Heinz G. Floss

Biosynthesis of the ansamycin antibiotic rifamycin : deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699

BACKGROUND The ansamycin class of antibiotics are produced by various Actinomycetes. Their carbon framework arises from the polyketide pathway via a polyketide synthase (PKS) that uses an unusual starter unit. Rifamycin (rif), produced by Amycolatopsis mediterranei, is the archetype ansamycin and it is medically important. Although its basic precursors (3-amino-5-hydroxy benzoic acid AHBA, and acetic and propionic acids) had been established, and several biosynthetic intermediates had been identified, very little was known about the origin of AHBA nor had the PKS and the various genes and enzymes that modify the initial intermediate been characterized. RESULTS A set of 34 genes clustered around the rifK gene encoding AHBA synthase were defined by sequencing all but 5 kilobases (kb) of a 95 kb contiguous region of DNA from A. mediterranei. The involvement of some of the genes in the biosynthesis of rifamycin B was examined. At least five genes were shown to be essential for the synthesis of AHBA, five genes were determined to encode the modular type I PKS that uses AHBA as the starter unit, and 20 or more genes appear to govern modification of the polyketide-derived framework, and rifamycin resistance and export. Putative regulatory genes were also identified. Disruption of the PKS genes at the end of rifA abolished rifamycin B production and resulted in the formation of P8/1-OG, a known shunt product of rifamycin biosynthesis, whereas disruption of the orf6 and orf9 genes, which may encode deoxysugar biosynthesis enzymes, had no apparent effect. CONCLUSIONS Rifamycin production in A. mediterranei is governed by a single gene cluster consisting of structural, resistance and export, and regulatory genes. The genes characterized here could be modified to produce novel forms of the rifamycins that may be effective against rifamycin-resistant microorganisms.

The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum

Maytansinoids are potent antitumor agents found in plants and microorganisms. To elucidate their biosynthesis at the biochemical and genetic level and to set the stage for their structure modification through genetic engineering, we have cloned two gene clusters required for the biosynthesis of the maytansinoid, ansamitocin, from a cosmid library of Actinosynnema pretiosum ssp. auranticum ATCC 31565. This is a rare case in which the genes involved in the formation of a secondary metabolite are dispersed in separate regions in an Actinomycete. A set of genes, asm22–24, asm43–45, and asm47, was identified for the biosynthesis of the starter unit, 3-amino-5-hydroxybenzoic acid (AHBA). Remarkably, there are two AHBA synthase gene homologues, which may have different functions in AHBA formation. Four type I polyketide synthase genes, asmA–D, followed by the downloading asm9, together encode eight homologous sets of enzyme activities (modules), each catalyzing a specific round of chain initiation, elongation, or termination steps, which assemble the ansamitocin polyketide backbone. Another set of genes, asm13–17, encodes the formation of an unusual “methoxymalonate” polyketide chain extension unit that, notably, seems to be synthesized on a dedicated acyl carrier protein rather than as a CoA thioester. Additional ORFs are involved in postsynthetic modifications of the initial polyketide synthase product, which include methylations, an epoxidation, an aromatic chlorination, and the introduction of acyl and carbamoyl groups. Tentative functions of several asm genes were confirmed by inactivation and heterologous expression.

The granaticin biosynthetic gene cluster of Streptomyces violaceoruber Tü22: sequence analysis and expression in a heterologous host

BACKGROUND The granaticins are members of the benzoisochromanequinone class of aromatic polyketides, the best known member of which is actinorhodin made by Streptomyces coelicolor A3(2). Genetic analysis of this class of compounds has played a major role in the development of hypotheses about the way in which aromatic polyketide synthases (PKSs) control product structure. Although the granaticin nascent polyketide is identical to that of actinorhodin, post-PKS steps involve different pyran-ring stereochemistry and glycosylation. Comparison of the complete gene clusters for the two metabolites is therefore of great interest. RESULTS The entire granaticin gene cluster (the gra cluster) from Streptomyces violaceoruber T-22 was cloned on either of two overlapping cosmids and expressed in the heterologous host, Streptomyces coelicolor A3(2), strain CH999. Chemical analysis of the recombinant strains demonstrated production of granaticin, granaticin B, dihydrogranaticin and dihydrogranaticin B, which are the four known metabolites of S. violaceoruber. Analysis of the complete 39,250 base pair sequence of the insert of one of the cosmids, pOJ466-22-24, revealed 37 complete open reading frames (ORFs), 15 of which resemble ORFs from the act (actinorhodin) gene cluster of S. coelicolor A3(2). Among the rest, nine resemble ORFs potentially involved in deoxysugar metabolism from Streptomyces spp. and other bacteria, and six resemble regulatory ORFs. CONCLUSIONS On the basis of these resemblances, putative functional assignments of the products of most of the newly discovered ORFs were made, including those of genes involved in the PKS and tailoring steps in the biosynthesis of the granaticin aglycone, steps in the deoxy sugar pathway, and putative regulatory and export functions.

