Improved Clostridia to produce building-block chemicals

Abstract

Microorganisms producing biofuels and building-block chemicals (i.e. organic acids) have the potential to be the basis of economically competitive bioprocesses. C. saccharoperbutylacetonicum N1-4HMT is one of the most valuable industrial organisms because of its butanol production, which produces organic acids during the first stage of fermentation, the acidogenic phase. One of the main challenges in producing organic acids is the low tolerance to the acidic environment generated by the organic acids. The project aimed to generate Clostridial strains that are more resistant to organic acid and hence generate improved bioprocesses. Several organisms can regulate their lipid membrane fluidity (saturation/unsaturation ratio) to increase their tolerance to their environment. From the different techniques available to detect unsaturation in the lipids, the iodine value was adapted to be used in biological membranes obtaining results that correlate with reported values. Another problem with organic acid production is the accumulation of protons inside the cell due to their dissociation. The cation/proton antiporters NhaA and Cpa1 were chosen candidates because they can alleviate H+ and Na+ accumulation. The glycerol facilitator GlpF was also chosen because its overexpression has been observed during the acidogenic phase of C. acetobutylicum. C. saccharoperbutylacetonicum N1-4HMT was genetically modified using the patented CLEAVETM technology to create +NhaA, +Cpa1 and ΔGlpF. Under conditions developed at Bio-Cleave Ltd, none of the mutated strains significantly improved organic acid production. 1 % (w/v) NaCl prevented the shift to the solventogenic phase in all the strains. A mutant strain ΔPssA that has the pssA gene knocked out, under Bio-Cleave Ltd conditions, did not produce solvents but only organic acids and was the strain that produced the highest butyric acid. The work in this thesis demonstrated that C. saccharoperbutylacetonicum N1-4HMT could be adapted to produce organic acids as end-products.