The potential advantages for fermentation production of chemicals at high temperatures are attractive, such as promoting the rate of biochemical reactions, reducing the risk of contamination and the energy consumption for fermenter cooling. In this work, we de novo engineered the thermophile Geobacillus thermoglucosidasius to produce riboflavin, since this bacterium can ferment diverse carbohydrates at an optimal temperature of 60°C with a high growth rate. We first introduced a heterogeneous riboflavin biosynthetic gene cluster and enabled the strain to produce detectable riboflavin (28.7 mg l⁻¹). Then, with the aid of an improved gene replacement method, we preformed metabolic engineering in this strain, including replacement of ribC_Gtg with a mutant allele to weaken the consumption of riboflavin, manipulation of purine pathway to enhance precursor supply, deletion of ccpN_Gtg to tune central carbon catabolism towards riboflavin production and elimination of the lactate dehydrogenase gene to block the dominating product lactic acid. Finally, the engineered strain could produce riboflavin with the titre of 1034.5 mg l⁻¹ after 12‐h fermentation in a mineral salt medium, indicating G. thermoglucosidasius is a promising host to develop high‐temperature cell factory of riboflavin production. This is the first demonstration of riboflavin production in thermophilic bacteria at an elevated temperature.

中文翻译：

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工程热嗜热葡糖芽孢杆菌用于生产核黄素

在高温下发酵生产化学药品的潜在优势是吸引人的，例如提高了生化反应的速度，降低了污染的风险以及降低了发酵罐冷却的能耗。在这项工作中，我们从头设计了嗜热性嗜热热地芽孢杆菌（Geobacillus thermoglucosidasius）以生产核黄素，因为该细菌可以在60°C的最佳温度下以高生长速率发酵各种碳水化合物。我们首先引入了异质核黄素生物合成基因簇，并使该菌株能够产生可检测的核黄素（28.7 mg l ^-1）。然后，借助改良的基因替换方法，我们对该菌株进行了代谢工程，包括替换了ribC。具有突变型等位基因的_Gtg可以减弱核黄素的消耗，操纵嘌呤途径以增强前体供应，删除ccpN _Gtg以调节中心碳分解代谢朝着核黄素的生成以及消除乳酸脱氢酶基因以阻断主要产物乳酸。最后，经过工程改造的菌株在无机盐培养基中发酵12小时后，可产生滴度为1034.5 mg l ^-1的核黄素，这表明热葡萄糖假单胞菌是发展核黄素生产高温细胞工厂的有希望的宿主。这是在高温下嗜热细菌中核黄素生成的首次证明。