| dc.description.abstract | 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. | en |
