Manipulation of storage polysaccharides in microorganisms
White, Joseph John
MetadataShow full item record
The rising demand for arable land, to meet the competing needs of food and energy for a growing population, will soon become unmanageable. There is therefore a pressing need to increase the efficiency with which these demands are met, or find alternative ways of meeting them. In this work, E. coli transformed with a copy of its own ADP-glucose pyrophosphorylase gene (glgC), under a lac promoter, was found to synthesise large inclusion bodies when grown in media supplemented with lactose and IPTG. Analysis of these inclusion bodies suggests that they are formed of polysaccharide, giving the cell a significantly higher total sugar content than a control grown under the same conditions. The inclusion bodies were found to react strongly with iodine, turning a blackish brown colour normally associated with iodine-starch reactions. Results also indicate that the bacteria are unable to digest these inclusion bodies once they have been formed. These findings suggest the presence of long α-helices, which would both bind with iodine and prohibit enzymatic digestion. It was therefore hypothesised that the extra GlgC enzymes were allowing glucan chains to extend at a faster rate during glycogen synthesis, leading to unbranched regions that were long enough to wind themselves into helices. The subsequent introduction of a copy of the E. coli branching enzyme gene (glgB), under the same lac promoter, was therefore expected to abolish the inclusion body phenotype, by allowing the branching of the polysaccharide to keep speed with the synthesis of its linear chains. This was indeed found to be the case. However, cells transformed with both additional gene copies were also found to accumulate a significantly higher total sugar content again: more than twice that of cells transformed with glgC alone and more than seven times that of a control, when grown under conditions designed to optimise polysaccharide synthesis. These transformants were also observed to grow to higher cell densities that a control, in various growth media. The results of both these transformations could be significant in meeting the demands of our growing society. In particular, the use of cyanobacterial glycogen as a carbon source for biofuels has recently been gaining interest, and the work presented here may well be applicable in this field, providing the possibility to significantly increase yields. Lastly, the effects of Isoamylases and Granule Bound Starch Synthase, taken from two starch producing organisms – Zea mays and Ostreococcus tauri – were investigated in an E. coli host. Results were inconclusive, but suggest many avenues for continuing the work.