Submerged fermentation of Streptomyces uncialis providing a biotechnology platform for uncialamycin biosynthesis, engineering, and production

Abstract

Uncialamycin (UCM) belongs to the anthraquinone-fused subfamily of 10-membered enediyne natural products that exhibits an extraordinary cytotoxicity against a wide spectrum of human cancer cell lines. Antibody-drug conjugates (ADCs), utilizing synthetic analogues of UCM as payloads, are in preclinical development. UCM is exclusively produced by Streptomyces uncialis DCA2648 on solid agar medium with low titers (∼0.019mg/ L), limiting its supply by microbial fermentation and hampering its biosynthetic and engineering studies by in vivo pathway manipulation. Here we report cultivation conditions that enable genetic manipulation of UCM biosynthesis in vivo and allow UCM production, with improved titers, by submerged fermentation of the engineered S. uncialis strains. Specifically, the titer of UCM was improved nearly 58-fold to ∼1.1mg/ L through the combination of deletion of biosynthetic gene clusters encoding unrelated metabolites from the S. uncialis wild-type, chemical mutagenesis and manipulation of pathway-specific regulators to generate the engineered S. uncialis strains, and finally medium optimization of the latter for UCM production. Genetic manipulation of UCM biosynthesis was demonstrated by inactivating selected genes in the engineered S. uncialis strains, one of which afforded a mutant strain accumulating tiancimycin B, a common biosynthetic intermediate known for the anthraquinone-fused subfamily of enediyne natural products. These findings highlight a biotechnology platform for UCM biosynthesis, engineering, and production that should facilitate both its fundamental studies and translational applications.

Anthraquinone-fused enediynes: discovery, biosynthesis and development

“…This result conrmed our previous hypothesis that the AFEs share a unied biosynthetic pathway, and TnmM/UcmM catalyze the oxidative reaction aer the ring opening in the biosynthesis of TNM A and UCM. 81 Moreover, the tnm, ucm and ypm BGCs each contains two genes (tnmK1/tnmK2, ucmK1/ ucmK2, and ypmK1/ypmK2) homologous to dynA4, which encodes a putative hydroxylase with an a/b-fold, while the dyn . 11 Considering that the ve AFEs are proposed to be derived from the same precursor and TNM A, UCM and YPM A lack the F-ring in DYN A, the extra genes in the tnm, ucm and ypm BGCs may be responsible for the late-stage modication steps in the biosynthesis of TNM A, UCM and YPM A.…”

“…This result confirmed our previous hypothesis that the AFEs share a unified biosynthetic pathway, and TnmM/UcmM catalyze the oxidative reaction after the ring opening in the biosynthesis of TNM A and UCM. 81 Moreover, the tnm , ucm and ypm BGCs each contains two genes ( tnmK1 / tnmK2 , ucmK1 / ucmK2 , and ypmK1 / ypmK2 ) homologous to dynA4 , which encodes a putative hydroxylase with an α/β-fold, while the dyn and the sgd BGCs each contains one gene. A recent work from the Liang group suggests that DynA4 may participate in C–C bond formation in the biosynthesis of DYN A.…”

“…A recent work published by the Shen group showed that TNM B ( 85 ) was also accumulated in the S. uncialis Δ ucmM mutant strain, further supporting the proposed unified pathway for AFEs. 81 Based on the results from Streptomyces sp. CB03234 and S. uncialis , the biosynthetic pathways for TNM A, UCM, YPM A and sealutomicin were proposed (Fig.…”

“…The yield of UCM in submerged fermentation was further increased to 1.1 mg L −1 by the combination of chemical mutagenesis, overexpression of transcriptional activators and medium optimization. 81 Ribosome engineering, 108,109 transcriptional analysis, 108 and genome shuffling 110 of Streptomyces sp. CB03234 were performed to enhance the production of TNM A. CB03234-GS26, a mutant strain resulting from the genome shuffling of a gentamycin-induced mutant and a streptomycin-induced mutant, exhibited the highest potential for TNMA production.…”

Introduction to the special issue: “Natural Product Discovery and Development in the Genomic Era: 2021”

“…Finally, Yoon and co-workers teach us how to further enhance clavulanic acid production titer in an industrial strain of Streptomyces clavuligerus by optimizing glycerol utilization, demonstrating reporter-guided mutant selection as an efficient method for further titer improvement of randomly mutagenized industrial strains (Shin et al, 2021 ). Shen and co-workers describe the development of submerged fermentation of engineered Streptomyces uncialis strains for uncialamycin production, highlighting a biotechnology platform that should greatly facilitate both fundamental studies and translational applications of uncialamycin as a promising anticancer drug lead (Hindra et al, 2021 ).…”