Abstract

The feasibility of increasing isoprenoid biosynthesis in the obligate photoautotrophic cyanobacterium Synechococcus elongatus PCC 7942 was explored through the use of a heterologous mevalonate pathway. Incorporation of heterologous genes that produce non-native metabolic intermediates has previously been demonstrated as a method of increasing product formation while bypassing endogenous regulatory mechanisms. While this design strategy has been exhaustively explored in model heterotrophic microbes, far less work has been carried out in photoautotrophs. Practically, this disparity is due to the preference of using faster growing heterotrophs, greater understanding of metabolic regulation in model heterotrophs, and more limited characterisation of genes and genetic regulatory elements in cyanobacterial hosts (particularly S. elongatus). Here the host response to incorporation of a heterologous mevalonate pathway under the control of non-native promoters is characterised. Analysis was focused on metabolic intermediates, endogenous isoprenoid products, and cell growth indicators, primarily using HPLC and GC-MS. The heterologous route for isoprenoid biosynthesis was found to be functional. An intermediate in this pathway, mevalonate, accumulated to 70μM under standard laboratory conditions. Recorded changes to endogenous isoprenoid products were smaller, but still apparent. Because of commercial interest in phototrophic production of isoprenoids, particularly for fuels, I also attempted to generate a previously demonstrated biofilm phenotype in S. elongatus. This phenotype is of interest for testing in evanescent field based photobioreactors, but I was unable to replicate it during the period of work.