Microbial Cell Factories BioMed Central Review

Specific complex interactions between soil bacteria belonging to Rhizobium, Sinorhizobium, Mesorhizobium, Phylorhizobium, Bradyrhizobium and Azorhizobium commonly known as rhizobia, and their host leguminous plants result in development of root nodules. Nodules are new organs that consist mainly of plant cells infected with bacteroids that provide the host plant with fixed nitrogen. Proper nodule development requires the synthesis and perception of signal molecules such as lipochitooligosaccharides, called Nod factors that are important for induction of nodule development. Bacterial surface polysaccharides are also crucial for establishment of successful symbiosis with legumes. Sugar polymers of rhizobia are composed of a number of different polysaccharides, such as lipopolysaccharides (LPS), capsular polysaccharides (CPS or K-antigens), neutral β-1, 2-glucans and acidic extracellular polysaccharides (EPS). Despite extensive research, the molecular function of the surface polysaccharides in symbiosis remains unclear. This review focuses on exopolysaccharides that are especially important for the invasion that leads to formation of indetermined (with persistent meristem) type of nodules on legumes such as clover, vetch, peas or alfalfa. The significance of EPS synthesis in symbiotic interactions of Rhizobium leguminosarum with clover is especially noticed. Accumulating data suggest that exopolysaccharides may be involved in invasion and nodule development, bacterial release from infection threads, bacteroid development, suppression of plant defense response and protection against plant antimicrobial compounds. Rhizobial exopolysaccharides are species-specific heteropolysaccharide polymers composed of common sugars that are substituted with non-carbohydrate residues. Synthesis of repeating units of exopolysaccharide, their modification, polymerization and export to the cell surface is controlled by clusters of genes, named exo/exs, exp or pss that are localized on rhizobial megaplasmids or chromosome. The function of these genes was identified by isolation and characterization of several mutants disabled in exopolysaccharide synthesis. The effect of exopolysaccharide deficiency on nodule development has been extensively studied. Production of exopolysaccharides is influenced by a complex network of environmental factors such as phosphate, nitrogen or sulphur. There is a strong suggestion that production of a variety of symbiotically active polysaccharides may allow rhizobial strains to adapt to changing environmental conditions and interact efficiently with legumes.


Review
Rapid industrialization has led to a dramatically accelerated consumption of fossil fuels with a consequent increase in atmospheric levels of the greenhouse gas carbon dioxide (CO 2 ). This sustained increase of atmospheric CO 2 has already initiated a chain of events with negative ecological consequences [1][2][3]. Failure to reduce these greenhouse gas emissions will have a catastrophic impact upon both the environment and the economy on a global scale [4,5]. The reduction has to be brought about by global concerted effort by all countries in order to be effective and meaningful.
At one end of the spectrum -that of generation and utilization of energy resulting in generation of carbon dioxide -hydrocarbons serve as intermediaries for energy storage. Hydrocarbons are not energy by themselves but store energy in their bonds, which is released during combustion. They are thus intermediates for obtaining stored bond energy within them and carbon dioxide is emitted as a consequence of combustion to extract this stored energy. In recent times hydrogen has received renewed attention as the potential replacement for hydrocarbons [6][7][8][9][10]. However, hydrogen too is an intermediary for obtaining stored bond energy. Recent reports suggest that hydrogen as intermediary may not be entirely free from problems. Also, the problems from use of hydrogen as fuel are yet to be fully realized or foreseen [11,12]. In all these endeavors a key question, that whether the hydrocarbons will be still retained as intermediaries in energy utilization and the problem of air pollution caused as a result of their combustion can be technologically ameliorated, has not been looked in as much detail as perhaps it should have been. This can possibly be achieved by contained handling of carbon dioxide. The contained handling and fixation of CO 2 can be achieved biotechnologically, chemically or by a combination of both.
Sugar binding proteins derived from microbial and other sources have been used for various applications such as diagnostics and affinity purification [13,14], however they have not been used in environmental biotechnological applications. The possibility of their potential application in environmental biotechnology and review of a few potential candidates is presented here.

