Engineering Cupriavidus necator H16 for 3-hydroxypropionic acid production

As chemical production is largely sustained by oil-based products and accounts for 10% of the global final energy consumed, it is an essential target in the fight against climate change and ecosystem destruction. Biological synthesis in microorganisms has been proposed as the choice alternative for production of commodity chemicals using non-petroleum energy sources. Among the many chassis organisms under investigation, Cupriavidus necator displays many characteristics that make it ideal for the biosynthesis of such chemicals. Notably, the organism can grow lithoautotrophically (using only CO2 and H2 as carbon and energy sources) and on a range of non-traditional substrates. The main aim of this study was to engineer C. necator to produce 3-hydroxypropionic acid (3-HP), a precursor to industrial chemicals such as acrylic acid, 1,3-propandiol and poly-3-HP, with a global market estimated to be worth over $1 billion. There are numerous pathways to produce 3-HP biologically, but the malonyl-CoA pathway has been described as one of the most promising, due to the small number of genes required and its thermodynamic feasibility. The pathway is based on only two enzymes, acetyl-CoA carboxylase (ACC) and malonyl-CoA reductase (MCR): ACC converts acetyl-CoA to malonyl-CoA, which is then reduced by the action of MCR to malonate semialdehyde and finally 3-HP. In the first step, the ACC subunit encoding genes from Corynebacterium glutamicum were combined into a synthetic operon controlled by an inducible promoter and expressed in this strain together with the mcr gene from Chloroflexus aurantiacus. However, no 3-HP production was achieved. To understand why no 3-HP was seen, strain choice was evaluated, and each step of the malonyl-CoA pathway was assessed. An enzyme-based colorimetric biosensor for malonyl-CoA in C. necator H16 was developed, and results showed that neither C. glutamicum ACC expression, nor gene deletions (mcd, mmsA1/2/3, phaCAB) had any effect on malonyl-CoA availability. The same assay also demonstrated the detrimental effect of overexpressing the ACC in C. necator, with even the lowest induction level reducing cell viability greatly. Although the MCR enzyme was found to be produced, its activity was very low (0.025 μmol min−1 mg−1). Modifications known to increase MCR activity were introduced, including splitting of the enzyme into malonyl-CoA reductase and MSA reductase domains, and overexpression of a further hydroxypropionate dehydrogenase gene (ydfGEc) to enhance conversion of malonate semialdehyde (MSA) to 3-HP. Again, a 3-HP producing strain could not be generated. It was found that 3-HP and its degradation intermediate MSA appear to have unforeseen effects on the catabolism of compounds consumed by the Entner-Doudoroff pathway. In wild type cells the acid is consumed preferentially over sugars thereby repressing the genes involved in fructose breakdown. It was uncovered that 3-HP presence had undesirable effects on strains of C. necator unable to degrade it, such as such as the inability to grow on fructose and N-acetylglucosamine, and slower growth on gluconate. This could be a contributing factor in the inability of engineered C. necator strains to produce 3-HP when grown on these substrates