State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan West Road, Haidian District, Beijing, 100193, China.
School of Life Science, Northeast Agricultural University, No. 59 Mucai Street, Xiangfang District, Harbin, 150030, China.
Appl Microbiol Biotechnol. 2020 Apr;104(7):2935-2946. doi: 10.1007/s00253-020-10410-8. Epub 2020 Feb 11.
Milbemycins and their semisynthetic derivatives are recognized as effective and eco-friendly pesticides, whereas the high price limits their widespread applications in agriculture. One of the pivotal questions is the accumulation of milbemycin-like by-products, which not only reduces the yield of the target products milbemycin A3/A4, but also brings difficulty to the purification. With other analogous by-products abolished, α9/α10 and β-family milbemycins remain to be eliminated. Herein, we solved these issues by engineering of post-modification steps. First, Cyp41, a CYP268 family cytochrome P450, was identified to participate in α9/α10 biosynthesis. By deleting cyp41, milbemycin α9/α10 was eliminated with an increase of milbemycin A3/A4 titer from 2382.5 ± 55.7 mg/L to 2625.6 ± 64.5 mg/L. Then, MilE, a CYP171 family cytochrome P450, was determined to be responsible for the generation of the furan ring between C6 and C8a of milbemycins. By further overexpression of milE, the production of β-family milbemycins was reduced by 77.2%. Finally, the titer of milbemycin A3/A4 was increased by 53.1% to 3646.9 ± 69.9 mg/L. Interestingly, overexpression of milE resulted in increased transcriptional levels of milbemycin biosynthetic genes and production of total milbemycins, which implied that the insufficient function of MilE was a limiting factor to milbemycin biosynthesis. Our research not only provides an efficient engineering strategy to improve the production of a commercially important product milbemycins, but also offers the clues for future study about transcriptional regulation of milbemycin biosynthesis.
米尔贝霉素及其半合成衍生物被认为是有效且环保的农药，但其高价格限制了其在农业中的广泛应用。其中一个关键问题是米尔贝霉素类似物副产物的积累，这不仅降低了目标产物米尔贝霉素 A3/A4 的产量，而且给纯化带来了困难。在消除了其他类似的副产物后，α9/α10 和 β-家族米尔贝霉素仍需要被消除。在此，我们通过后修饰步骤的工程改造解决了这些问题。首先，鉴定出 CYP41 是一种 CYP268 家族细胞色素 P450，参与α9/α10 的生物合成。通过删除 cyp41，消除了米尔贝霉素 α9/α10，米尔贝霉素 A3/A4 的产量从 2382.5±55.7 mg/L 增加到 2625.6±64.5 mg/L。然后，确定 MilE 是一种 CYP171 家族细胞色素 P450，负责米尔贝霉素中 C6 和 C8a 之间呋喃环的生成。通过进一步过表达 milE，β-家族米尔贝霉素的产量减少了 77.2%。最终，米尔贝霉素 A3/A4 的产量增加了 53.1%，达到 3646.9±69.9 mg/L。有趣的是，milE 的过表达导致米尔贝霉素生物合成基因的转录水平增加和总米尔贝霉素的产量增加，这表明 MilE 的功能不足是米尔贝霉素生物合成的限制因素。我们的研究不仅为提高一种商业上重要的产品米尔贝霉素的产量提供了一种有效的工程策略，也为米尔贝霉素生物合成的转录调控提供了线索。