3-Phosphoglyceraldehyde
Fructose-1,6-bisphosphate + + +   (Figure 1), which proceeds with no loss of CO 2 (unlike cellular biochemical systems) in 11 steps in a series of bioreactors [20]. This scheme is very different from generation of RuBP from D-glucose for start-up process [21] and employing 11 steps in different reactors requiring large volume and weight. The linear combination of reactors with large volume and weight are unsuit-An alternate arrangement of enzymes in the scheme outlined in Fig. 1

Figure 2
An alternate arrangement of enzymes in the scheme outlined in Fig. 1. This schemes harbors four reactors with indicated enzyme complexes enabling internal channeling, greatly reduces volume and weight for regenerating reactors with faster overall conversion rate to RuBP starting with 3PGA making the system compatible for application in mobile devices in addition to stationary emitters. The reactors may use the sugar binding entities at indicated positions, the hollow and solid symbols represent binding and release phase of the binding-molecules, the plus, circle, cylinder and box are symbols for 3PGA, DHAP, X5P and RuBP binders respectively.  In-situ separation Matrix able for use with mobile CO 2 emitters leaving only the stationary source of emission to be controlled using this technology [17]. To circumvent these problems we have devised a new scheme presented in Figure 2[22]. Based on this scheme, we have designed enzymes as functionally interacting complexes/interactomes or successive conversion in radial flow with layers of uniformly oriented enzymes in concentric circle with axial collection flow system for three enzymes in first reactor for the scheme presented in Figure 2. The four reactors harboring enzymatic complexes/mixtures replace the current 11 reactors. This leads to a faster conversion rate and requires less volume and material weight. However, 4 sugar moieties [3-phosphoglyceraldehyde (3PGAL), Dihydroxyacetone phosphate (DHAP), Xylulose-5-phosphate (X5P) and Ribulose-1, 5-bisphosphate (RuBP)] must be separated at four key steps, as illustrated in Figure 2. In figure 2, using four symbols with solid for bound state and empty for released state, for potential binders: plus for 3PGA, circle for DHAP, cylinder for X5P and box for RuBP, the possible place for utility of these binders have been depicted. In the course of this review, we will consider the availability of enzymatic proteins and non-enzymatic proteins that would be potentially useful as specific binders for these sugar molecules. With a recombinant mutant enzyme we illustrate that such an approach has potential to be used as an in-situ reversible binding matrix for sugar binding and release.

Potential utilizable sugar binding proteins in RuBP regeneration
Three categories of binding proteins can be potentially employed for differential absorption of sugars and for subsequent elution and feeding the reactors downstream in conversion cascade. These are: mutant enzymatic proteins that retain the ability of binding but completely lack any catalytic activity, lectins or proteins of non-immunogenic origin [23] having more than one binding site for the sugar (in nature they cause agglutination of due to sugar binding at multiple sites) and mutant or wild type receptors that binds sugars but are incapable of eliciting further biological activities. The desirable proteins in all these categories are those for which binding affinity is high in a condition close to pH of the emanating solution from the reactor and other conditions for reactor effluent, ability to bind reversibly with respect to some simple but easily manipulable physicochemical parameter (such as temperature, pH, salt concentration), and the ability to be easily attached to a matrix using simple chemistry without loss of binding ability and a long shelf life.  We undertook this review because, although the comprehensive information on a large number of enzymes have been accumulated in BRENDA database [24,25], but the systematic information on their mutants is lacking and non-enzymatic binders of sugar ligands are not identified / listed in the database.

