National Engineering Research Center of Seafood, School of Food Science and Technology, Dalian Polytechnic University, Dalian, People's Republic of China.
Biotechnol Appl Biochem. 2021 Feb;68(1):71-81. doi: 10.1002/bab.1895. Epub 2020 May 22.
Rhodosporidium toruloides has been reported as a potential biotechnological microorganism to produce carotenoids. The most commonly used molecular and genetic manipulation methods based on Agrobacterium-mediated transformation (ATMT). However, this method was of relatively lower transformation efficiency. In this study, we optimized the ATMT method for R. toruloides on account of the promoter on T-DNA, the ratio of A. tumefaciens to R. toruloides NP11, acetosyringone concentration, cocultivation temperature and time, and a transformation efficiency of 2,369 cells per 10 recipient cells was obtained and was 24 times as that of the previous report. With this optimized method, four redder mutants and four yellower mutants were selected out with torularhodin and β-carotene production preference, respectively. The highest torularhodin production was 1,638.15 µg/g dry cell weight in A1-13. The yellower mutants were found to divert the metabolic flux from torularhodin and torulene to γ-carotene and β-carotene, and the proportion of γ-carotene and β-carotene were all over 92%. TAIL-PCR was carried out to found T-DNA insertion in these mutants, and insertion hotspot was found. RT-qPCR results showed that CTA1 genes in these mutants were closely related to the synthesis of total carotenoids, especially torularhodin, and was a potenial metabolic engineering site in the future.
红色红酵母已被报道为一种有潜力的生产类胡萝卜素的生物技术微生物。最常用的基于农杆菌介导转化（ATMT）的分子和遗传操作方法。然而，这种方法的转化效率相对较低。在本研究中，我们优化了红色红酵母的 ATMT 方法，考虑到 T-DNA 上的启动子、农杆菌与红色红酵母 NP11 的比例、乙酰丁香酮浓度、共培养温度和时间，转化效率达到了每 10 个受体细胞 2369 个细胞，是之前报道的 24 倍。利用这种优化的方法，分别选择了四个产番茄红素产量更高和四个β-胡萝卜素产量更高的突变体。A1-13 中番茄红素产量最高，达到 1638.15µg/g 干细胞重量。黄色突变体被发现将代谢通量从番茄红素和 torulene 转移到γ-胡萝卜素和β-胡萝卜素，γ-胡萝卜素和β-胡萝卜素的比例都超过 92%。TAIL-PCR 用于发现这些突变体中的 T-DNA 插入，并发现了插入热点。RT-qPCR 结果表明，这些突变体中的 CTA1 基因与总类胡萝卜素的合成密切相关，特别是番茄红素，是未来潜在的代谢工程靶点。
