Lactococcus lactis suffers from oxidative stress and riboflavin starvation at elevated temperatures due to dissolved oxygen, which can be relieved partially by exogenously supplied riboflavin. Here we explore whether this phenomenon can be harnessed to obtain riboflavin overproducing mutants. Using a riboflavin auxotrophic L. lactis strain as a riboflavin biosensor, we screened L. lactis cultures that had been exposed to temperature induced oxidative stress for up to one year. Riboflavin secreting mutants could readily be identified, some of which had arisen after just two weeks of exposure to oxidative stress. Whole genome sequencing revealed mutations in the riboswitch, which regulate riboflavin biosynthesis. Riboflavin secretion conferred a significant increase in tolerance to oxidative stress and enabled growth at high temperatures in the presence of dissolved oxygen. It was subsequently demonstrated that vigorous aeration at high temperature (37 °C) could prompt rapid emergence of riboflavin secreting mutants. The protective effect provided by riboflavin against oxidative stress may explain the natural occurrence of lactic acid bacteria (LAB) secreting riboflavin. By optimizing fermentation conditions and eliminating lactate formation, we achieved 64 mg/L riboflavin, the highest reported titer so far for LAB, which indicates great potential for use as a riboflavin fortification agent in food.

中文翻译：

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利用氧化应激获得天然分泌核黄素的乳酸菌，用于生物强化


乳酸乳球菌在高温下由于溶解氧而遭受氧化应激和核黄素饥饿，这可以通过外源性供应的核黄素部分缓解。在这里，我们探讨了是否可以利用这种现象来获得核黄素过量产生的突变体。使用核黄素营养缺陷型乳酸乳杆菌菌株作为核黄素生物传感器，我们筛选了暴露于温度诱导氧化应激长达一年的乳酸乳杆菌培养物。分泌核黄素的突变体可以很容易地识别出来，其中一些突变体是在暴露于氧化应激仅两周后出现的。全基因组测序揭示了调节核黄素生物合成的核糖开关突变。核黄素分泌显著提高了对氧化应激的耐受性，并在溶解氧存在下能够在高温下生长。随后证明，在高温 （37 °C） 下剧烈曝气可以促使分泌核黄素的突变体迅速出现。核黄素对氧化应激提供的保护作用可以解释乳酸菌 （LAB） 分泌核黄素的自然存在。通过优化发酵条件和消除乳酸形成，我们实现了 64 mg/L 核黄素，这是迄今为止 LAB 报道的最高滴度，这表明在食品中用作核黄素强化剂具有巨大潜力。