Metabolic engineering of Clostridium autoethanogenum for the production of 2,3-butanediol from carbon monoxide

Abstract

Clostridium autoethanogenum belongs to the group of acetogenic bacteria that utilizes carbon monoxide as carbon and energy source to grow and produce ethanol and 2,3-butanediol as well as acetic acid. Ethanol has an application as fuel, while 2,3-butanediol has value as an important precursor molecule for industrial chemicals produced at high volumes. The purpose of this thesis was to utilize a metabolic engineering approach by overexpressing genes in the 2,3-butanediol biosynthetic pathway and by disrupting genes in other competing pathways in order to maximise the carbon ux to 2,3-butanediol. A C. autoethanogenum strain which had been performing to produce ethanol and 2,3-butanediol in large-scale gas fermentation applications was used as the parent strain. The overexpression experiments targeted the three native genes, PFOR, alsS , and alsD. Recombinant plasmids were used to carry individual genes or gene combinations into the cell. The genes with the greatest potential to disrupt in order to increase the 2,3-butanediol selectivity were selected based on a genome-scale model for this organism. The six selected genes, aor1 , aor2 , pheA, idh, leuA and pck, were disrupted using the ClosTron technology. Initial screening data from the overexpression experiments indicated that the clear increase in 2,3-butanediol may be mainly associated with the overexpression of the alsD gene. A positive contribution of the alsS gene may be argued when both alsS and alsD gene were overexpressed although the results were not statistically signi cant. Further, overexpressing the PFOR gene appeared to have contributed to an increase in 2,3-butanediol production during the stationary phase of the fermentation, when no more growth was observed. The strain with all three genes overexpressed showed an increase in 2,3-butanediol selectivity from 14.5% in the control strain to 22.5% when characterized under continuous conditions in a continuously-stirred tank reactor. The gene disruption set of experiments did not show any improvement in 2,3-butanediol selectivity. Unexpectedly, metabolite analysis of a mutant in which the pheA gene was knocked out revealed the synthesis of three new compounds. These also were cis-4-hydroxycyclohexanecarboxylic acid, 3,4-dihydroxybenzoic acid and 2-aminobenzoic acid. A relatively high concentration of cis-4-hydroxy cyclohexanecarboxylic acid (880 mg/L) was measured. Furthermore, the fact that the pathway is not yet known makes it an interesting strain to be further investigated. Key words: Carbon Monoxide, 2,3-Butanediol, Clostridium autoethanogenum, Continuous Fermentation, Gas Fermentation, ClosTron, Overexpression