Abstract
Milbemycins are used commercially as insect repellents and acaricides; however, their high cost remains a significant challenge to commercial production. Hence, improving the titer of milbemycins for commercial application is an urgent priority. The present study aimed to effectively increase the titer of milbemycins using a combination of genome re-sequencing and metabolic engineering. First, 133 mutation sites were identified by genome re-sequencing in the mutagenized high-yielding strain BC04. Among them, three modifiable candidate genes (sbi_04868 encoding citrate synthase, sbi_06921 and sbi_06922 encoding alpha and beta subunits of acetyl-CoA carboxylase, and sbi_04683 encoding carbon uptake system gluconate transporter) related to primary metabolism were screened and identified. Next, the DNase-deactivated Cpf1–based integrative CRISPRi system was used in S. bingchenggensis to downregulate the transcription level of gene sbi_04868. Then, overexpression of the potential targets sbi_06921-06922 and sbi_04683 further facilitated milbemycin biosynthesis. Finally, those candidate genes were engineered to produce strains with combinatorial downregulation and overexpression, which resulted in the titer of milbemycin A3/A4 increased by 27.6% to 3164.5 mg/L. Our research not only identified three genes in S. bingchenggensis that are closely related to the production of milbemycins, but also offered an efficient engineering strategy to improve the titer of milbemycins using genome re-sequencing.
Key points
• We compared the genomes of two strains with different titers of milbemycins.
• We found three genes belonging to primary metabolism influence milbemycin production.
• We improved titer of milbemycins by a combinatorial engineering of three targets.
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Data availability
The re-sequence data related to this article were deposited in NCBI BioProject database with an accession number PRJNA664656.
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Acknowledgements
We would like to thank Professor Weihong Jiang (Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China) and Professor Yinhua Lu (Shanghai Normal University, Shanghai, China) for providing pSET-ddCpf1, Professor Mark Buttner (John Innes Centre, Norwich, UK) for providing plasmid pIJ10500, and Professor Weishan Wang (Chinese Academy of Sciences, Beijing, China) for providing plasmid PIJ-Potr and promoter kasOp*.
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This work received financial support from the National Natural Science Foundation of China (Grant Nos: 31972291, 31872936 and 31572070).
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WX, XW, and YL designed the research. Material preparation, data collection, and analysis were performed by YL and HW. XC assisted with experiments. The first draft of the manuscript was written by YL, WX, XW, HW, and SL, and YZ modified the manuscript. All authors read and approved the final manuscript.
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Liu, Y., Wang, H., Li, S. et al. Engineering of primary metabolic pathways for titer improvement of milbemycins in Streptomyces bingchenggensis. Appl Microbiol Biotechnol 105, 1875–1887 (2021). https://doi.org/10.1007/s00253-021-11164-7
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DOI: https://doi.org/10.1007/s00253-021-11164-7