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Nisin is commonly used as a biopreservative in foods. For industrial production, nisin-producing Lactococcus lactisstrains are usually grown to high cell densities to achieve the highest possible nisin titer. However, accumulation of lactic acid eventually halts production, even in pH-controlled fermentations. Here, we describe a nisin-producing L. lactisstrain Ge001, which was obtained after transferring the nisin gene cluster from L. lactisATCC 11454, by conjugation, into the natural mutant L. lactisRD1M5, with low lactate dehydrogenase activity. The ability of Ge001 to produce nisin was tested using dairy waste as the fermentation substrate. To accommodate redox cofactor regeneration, respiration conditions were used, and to alleviate oxidative stress and to reduce adsorption of nisin onto the producing cells, we found it to be beneficial to add 1 mM Mn2+and 100 mM Ca2+, respectively. A high titer of 12 084 IU/mL nisin could be reached, which is comparable to the highest titers reported using expensive, rich media. Summing up, we here present a 100% natural, robust, and sustainable approach for producing food-grade nisin and acetoin from readily available dairy waste.