Biological production of 2-butanol

Harnessing the machinery of microorganisms for producing useful chemicals has great potential. In this project, we explore if lactic acid bacteria (LAB) are suitable for producing 2-butanol, an alcohol that is currently manufactured by chemical synthesis from compounds derived from crude oil. LAB are best known for their application in dairy fermentations, but demonstrate great potential for use in other biotechnological processes. Microbial production of 2 butanol have so far met little success with titers far below those needed for industrial production. The usual precursor of biologically produced 2-butanol is meso-2,3-butanediol (mBDO), a compound derived from pyruvate. mBDO is dehydrated to 2-butanone by coenzyme B12 dependent dehydratases, and subsequently reduced to 2-butanol by alcohol dehydrogenases. In this project, I start out by evaluating if the LAB model organism Lactococcus lactis, a natural producer of mBDO is a suitable production platform for 2-butanol. I do this by introducing a coenzyme B12 dependent diol dehydratase from Klebsiella oxytoca and a 2-butanol dehydrogenase from Achromobacter xylosoxidans. Despite successful heterologous expression of the two enzyme activities, 2-butanol was not formed, and this I attributed to lack of an uptake system for coenzyme B12 in L. lactis. Secondly, the potential of transforming Lactobacillus brevis SE20 into a cell factory for production of 2-butanol is assessed. The strain was found to exhibit several traits favorable to 2-butanol production e.g. it is quite robust, is capable of producing 2-butanol from α-acetolactate, and contain the coenzyme B12 salvage pathway. Thus, expression of a single enzyme, an α-acetolactate synthase should allow 2-butanol production from sugar. Next, I investigated whether the essential mBDO dehydratase activity could be provided to L. lactis in-trans; a more simple strategy than introducing the B12 uptake system in L. lactis, or engineer the 2-butanol pathway in L. brevis. Using a co-cultivation strategy, where the diol dehydratase of L. brevis SE20 complemented an incomplete 2-butanol biosynthetic pathway in an engineered L. lactis strain, I demonstrated that 2 butanol formation was possible. A titer of 5.9 g/L and a yield of 0.58 mol/mol was achieved using a simple small-scale setup (50 mL), which are the highest reported from glucose in a one-step fermentation process. Initial attempts for scaling-up the production identified issues regarding process stability. The potential for scalingup the process is therefore still being assessed with testing of different approaches for increasing the 2-butanol titer.