Rewiring the metabolism of Synechocystis sp. PCC 6803 for sustainable production of acetone

Sammanfattning

The ongoing climate crisis urges us to reduce our net greenhouse gas emissions and adapt to a reality in which we are less dependent on fossil-based materials and chemicals. Cyanobacteria are photoautotrophic microbes that can be used as a carbon-neutral production platform for chemicals and fuels which are otherwise derived from fossil resources. By engineering the metabolism of these microbes, they can synthesise products of industrial interest directly from solar energy, water and carbon dioxide. The current thesis project aimed to create new strains of Synechocystis sp. PCC 6803 capable of acetone biosynthesis. As acetone is a widely used chemical derived from petroleum, it is of great interest to find ways to efficiently produce acetone in cyanobacteria instead. The first objective of the thesis project was to introduce a heterologous acetone biosynthesis pathway consisting of three enzymes: acetoacetyl-CoA synthase (NphT7), acetate-CoA transferase (AtoDA) and acetoacetate decarboxylase (Adc). Several attempts were made to introduce the genes of these enzymes to Synehcocystis, both through genome integration and expression from a replicative plasmid, but to no avail. To address the potential toxicity of either acetoacetate, an intermediate in the acetone pathway catalysed by AtoDA, or of acetone, the constitutive promoter upstream of the atoDA operon was exchanged for a metal ion inducible promoter to limit the production of acetoacetate and acetone until expression was induced. Still, no acetone production could be detected with this strain. The second objective was to create acetone-enchancing constructs targeting the upstream metabolism. Five constructs expressing enzymes of a heterologous phosphoketolase-phosphostransacetylase (PK-Pta) pathway was constructed. However, without an acetone-producing strain of cyanobacteria, the impact these modifications have on acetone synthesis in cyanobacteria could not be investigated. To get a rough estimate of the performance of the constructs and to illustrate the potential impact the PK-Pta pathway may have on acetone biosynthesis, Escherichia coli DH5α-Z1 was engineered to express these pathways and used in subsequent acetone screening with gas chromatography. The addition of the PK-Pta pathway significantly increased acetone production in E. coli. The results from the thesis project highlight the challenges of introducing an acetone pathway in Synechocystis. Future work should focus on investigating why the approaches taken in the project did not work. Aside from the potential toxicity issue, another possibility is that the problem stemmed from inefficient expression and/or activity of the enzymes. Once an acetone-platform is in place, the addition of a PK-Pta pathway can be evaluated for an increased production of acetone.