Discovery of 16-Demethylrifamycins by Removing the Predominant Polyketide Biosynthesis Pathway in Micromonospora sp. Strain TP-A0468

Abstract

A number of strategies have been developed to mine novel natural products based on biosynthetic gene clusters and there have been dozens of successful cases facilitated by the development of genomic sequencing. During our study on biosynthesis of the antitumor polyketide kosinostatin (KST), we found that the genome of Micromonospora sp. strain TP-A0468, the producer of KST, contains other potential polyketide gene clusters, with no encoded products detected. Deletion of kst cluster led to abolishment of KST and the enrichment of several new compounds, which were isolated and characterized as 16-demethylrifamycins (referred to here as compounds 3 to 6). Transcriptional analysis demonstrated that the expression of the essential genes related to the biosynthesis of compounds 3 to 6 was comparable to the level in the wild-type and in the kst cluster deletion strain. This indicates that the accumulation of these compounds was due to the redirection of metabolic flux rather than transcriptional activation. Genetic disruption, chemical complementation, and bioinformatic analysis revealed that the production of compounds 3 to 6 was accomplished by cross talk between the two distantly placed polyketide gene clusters pks3 and M-rif. This finding not only enriches the analogue pool and the biosynthetic diversity of rifamycins but also provides an auxiliary strategy for natural product discovery through genome mining in polyketide-producing microorganisms. IMPORTANCE Natural products are essential in the development of novel clinically used drugs. Discovering new natural products and modifying known compounds are still the two main ways to generate new candidates. Here, we have discovered several rifamycins with varied skeleton structures by redirecting the metabolic flux from the predominant polyketide biosynthetic pathway to the rifamycin pathway in the marine actinomycetes species Micromonospora sp. strain TP-A0468. Rifamycins are indispensable chemotherapeutics in the treatment of various diseases such as tuberculosis, leprosy, and AIDS-related mycobacterial infections. This study exemplifies a useful method for the discovery of cryptic natural products in genome-sequenced microbes. Moreover, the 16-demethylrifamycins and their genetically manipulable producer provide a new opportunity in the construction of novel rifamycin derivates to aid in the defense against the ever-growing drug resistance of Mycobacterium tuberculosis.

A New Micromonospora Strain with Antibiotic Activity Isolated from the Microbiome of a Mid-Atlantic Deep-Sea Sponge

“…Work will continue to mine these clusters, particularly those with low sequence similarity to known clusters, for novel, antimicrobial compounds. Recently, Zhou et al (2019) [32] forced the production of a different secondary metabolite in Micromonospora sp. TP-A0468 by deletion of the kosinostatin-producing cluster.…”

34a-Hydroxylation in Rifamycin Biosynthesis Catalyzed by Cytochrome P450 Encoded by rif-orf13

“…TP-A0468. [12] These compounds are structurally identical to the known rifamycins except for the absence of the methyl substitute on C16. Homologous genes of orf11 and orf17 are missing in the biosynthetic gene cluster of 16-demethylrifamycins, while the 34a-hydroxylated intermediate 2 is produced.…”

“…Given the fact that rifamycin W is accumulated in orf13-missing strains (S. arenicola CNS-205 and Micromonospora sp. TP-A0468) [4,12] but not in orf13-harboring strains (A. mediterranei and A. tolypomycina), [5,17] it could be deduced that orf13 is a reduplicate copy evolved from orf5 to promote the biosynthetic rate of rifamycins. Our work therefore clarifies the function of rif-orf13 in the biosynthesis of rifamycin and explains the contradictory results of previous studies.…”

Novel Bioactive Polyketides Isolated from Marine Actinomycetes: An Update Review from 2013 to 2019

“…strain TP-A0468. [17] Early research on the structure-activity relationship of rifamycin [18] revealed that the oxygen atoms at C-1, C-8, C-23, and C-25 were essential for antibacterial activity. Moreover, the ether bond from oxidative cleavage of C-12 29 μM, respectively.…”

“…The results showed that compound 7 possessed the similar antibacterial activity to rifamycin S, however, compounds 4 -6 were almost inactive under the test conditions. [17] The ether bond from oxidative cleavage of the C-12 (29) double bond was essential for antibacterial activity. The antibacterial activity between 16-demethylrifamycin 7 and rifamycin S showed that the absence of C-16 methyl group does not significantly affect the bioactivity of 16-demethylrifamycins.…”