Proteins binding D-Ribulose-1,5-bisphosphate
A number of Ribulose-1,5-bisphosphate and metabolizing enzymes such as Ribulose phosphate kinase and their mutants binds D-ribulose-1,5-bisphosphate. The RuBP binding entities devoid of any enzymatic activities are very valuable in reactors necessitating extraction and separation of RuBP from other sugar compounds (Table  4). Very few non-enzymatic proteins bind RuBP and none of them are microbial sources, and hence have not been The recombinant his-tagged wild-type and R38Q mutant 3-phosphoglycerate kinase was subjected to affinity purification on Ni-NTA column as described previously [20] Figure 3 The recombinant his-tagged wild-type and R38Q mutant 3-phosphoglycerate kinase was subjected to affinity purification on Ni-NTA column as described previously [20]. A. SDS-PAGE of recombinant wild-type and R38Q mutant S. cerevisiae 3-phosphoglycerate kinase. The proteins (1 and 1.8 µg respectively) was separated in 10% polyacrylamide gel and stained with Coommassie blue R250. B. TLC analysis of sugars prior to and after in-situ separation with R38Q. The recombinant R38Q mutant (R38Q-PGK) was coupled with Protein A sepharose beads and incubated overnight with a mixture of sugars, 3-phosphoglycerate (3PGA), ribulose-5-phosphate (R5P), Glucose-6-phosphate (G6P) and Fructose-6-posphate (F1,6-bP). After washing with 180 mM NaCl, the sugars were eluted with 1 M NaCl. Lane 1, mixture of sugar prior to incubation with R38Q-PGK and Lane-2 after elution with 1 M NaCl.  incorporated in this review, Rubisco associated protein from soybean is one of them, that show significant RuBP binding [137].

Illustrating example
In order to illustrate the utility of non-catalytic enzymatic mutants as specific sugar binders for in-situ separation in reactors, recombinant Saccharomyces cerevisiae 3-phosphoglycerate kinase mutant R38Q [41] was prepared. Mutagenesis was carried out using wild type protein construct in plasmid pET19b as a template. The R38Q mutant was constructed with the Quickchange/Chameleon sitedirected mutagenesis kit from stategene using primers as described elsewhere [41]. DNA sequencing of the plasmid identified the mutant. Recombinant wild-type and mutant (R38Q) 3-phosphoglycerate kinase (PGK) were purified to apparent homogeneity as described previously [20] have been shown in Figure 3A. The wild-type and mutant protein was incubated with 10 mM 3-phosphoglycerate barium salt (3PGA) in 50 mM Tris-Cl buffer, pH 7.5 containing 50 mM NaCl for overnight at room temperature. No modification of 3PGA was observed after incubation with R38Q mutant protein (data not shown). The R38Q was coupled with Protein A sepharose beads using dimethylpimelimidate. The recombinant R38Q mutant protein beads (R38Q-PGK) was incubated overnight at room temperature with a mixture of sugars, 3phosphoglycerate, barium salt (3PGA), ribulose-5-phosphate (R5P), Glucose-6-phosphate (G6P) and Fructose-6posphate (F1,6-bP) each at a concentration of 10 mM in a volume of 200 µl. After incubation they were washed with 1.5 ml of 180 mM NaCl in 50 mM Tris-Cl buffer, pH 7.5. They were subjected to elution with 1 M NaCl. Lane 1, mixture of sugar prior to incubation with R38Q-PGK and Lane-2 after elution with 1 M NaCl.

Conclusion
The enzyme-mutants lacking catalytic activity represent an important group of proteins that could be used for development of sugar-binding proteins reversible with respect to physicochemical parameters such as pH or salt concentration. Nevertheless, the non-enzymatic proteins also represent a suitable repertoire of such potential scaffolds, which could be used for development as sugar-binding proteins to be used in reactors for simultaneous separation of sugars that would be used in subsequent conversion steps. We have developed a RuBP production scheme from 3PGA [16,17] and also a de novo RuBP production scheme from D-glucose [21] for continuous CO 2 fixation and for start-up of the fixation respectively employing series of reactors. Both systems for production of RuBP will benefit from specific sugar binders but besides their use in environmental biotechnology, they will find application in diagnostics, separation technologies and also as research reagents.