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 P_trc, 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.