Autotrophic CO2 fixation via the CBB cycle in R. eutropha is genetically determined by two operons (cbbLSXYEFPTZGKA), one of which is located on chromosome 2 (H16_B1383 to H16_B1396) and the other on megaplasmid pHG1 (PHG416 to PHG427). The chromosomal operon (cbbc) of the strain has a length of about

15.2 kilobase (kb) pairs comprising 13 genes. The second, highly homologous operon (cbbp) is located on the megaplasmid pHG1 and contains only 12 genes, totaling approximately 12.8 kb. With the exceptions of the triose-3-phosphate isomerase (tpiA, H16_A1047) and ribose-5-phosphate isomerase (rpiA, H16_ A2345), all enzymes of the CBB cycle are encoded in the cbb operons [52, 53]. The chromosomal and plasmid-borne cbb promoters in R. eutropha are functionally equivalent despite minor structural differences [54] . Transcription of all genes in either cbb operon depends on a single promoter upstream of cbbL [52, 54-56] that is subject to strong regulation [57]. Growth of R. eutropha under autotrophic condi­tions leads to high expression of the cbb operon genes [58]. The cbbR gene encodes for the transcriptional regulatory protein of the operon [55, 56]. In many organisms, the cbbR gene is typically located adjacent and in divergent orientation to its cognate operon. Inactivation or deletion of the cbbR prevents cbb operon transcription [55, 59]. The activating function of CbbR appears to be modulated by metabolites that signal the nutritional state of the cell to the cbb system. CbbR from R. eutropha is a sensor of the intracellular phosphoenol pyruvate (PEP) concentration. PEP increases the affinity of the activator to its operator target site, resulting in a decreased activat­ing potential of the CbbR protein in vitro [60] . This observation suggests that the role of CbbR in cbb operon transcription can thus be as an activator or a repressor.

The existence of subpromoters within the operons was excluded, and premature transcription termination thus represents an important mechanism leading to dif­ferential gene expression within the cbb operons of R. eutropha [61]. There is evi­dence for the participation of additional regulators in cbb control [59].

The enzymes of the entire CBB cycle are represented in Fig. 3. Additionally, the cbb operons contain cbbX and cbbY, which have no known function in R. eutropha. However, the gene rbcX from cyanobacteria, located between the two RuBisCO subunit genes, is responsible for the assembling the RuBisCO holoenzyme, together with chaperones GroEL/ES [62]. The CbbX protein product presents no homology to RbcX, but has conserved domains of the AAA family proteins, ATPase proteins
that often perform chaperone-like functions assisting in the assembly, operation, or disassembly of protein complexes [63].

The structure of the RuBisCO from R. eutropha was published (Protein Data Base 1BXN). As in most bacteria and higher plants with form I of RuBisCO, the enzyme complex is built up from eight large subunits and eight small subunits (L8S8) [64].

Distinct residues from each subunit of RuBisCO thus comprise the active site required for both carboxylation by CO2 and oxidation by O2, with the two gaseous substrates clearly competing for the same active site. The specificity factor (SF) is calculated as follows:

V K

CO2 O2

V K

O2 CO2

In (4), VCO, VO, KCO, KO refer to the Michaelis-Menten constants of

RuBisCO for the different substrates. SF defines the ratio between carboxylation and oxidation rates performed by each enzyme [65], but does not provide a direct measure of the rates, rate constants, or catalytic efficiencies of either carboxylation or oxygenation [66]. In R. eutropha SF = 75 [67].

The rate-limiting step in CO2 assimilation is catalyzed by RuBisCO, which is a very poor catalyst, exhibiting low affinity for CO2 and using O2 as an alternative substrate. Protein engineering could potentially be used to increase RuBisCO’s CO2 carboxylation activity, thereby increasing SF [68]. However, because both CO2 and O2 compete for the same active site [66], a short-term strategy to enhance IBT pro­duction would be to increase the intracellular CO2 concentration.

RuBisCO’s oxygenation reaction produces one molecule of 3-PGA and one mol­ecule of 2-phosphoglycolate [69]. The cbbZ gene, which encodes a phosphogyco — late phosphatase on the cbb operon might be an evolutionary adaptation in response to the presence of the 2-phosphoglycolate produced by RuBisCO during the oxida­tion reaction. CbbZ would prevent the accumulation of potentially toxic concentra­tions of 2-phosphoglycolate and rescue part of the carbon that would otherwise be lost through the glycolate metabolism [56]. In summary, the CBB cycle will play an important role in autotrophic IBT biosynthesis by R. eutropha and increasing car­bon flux through the CBB cycle is likely to enhance the IBT production rate.