Analysis of carotenoid profile changes and carotenogenic genes transcript levels in Rhodosporidium toruloides mutants from an optimized Agrobacterium tumefaciens‐mediated transformation method
Analysis of carotenoid profile changes and carotenogenic genes transcript levels in Rhodosporidium toruloides mutants from an optimized Agrobacterium tumefaciens‐mediated transformation method
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DOI：
10.1002/bab.1895
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发表时间：
2020-02
影响因子：
2.8
通讯作者：
Zeping Sun;Jing Lv;Chaofan Ji;Huipeng Liang;Shengjie Li;Zhaoxia Yang;Wenhuan Xu;Sufang Zhang;Xinping Lin
中科院分区：
文献类型：
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作者：
Zeping Sun;Jing Lv;Chaofan Ji;Huipeng Liang;Shengjie Li;Zhaoxia Yang;Wenhuan Xu;Sufang Zhang;Xinping Lin
圆红冬孢酵母是一种很有潜力的生产类胡萝卜素的生物工程微生物。最常用的分子和遗传操作方法基于农杆菌介导的转化（ATMT）。但这种方法的转化效率相对较低。在这项研究中，我们优化了ATMT方法的R。由于T-DNA上的启动子，A. tumefaciens到R.在酵母NP 11、乙酰丁香酮浓度、共培养温度和共培养时间的条件下，获得了2,369个细胞/105个受体细胞的转化效率，是以前报道的24倍。利用该优化方法，分别筛选出4株红色突变株和4株黄色突变株，分别具有红曲霉素和β-胡萝卜素的生产优势。A1 - 13中的最高红曲霉素产量为1，638.15 µg/g干细胞重量。结果表明，变黄突变体将红曲霉素和红曲霉烯的代谢流转向γ-胡萝卜素和β-胡萝卜素，γ-胡萝卜素和β-胡萝卜素的比例均在92%以上。通过TAIL-PCR检测发现突变体中存在T-DNA插入，并发现插入热点，RT-qPCR结果表明突变体中的CTA 1基因与总类胡萝卜素尤其是红曲霉素的合成密切相关，是潜在的代谢工程位点。
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 105 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.