Tero Kunnari

Engineering Anthracycline Biosynthesis toward Angucyclines

ABSTRACT The biosynthesis pathways of two anthracyclines, nogalamycin and aclacinomycin, were directed toward angucyclines by using an angucycline-specific cyclase, pgaF, isolated from a silent antibiotic biosynthesis gene cluster. Addition of pgaF to a gene cassette that harbored the early biosynthesis genes of nogalamycin resulted in the production of two known angucyclinone metabolites, rabelomycin and its precursor, UWM6. Substrate flexibility of pgaF was demonstrated by replacement of the nogalamycin minimal polyketide synthase genes in the gene cassette with the equivalent aclacinomycin genes together with aknE2 and aknF, which specify the unusual propionate starter unit in aclacinomycin biosynthesis. This modification led to the production of a novel angucyclinone, MM2002, in which the expected ethyl side chain was incorporated into the fourth ring.

Production of hybrid anthracycline antibiotics by heterologous expression of Streptomyces nogalater nogalamycin biosynthesis genes.

A cluster of anthracycline biosynthetic genes isolated from Streptomyces nogalater was expressed in Streptomyces lividans and in Streptomyces galilaeus. A 12 kb DNA fragment cloned from this cluster in pIJ486 caused the production of a novel compound when introduced into S. lividans. The compound is derived from nogalonic acid methyl ester, an early intermediate in nogalamycin biosynthesis. Complementation with the cloned 12 kb fragment of S. galilaeus mutants blocked in aclacinomycin biosynthesis caused the production of hybrid anthracyclines. Cloning of the nogalamycin gene cluster should make possible a detailed study of the biosynthesis of this interesting antibiotic, as well as the production of novel anthracyclines of potential value as cytostatic drugs.

Identification of a Cyclase Gene Dictating the C-9 Stereochemistry of Anthracyclines from Streptomyces nogalater

ABSTRACT Nogalamycin is an anthracycline antibiotic produced byStreptomyces nogalater. Its aglycone has a unique stereochemistry (7S, 9S, 10R) compared to that of most other anthracyclines (7S, 9R, 10R). The gene snoaL, encoding a nogalonic acid methyl ester cyclase for nogalamycin, was used to generate nogalamycinone, demonstrating that the single cyclase dictates the C-9 stereochemistry of anthracyclines.

Elucidation of anthracyclinone biosynthesis by stepwise cloning of genes for anthracyclines from three different Streptomyces spp.

The anthracycline skeleton is biosynthesized by aromatic (type II) polyketide synthases. Furthermore, three post-polyketide steps are needed to form the basic aglycone of anthracyclines. Auramycinone was produced in Streptomyces lividans by introducing nine structural genes from three different anthracycline-producing Streptomyces species. The genes used to construct the auramycinone biosynthesis cluster were derived from nogalamycin-, daunomycin- and aclacinomycin-producing Streptomyces strains. The biosynthetic stages were divided into polyketide and post-polyketide steps on the assumption that the first stable intermediate would be nogalonic acid, named analogously to aklanonic acid, the precursor of several anthracyclines. Single genes were cloned in the expression construct in the order determined by the proposed biosynthetic pathway. This facilitated investigation of the products formed in the heterologous host after addition of each separate gene to the construct. The results thus elucidate the biosynthesis steps, products and the genes responsible for the reactions needed to build up an anthracyclinone.

Folding of the polyketide chain is not dictated by minimal polyketide synthase in the biosynthesis of mithramycin and anthracycline

