The demand for bio-based retinol (vitamin A) is currently increasing, however its instability represents a major bottleneck in microbial production. Here, we developed an efficient method to selectively produce retinol in Yarrowia lipolytica. The β-carotene 15,15′-dioxygenase (BCO) cleaves β-carotene into retinal, which is reduced to retinol by retinol dehydrogenase (RDH). Therefore, to produce retinol, we first generated β-carotene-producing strain based on a high-lipid-producer via overexpressing genes including heterologous β-carotene biosynthetic genes, GGS1^F43I mutant of endogenous geranylgeranyl pyrophosphate synthase isolated by directed evolution, and FAD1 encoding flavin adenine dinucleotide synthetase, while deleting several genes previously known to be beneficial for carotenoid production. To produce retinol, 11 copies of BCO gene from marine bacterium 66A03 (Mb.Blh) were integrated into the rDNA sites of the β-carotene overproducer. The resulting strain produced more retinol than retinal, suggesting strong endogenous promiscuous RDH activity in Y. lipolytica. The introduction of Mb.Blh led to a considerable reduction in β-carotene level, but less than 5% of the consumed β-carotene could be detected in the form of retinal or retinol, implying severe degradation of the produced retinoids. However, addition of the antioxidant butylated hydroxytoluene (BHT) led to a >20-fold increase in retinol production, suggesting oxidative damage is the main cause of intracellular retinol degradation. Overexpression of GSH2 encoding glutathione synthetase further improved retinol production. Raman imaging revealed co-localization of retinol with lipid droplets, and extraction of retinol using Tween 80 was effective in improving retinol production. By combining BHT treatment and extraction using Tween 80, the final strain CJ2104 produced 4.86 g/L retinol and 0.26 g/L retinal in fed-batch fermentation in a 5-L bioreactor, which is the highest retinol production titer ever reported. This study demonstrates that Y. lipolytica is a suitable host for the industrial production of bio-based retinol.

目前对生物基视黄醇（维生素 A）的需求正在增加，但其不稳定性是微生物生产的主要瓶颈。在这里，我们开发了一种在解脂耶氏酵母中选择性生产视黄醇的有效方法。β-胡萝卜素 15,15'-双加氧酶 (BCO) 将 β-胡萝卜素切割成视黄醛，视黄醇被视黄醇脱氢酶 (RDH) 还原为视黄醇。因此，为了生产视黄醇，我们首先通过过表达包括异源β-胡萝卜素生物合成基因、定向进化分离的内源香叶基香叶基焦磷酸合酶突变体GGS1 ^F43I和FAD1在内的高脂质生产者产生β-胡萝卜素生产菌株。编码黄素腺嘌呤二核苷酸合成酶，同时删除几个以前已知对类胡萝卜素生产有益的基因。为了生产视黄醇，将来自海洋细菌 66A03 ( Mb.Blh ) 的11 个 BCO 基因拷贝整合到 β-胡萝卜素过度生产者的 rDNA 位点中。所得菌株产生的视黄醇比视黄醛多，表明解脂耶氏酵母中具有强烈的内源性混杂 RDH 活性。Mb.Blh的介绍导致β-胡萝卜素水平显着降低，但不到5%的消耗的β-胡萝卜素可以以视黄醛或视黄醇的形式检测到，这意味着产生的类视黄醇严重降解。然而，添加抗氧化剂丁基化羟基甲苯 (BHT) 导致视黄醇产量增加 20 倍以上，这表明氧化损伤是细胞内视黄醇降解的主要原因。GSH2的过表达编码谷胱甘肽合成酶进一步提高了视黄醇的产生。拉曼成像显示视黄醇与脂滴共定位，使用 Tween 80 提取视黄醇可有效提高视黄醇的产生。通过将 BHT 处理和使用 Tween 80 提取相结合，最终菌株 CJ2104 在 5 L 生物反应器中的补料分批发酵中产生了 4.86 g/L 视黄醇和 0.26 g/L 视黄醛，这是有史以来报告的最高视黄醇生产滴度。该研究表明解脂耶氏酵母是生物基视黄醇工业生产的合适宿主。