Engineering Pseudomonas putida for the Production of 2,3-Butanediol Isomers and Acetoin.

The strain Pseudomonas putida KT2440 has been genetically modified to synthesize 2,3-butanediol (2,3-BDO). To achieve this goal, we have used as a host the P. putida KT2440 acoB::mini-Tn5 mutant unable to mineralize acetoin and 2,3-BDO. This strain has been further engineered by overexpressing in the pIZ2 plasmid, using the inducible promoter Ptrc, the budABC operon from Klebsiella oxytoca, which encodes the three enzymes required to transform pyruvate into 2,3-BDO. Flask and bioreactor experiments were carried out for the optimization of the 2,3-BDO production using acetate or glucose as substrates, which was highly dependent on the initial substrate concentration and the optimal oxygen supply. Using glucose in a fed-batch bioreactor enabled the highest diol (i.e., 2,3-BDO stereoisomers and acetoin) yield, attaining a concentration of 16 g/L, while the maximum production of diols in a fed-batch using acetate as a substrate was 1 g/L. The recombinant P. putida KT2440 acoB::mini-Tn5 (pIZ2Bud) showed a high tolerance to furfural, a contaminant produced after lignocellulose pretreatment, which can facilitate the production of 2,3-BDO from this residue. This is the first report on P. putida for the production of 2,3-BDO, which may contribute to facilitating the use of this chassis at an industrial scale.