B-pinene synthesis in synechococcus sp. pcc 7002 cyanobacteria: metabolic engineering for high-density biofuel applications
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In light of escalating concern over global climate change and the declining availability of non-renewable fossil fuels, a significant body of research has accumulated which explores renewable sources for comparable products, including direct biosynthesis of high-energy compounds by microorganisms. While Escherichia coli is an obvious host due to its genetic malleability, cyanobacteria are both genetically tractable and photosynthetic, and thus present an attractive option for sustainable biosynthesis. Pinenes (α and β forms) are bicyclic monoterpenes with an energy density comparable to some high-performance aviation fuels; when dimerized, their energetic properties are functionally identical to JP-10, a jet propellant employed by the US Navy. Due to its high cost of manufacture, JP-10 is presently limited to use in ramjet missiles, but microbial synthesis of an alternative could reduce the cost and open it up to a wider array of functions. Proof-of-concept production has been demonstrated in E. coli and the model cyanobacterium Synechocystis sp. PCC 6803 using pinene synthases whose majority product is the α enantiomer. The β form, in addition to being less common in nature, has a higher energy density and thus is potentially more valuable. This study aimed to demonstrate that Synechococcus sp. PCC 7002, a fast-growing cyanobacterium, could manufacture β-pinene by supplementing the methyl-erythritol-phosphate (MEP) pathway with two enzymes: a geranyl diphosphate synthase (GPPS, adapted from Abies grandis) and a β-pinene synthase (bPinS, sourced from Artemisia annua). Four transgenic strains, carrying codon-optimized genes for these enzymes, have been produced. While sequencing and reverse transcriptase-quantitative PCR (RT-qPCR) analyses verify that the genes are unadultered and being expressed at a high level, no β- pinene output has been detected. In addition to attempting a proof-of-concept of β-pinene production by PCC 7002, this study examined possible improvements to the strain’s commercial viability, in terms of metabolic requirements and transgene maintenance. The terminal step in the methionine biosynthesis pathway may be carried out by two enzymes: MetH, which requires a vitamin B12 cofactor to proceed, and MetE, which operates independently of vitamin B12; PCC 7002 natively employs the former. By transforming with a construct which targeted an E. coli gene for MetE to the metH site, this study attempted to produce a B12-independent strain of PCC 7002 and demonstrate that metE is suitable as a nonantibiotic selectable marker. Isolating such a strain proved to be impossible by methods employed, as metE provided insufficient selective power to overcome the cellular retention of vitamin B12.
A Thesis Submitted In Partial Fulfillment of the Requirements For the Degree of Master of Science of Biology in Microbiology