Abstract
Tiancimycin-A (TNM-A) is an anthraquinone-fused ten-membered enediyne produced by Streptomyces sp. CB03234, which is very promising for the development of anticancer antibody-drug conjugates (ADCs). To improve the titer of TNM-A, we have generated high-producing mutants CB03234-S and CB03234-R through ribosome engineering, but still not sufficient for pilot production of TNM-A. As the follow-up work, gentamycin-induced ribosome engineering was further adopted here to generate the mutant CB03234-G, which produced similar level of TNM-A as in CB03234-S and CB03234-R. Benefiting from the distinct antibiotic resistances of three ribosome engineering mutants, genome shuffling between any two of them was respectively carried out, and finally obtained the recombinant CB03234-GS26. Under optimal conditions, CB03234-GS26 produced 40.6 ± 1.0 mg/L TNM-A in shaking flasks and 20.8 ± 0.4 mg/L in a scaled-up 30-L fermentor. Comparing with the parental high-producing mutants, the over 1.6-fold titer improvement of CB03234-GS26 in fermentor was more promising for pilot production of TNM-A. Besides the distinctive morphological features, genetic characterization revealed that CB03234-GS26 possessed 1.8 kb rsmG related deletion just the same as CB03234-S, but no mutation was found in rpsL. Subsequent knockouts proved that rsmG was unrelated to titer improvement of TNM-A, which implied other genomic variations and mechanisms rather than ribosome engineering to enhance the biosynthesis of TNM-A. Therefore, CB03234-GS26 provided a basis to locate potential novel genetic targets, and explore the interactions between complex metabolic network and TNM biosynthetic pathway, which shall promote future construction of high-yielding systems for TNM-A and other anthraquinone-fused enediynes.
Key Points
•United genome shuffling and ribosome engineering help further strain improvement.
•CB03234-GS26 with improved titer is practical for the pilot production of TNM-A.
•Enhanced TNM-A production should attribute to novel genetic features/mechanisms.
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Funding
This work was supported in part by grants from the National Natural Science Foundation of China Grants 81530092 and 81872779, the Chinese Ministry of Education 111 Project B0803420, and the Fundamental Research Funds for the Central Universities of Central South University (CSU) 2019zzts773.
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ZXC and DYW conceived and designed this project. KWP, HY, and LL contributed to the ribosome engineering of various mutants. LHM and JCZ conducted the genome shuffling of various mutants and fermentation validation. LHM and LJ conducted genetic characterization and analysis. LHM and ZZK performed the scaled-up studies. LHM and ZXC analyzed data. LHM, JCZ, and ZXC co-wrote the manuscript. All authors read and approved the final manuscript.
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Liu, H., Jiang, C., Lin, J. et al. Genome shuffling based on different types of ribosome engineering mutants for enhanced production of 10-membered enediyne tiancimycin-A. Appl Microbiol Biotechnol 104, 4359–4369 (2020). https://doi.org/10.1007/s00253-020-10583-2
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DOI: https://doi.org/10.1007/s00253-020-10583-2