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Highlights A new strain, L. lactis CML B4, overproducing acetoin was obtained by mutagenesis.  This ldh-mutated strain showed reduced LDH and increased NOX activities.  Fermentations yielded >40 and 59 gL −1 acetoin in batch and fed-batch, respectively.  Maximum yields were close to 88% and productivities exceeded 2 gL −1 h −1 .  Potential use of the strain for industrial production of acetoin from bioresources. AbstractWith the aim of applying biotechnology to produce acetoin, a chemical that can be used as an aroma and as a building block for other compounds, several putative mutants with reduced lactic acid synthesis were obtained from a wild-type homolactic strain of L. lactis subjected to chemical mutagenesis. Among these mutants, a strain was isolated, CML B4, that showed reduced lactate dehydrogenase (LDH) and increased NADH oxidase (NOX) activities. Shaken flask cultures of this mutant strain mainly produced acetoin, increasing the levels produced compared to the wild-type strain by 15-fold. A point mutation detected in the ldh gene encoding LDH was probably the genetic defect responsible for this phenotype. In pH-controlled aerobic batch fermentation, the CML B4 strain produced more than 40 gL −1 acetoin, which was increased by up to 59 gL −1 in fed-batch fermentations, with yields close to 88 and 74%, respectively, and productivities exceeding 2 gL −1 h −1 . These results indicate that this strain could be used industrially as a cell factory for the production of acetoin from bioresources.