8. Conclusions and future prospects
The production of alginate in A. vinelandii seems to be linked to metabolic signals indicating cell damage. This observation is supported by the fact that stress sigma factors AlgU and RpoS are key regulators for alginate synthesis and that a signal derived from cell wall damage triggers alginate production [30,33,42]. Other mutations which have a stimulatory effect on alginate yield (per cell basis), such as the one blocking PHB synthesis [46], also have a negative effect on the growth capacity of A. vinelandii. Therefore, it is likely that any mutation increasing alginate production will have a negative effect upon cell growth, and consequently on the volumetric yield of alginate. It will thus be necessary to implement a different approach, in order to overcome this fact. One of these strategies might consist in developing new fermentation schemes, such as multistage fermentations, which promote better growth, in order to take advantage of the higher specific alginate production capacities of such strains. Other strategies might involve metabolic engineering of A. vinelandii in order to improve, for example, the availability of fructose 6-P, the precursor of the activated monomer GDP-mannose, by producing this metabolite directly from glucose-6-P instead of making it from trioses when growing on substrates yielding glucose [86]. On the other hand, it was previously reported that in P. aeruginosa, the introduction of multiple copy numbers of alg8 dramatically increased alginate production, suggesting that the polymerization step constitutes a bottleneck in the production of alginate [9]. It would thus be interesting to test, whether this is also the case for A. vinelandii. The existence of seven C-5 epimerases in A. vinelandii, showing a variety of epimerization patterns indicates that this bacterium is capable of producing alginates with a great variety of characteristics [11]. It would be interesting to investigate the environmental conditions which might be affecting these epimerization activities, so as to be able to produce alginates with a specific degree of epimerization and sequence distribution. From the fermentation/bioengineering side, it is evident that relatively minor improvements have been achieved in terms of the volumetric yield of alginate; however significant improvements have been achieved in terms of the molecular characteristics of the polymer, by manipulating environmental conditions. In particular, it has been shown that dissolved oxygen tension and the specific bacterial growth rate play a key role in defining the molecular weight distribution. In addition, the manipulation of culture broth components (such as MOPS) influences the acetylation degree of the polymer. This knowledge opens up many possibilities for designing processes to produce tailor-made alginates. Although recent research concerning fermentation strategies using A. vinelandii strains for the production of PHAs production is scarce, a mutant strain of this organism has shown to be potentially useful for the production of PHB and its copolymers [58]. Our understanding about the regulation of PHAs synthesis in A. vinelandii and of its metabolic relationships with other pathways has grown considerably. However this information has not been used for designing improved strains or new fermentation procedures in order to increase PHA productivity. It would be interesting to test thoroughly for the PHB production capacity among strains such as the algA or the pycA mutants, which have been shown to significantly increase the amount of accumulated PHB.
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