5. From plate to fermentor
One of the aspects frequently considered during the selection of polysaccharide-overproducing strains on plates is the mucoid phenotype of the colonies. This is the case for the A. vinelandii mutant strain impaired in PHB synthesis (AT6). This strain was isolated in our research group and presented a highly mucoid phenotype when compared to the parental strain (ATCC 9046) (Figure 3). It is important to point out that mucoidy is determined both by the concentration of the polymer and/or its chemical characteristics, specifically its molecular mass. For this reason, both characteristics were evaluated in plates as well as in submerged cultures. Although the mutant AT6 showed a high yield in plate as well as in submerged culture [46], the alginate volumetric production and the molecular mass of the polymer were lower than the values exhibited by the parental strain (Table 1).
A similar problem can be observed when seeking for improved strains for the purpose of PHA production. The A. vinelandii pyruvate carboxylase mutant (AJ1678), characterized as PHB overproducer strain, was identified because its colonies have a higher opacity on plates containing sucrose. As expected, the opacity was due to a higher amount of PHB granules in the cells. However, when this strain was grown in shake flasks, it produced the same amount of PHB as that obtained using the wild type, probably due to the presence of the alternative enzyme, PEP carboxylase, under this condition [57].
Overall, our results reveal the limitations of selecting alginate or PHB over-producing strains, using only the phenotype observed on plates as criterion and emphasize the necessity to characterize the mutant strains, using controlled culture conditions.
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