Early Steps in the Biosynthetic Pathway of Rishirilide B

Abstract

The biological active compound rishirilide B is produced by Streptomyces bottropensis. The cosmid cos4 contains the complete rishirilide B biosynthesis gene cluster. Its heterologous expression in the host Streptomyces albus J1074 led to the production of rishirilide B as a major compound and to small amounts of rishirilide A, rishirilide D and lupinacidin A. In order to gain more insights into the biosynthesis, gene inactivation experiments and gene expression experiments were carried out. This study lays the focus on the functional elucidation of the genes involved in the early biosynthetic pathway. A total of eight genes were deleted and six gene cassettes were generated. Rishirilide production was not strongly affected by mutations in rslO2, rslO6 and rslH. The deletion of rslK4 and rslO3 led to the formation of polyketides with novel structures. These results indicated that RslK4 and RslO3 are involved in the generation or selection of the starter unit for rishirilide biosynthesis. In the rslO10 mutant strain, two novel compounds were detected, which were also produced by a strain containing solely the genes rslK1, rslK2, rslK3, rslK4, and rslA. rslO1 and rslO4 mutants predominately produce galvaquinones. Therefore, the ketoreductase RslO10 is involved in an early step of rishirilide biosynthesis and the oxygenases RslO1 and RslO4 are most probably acting on an anthracene moiety. This study led to the functional elucidation of several genes of the rishirilide pathway, including rslK4, which is involved in selecting the unusual starter unit for polyketide synthesis.Molecules 2020, 25, 1955 2 of 15 the initiation module, can be found, consisting of a ketosynthase, a malonyl CoA:ACP transferase and an acyl carrier protein (ACP). The initiation module selects the starter unit and catalyzes the first elongation of the growing polyketide, before the molecule is loaded onto the minPKS for further elongation reactions. Additional enzymes, like ketoreductases, cyclases and aromatases complement the minPKS and determine the folding of the respective polyketide [1]. The aromatic polyketide rishirilide B (Figure 1) was first discovered by Iwaki et al. in 1984. Rishirilide B was isolated from Streptomyces rishiriensis OFR-1056 in the course of screening for new α2-macroglobulin inhibitors [2]. This compound was also described as a product of S. olivaceus SCSIO T05, which also produced rishirilide C, lupinacidin A and galvaquinone A and B [3] (Figure 1). The biosynthetic gene cluster of this strain responsible for the formation of all four compounds was cloned and biosynthetic studies were performed [3]. S. bottropensis (formerly S. sp. Gö C4/4) also produces the compounds rishirilide A, B, D and lupinacidin A (Figure 1). The corresponding rishirilide biosynthetic gene cluster was cloned on a cosmid (cos4), which was subsequently expressed in the heterologous host S. albus J1074. Rishirilide B was produced as a major compound. Moreover, rishirilide A, D and lupinacidin A were also produced in small amounts [3] (Figure 1)....

Regulatory Control of Rishirilide(s) Biosynthesis in Streptomyces bottropensis

“…The following concentrations of the ligands were used: 1, 10, 50, 100 µM and 0.25, 0.5, 1 mM. The tested ligands were purified as described before [ 33 , 35 ] or in Supplementary Materials (information about structure elucidation of compounds isolated from ∆ rslO7 mutant).…”

“…RNA, genomic DNA (C), and cDNA were used as a template in PCR reactions. The functions of rsl -genes are described in [ 30 , 33 , 35 ].…”

“…Rishirilide A and B act as inhibitors of α2-macroglobulin, whereas rishirilide B exhibits an additional inhibition activity towards glutathione-S-transferase [ 31 , 32 ]. Genes for rishirilides biosynthesis ( rsl -genes) are clustered together and were recently functionally characterized [ 33 ].…”

“…Rishirilides are synthesized from the valine-derived isobutyl-CoA starter unit and malonyl-CoA extenders, followed by decarboxylation of one acetate unit giving a methyl group. The minimal PKS complex derives a linear tricyclic intermediate, which is further modified by cluster-encoded tailoring enzymes [ 33 , 34 ]. Interestingly, the rishirilide scaffold is formed in two key steps: a reductive ring opening of an epoxide moiety on polycyclic intermediate by the flavin-dependent reductase RslO5, followed by an extraordinary oxidative carbon backbone rearrangement catalyzed by flavin-dependent Baeyer-Villiger monooxygenase RslO9 [ 35 ].…”

Biosynthesis of the Tricyclic Aromatic Type II Polyketide Rishirilide: New Potential Third Ring Oxygenation after Three Cyclization Steps

“…In the course of our rishirilide biosynthetic pathway studies, a remarkable amount of information on early and tailoring steps has been recently elucidated and published. The polyketide synthase RslK4 and the putative 3-oxoacyl-ACP reductase RslO3 are involved in the generation of the unusual starter unit, and the early acting ketoreductase RslO10 is involved in first ring cyclization/aromatization [ 5 ].…”