tightly regu­lated, for example through the PEP-pyruvate-oxaloacetate node [27, 85]. The high PHB accumulation during nutrient starvation suggests that the stringent response of R. eutropha keeps a tight control on any sink but PHB synthesis. This notion is confirmed by the excretion of pyruvate when the PHB synthesis operon is disrupted [35]. This control of pyruvate sinks can be used to optimize IBT production using a two-stage fermentation strategy (see Sect. 4.2).

pathway and their roles in biosyn­theses of other molecules. (a) Pyruvate and (b) 2-KIV. Shown in bold are enzymes that utilize these key intermediates as substrates

The final precursor of valine, 2-KIV, can be decarboxylated by a heterologous Kivd to yield isobutyraldehyde, the direct precursor of IBT. However, KIV is also a substrate of seven additional enzymes in R. eutropha (Fig. 6). To optimize the flux from KIV to IBT, alternate utilization of KIV needs to be minimized.

Of the seven enzymes representing KIV sinks, at least three are likely to be essential for survival of R. eutropha without auxotrophy. Isopropylmalate synthase (LeuA1, Fig. 6) is essential for leucine synthesis, BCAA aminotransferase (IlvE, Fig. 6) catalyzes the final step of valine synthesis, and 3-methyl-2-oxobutanoate
hydroxymethyltransferase (PanB, Fig. 6) catalyzes the formation of 2-dehydropan — tanoate, a precursor of Coenzyme A (CoA) [86]. Since use of an auxotrophic strain for IBT production would increase the cost of production, the promoter exchange strategy discussed previously could be used to minimize the activity of the above enzymes.

The remaining four enzymes representing KIV sinks can likely be deleted from the genome without auxotrophy, either because the enzyme is nonessential or has a redundant function. Leucine dehydrogenase (B0449, Fig. 6) and aminotransferase AlaT (Fig. 6) catalyze the conversion of KIV to valine and are thus redundant with BCAA aminotransferase [ 86] . Additionally, the second copy of isopropylmalate synthase (LeuA2, Fig. 6) will be removed from the genome to minimize the amount of protein available. Finally, 2-ketoisovalerate dehydrogenase (BkdAB, Fig. 6) is involved in valine degradation [86]. Minimizing degradation of valine will reduce the necessity to synthesize it from KIV; thus removing the genes encoding this enzyme from the genome offers two potential benefits.