BACKGROUND Mithramycin, nogalamycin and aclacinomycins are aromatic polyketide antibiotics that exhibit antitumour activity. The precursors of these antibiotics are formed via a polyketide biosynthetic pathway in which acetate (for mithramycinone and nogalamycinone) or propionate (for aklavinone) is used as a starter unit and nine acetates are used as extender units. The assembly of building blocks is catalyzed by the minimal polyketide synthase (PKS). Further steps include regiospecific reductions (if any) and cyclization. In the biosynthesis of mithramycin, however, ketoreduction is omitted and the regiospecificity of the first cyclization differs from that of anthracycline antibiotics (e.g. nogalamycin and aclacinomycins). These significant differences provide a convenient means to analyze the determinants for the regiospecificity of the first cyclization step. RESULTS In order to analyze a possible role of the minimal PKS in the regiospecificity of the first cyclization in polyketide biosynthesis, we expressed the mtm locus, which includes mithramycin minimal PKS genes, in Streptomyces galilaeus, which normally makes aclacinomycins, and the sno locus, which includes nogalamycin minimal PKS genes, in Streptomyces argillaceus, which normally makes mithramycin. The host strains are defective in the minimal PKS, but they express other antibiotic biosynthesis genes. Expression of the sno minimal PKS in the S. argillaceus polyketide-deficient strain generated mithramycin production. Auramycins, instead of aclacinomycins, accumulated in the recombinant S. galilaeus strains, suggesting that the mithramycin minimal PKS is responsible for the choice of starter unit. We also describe structural analysis of the compounds accumulated by a ketoreductase-deficient S. galilaeus mutant; spectroscopic studies on the major polyketide compound that accumulated revealed a first ring closure which is not typical of anthracyclines, suggesting an important role for the ketoreductase in the regiospecificity of the first cyclization. CONCLUSIONS These experiments clearly support the involvement of ketoreductase and a cyclase in the regiospecific cyclization of the biosynthetic pathway for aromatic polyketides.

Hybrid compounds derived from the combination of anthracycline and actinorhodin biosynthetic pathways

A new approach in the field of polyketide biosynthetic engineering, the combination of the biosynthetic routes of two different sources, is introduced. Streptomyces nogalater genes expressed in S. lividans TK24 yield the hybrid strain TK24/pSY15. Structural analysis of the products isolated from cultivation of the hybrid strain revealed the ability of the hybrid to produce novel compounds. Instead of accumulating characteristic products (e.g. actinorhodin) of the host S. lividans TK24, or intermediate compounds expected to be generated by the plasmid pSY15 (e.g. nogalamycin precursor), the hybrid strain produces novel compounds reflecting the enzymatic activity of both the host and the expressed plasmid. This implies that genes from two different types of aromatic polyketide biosynthesis are working together. The method described in this work complements earlier targeted biosyntheses.

Incorrectly folded aromatic polyketides from polyketide reductase deficient mutants

Compounds produced by the polyketide ketoreductase deficient Streptomyces mutants HO61 and P67 are described. The structures of the compounds indicate that ketoreductase activity is required for correct condensation of the polyketide chain in the biosynthesis of aromatic polyketides.

Hybrid anthracyclines by a genetically engineered Streptomyces galilaeus mutant

Abstract A Streptomyces galilaeus mutant carrying genes transfered from S. purpurascens is reported to produce two hybrid anthracyclines. This study demonstrates the possibility of producing hybrid anthracyclines in a combinatorial way by genetic engineering of anthracycline producing strains.

Hybrid compounds generated by the introduction of a nogalamycin-producing plasmid into Streptomyces argillaceus

The combination of genetic material from different antibiotic-producing organisms is a versatile and ever-expanding approach for the production of novel, hybrid compounds possessing bioactivity. The introduction of a plasmid (pSY21b) derived from Streptomyces nogalater and encoding PKS for nogalamycin production into the host strain S. argillaceus A43 led to the production of three new compounds in addition to the normally produced mithramycin. The new compounds are hybrids in the sense that they share features associated with the genes of both the host and the introduced plasmid. The structural elucidation of the novel compounds relied primarily on NMR spectroscopy, which revealed the three hybrids to be glycosides with the same aglycone common to all. Determination of the relative stereochemistry within the aglycone unit was confirmed by single-crystal X-ray analysis of the aglycone, which also revealed the tautomeric equilibrium to be in a very different position in comparison to that in the solution state. The glycosylation profile was clearly determined by the host, as the typical mithramycin sugars, D-oliose, D-olivose, and D-mycarose, were all expressed. Notable for the mutant was the high titre of the shunt products, 60% of the metabolic output, together with a lack of structural diversity of the hybrid aglycone present in the products, two features which are not normally observed.