Engineering the Cyanobacterium Synechococcus Sp. PCC 7002 to Produce Commodity Chemicals

Metabolic engineering of cyanobacteria allows for the direct conversion of CO₂ and sun light into biofuels and petrochemical replacements without the need for plant derived sugars.  In addition to the photosynthetic abilities of these organisms, their inherent physiology and metabolism may make them more effective hosts for certain pathways than heterotrophic bacteria.  The goal of this research is to demonstrate a pathway for the production of 3-hydroxypropionate in which cyanobacteria are a more effective host than E. coli.  3-hydroxypropionate (3HP) is a compound of interest because it can be easily converted to a variety of commodity chemicals including acrylic acid.  The cyanobacterium Synechococcus sp. PCC 7002 was chosen as a potential host because of its fast growth rate, tractable genetics systems, and wide tolerance range to salinity and light intensity.   The first hurdle in engineering this system was addressing end product toxicity.  A loss of function mutation was identified that increases the minimum inhibitory concentration of 3HP 50-fold.  This mutation was in the gene acsA, which was annotated as any acetyl-CoA ligase.  Knocking out acsA resulted in increased tolerance to 3HP, acrylic acid, and propionate.  An enzyme assay of the purified protein demonstrated that acsA has activity towards these compounds.  The acsA knock out strain was used as a starting platform for 3HP production.  The pathway chosen for 3HP production involves the heterologous expression of a malonyl-CoA reductase from Chloroflexus aurantiacus.  This enzyme converts malonyl-CoA to 3HP.  This reaction requires the cofactor NADPH, which is at a high concentration in cyanobacteria relative to E. coli.  Expression of this gene from a native multi-copy plasmid resulted in the accumulation of 3HP.  In order to increase the production of 3HP from malonyl-CoA, additional acetyl-CoA carboxylase genes will be expressed to increase the intracellular concentration of malonyl-CoA.  The engineered strains from these experiments will be grown in a photobioreactor to determine how 3HP yields compare to E. coli strains expressing the same pathway.