3-Amino-5-hydroxybenzoic Acid Synthase, the Terminal Enzyme in the Formation of the Precursor of mC7N Units in Rifamycin and Related Antibiotics

The biosynthesis of ansamycin antibiotics, like rifamycin B, involves formation of 3-amino-5-hydroxybenzoic acid (AHBA) by a novel variant of the shikimate pathway. AHBA then serves as the starter unit for the assembly of a polyketide which eventually links back to the amino group of AHBA to form the macrolactam ring. The terminal enzyme of AHBA formation, which catalyzes the aromatization of 5-deoxy-5-amino-3-dehydroshikimic acid, has been purified to homogeneity from Amycolatopsis mediterranei, the encoding gene has been cloned, sequenced, and overexpressed in Escherichia coli. The recombinant enzyme, a (His)6 fusion protein, as well as the native one, are dimers containing one molecule of pyridoxal phosphate per subunit. Mechanistic studies showed that the enzyme-bound pyridoxal phosphate forms a Schiff’s base with the amino group of 5-deoxy-5-amino-3-dehydroshikimic acid and catalyzes both an α,β-dehydration and a stereospecific 1,4-enolization of the substrate. Inactivation of the gene encoding AHBA synthase in theA. mediterranei genome results in loss of rifamycin formation; production of the antibiotic is restored when the mutant is supplemented with AHBA.

Chapter 5 Biochemistry of Ergot Alkaloids—Achievements and Challenges

Publisher Summary The chapter discusses the biochemistry of ergot alkaloids, its achievements, and challenges and gives a clear picture of the current knowledge of the formation of ergot alkaloids in nature. It covers the biotechnological aspects and some current trends in ergot alkaloid pharmacology. Ergot alkaloids comprise a group of indole alkaloids that are predominantly found in various species of the ascomycete Claviceps. Some general information on ergot alkaloids and new alkaloids that were isolated during 1989 is summarized in the chapter. Ergot alkaloids are 3,4-substituted indole derivatives. An essential structural element of ergot alkaloids is the tetracyclic ergoline ring system. The chapter discusses methods for the commercial production of medicinally important ergot alkaloids that include isolation from sclerotia of C. purpurea, extraction from saprophytic cultures of different Claviceps species, mainly C. purpurea, C. paspali, and C. fusiformis, and partial synthesis.

Purification and properties of dimethylallylpyrophosphate: Tryptophan dimethylallyl transferase, the first enzyme of ergot alkaloid biosynthesis in Claviceps. sp. SD 58

Abstract Dimethylallylpyrophosphate: l -tryptophan dimethylallyltransferase (DMAT synthetase), the first pathway-specific enzyme of ergot alkaloid biosynthesis, has been isolated from mycelia of Claviceps sp., strain SD 58, and purified to apparent homogeneity. The enzyme reaction products were identified as l -4-(γ,γ-dimethylallyl)tryptophan and inorganic pyrophosphate. DMAT synthetase is a single subunit protein of molecular weight 70,000–73,000 and has an isoelectric point at pH 5.8. The enzyme is activated by Fe2+, Mg2+, and particularly Ca2+; Km values for l -tryptophan and dimethylallylpyrophosphate were determined to be 0.067 and 0.2 m m , respectively. Kinetic analysis indicated that the DMAT synthetase reaction proceeds by a sequential rather than a ping-pong mechanism.

Gene Cluster Responsible for Validamycin Biosynthesis in Streptomyces hygroscopicus subsp. jinggangensis 5008

ABSTRACT A gene cluster responsible for the biosynthesis of validamycin, an aminocyclitol antibiotic widely used as a control agent for sheath blight disease of rice plants, was identified from Streptomyces hygroscopicus subsp. jinggangensis 5008 using heterologous probe acbC, a gene involved in the cyclization of d-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone of the acarbose biosynthetic gene cluster originated from Actinoplanes sp. strain SE50/110. Deletion of a 30-kb DNA fragment from this cluster in the chromosome resulted in loss of validamycin production, confirming a direct involvement of the gene cluster in the biosynthesis of this important plant protectant. A sequenced 6-kb fragment contained valA (an acbC homologue encoding a putative cyclase) as well as two additional complete open reading frames (valB and valC, encoding a putative adenyltransferase and a kinase, respectively), which are organized as an operon. The function of ValA was genetically demonstrated to be essential for validamycin production and biochemically shown to be responsible specifically for the cyclization of d-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone in vitro using the ValA protein heterologously overexpressed in E. coli. The information obtained should pave the way for further detailed analysis of the complete biosynthetic pathway, which would lead to a complete understanding of validamycin biosynthesis.

Isolation of dimethylallylpyrophosphate: Tryptophan dimethylallyl transferase from the ergot fungus (Claviceps spec.)

Summary Cell-free extracts of Claviceps spec. , strain SD 58, were obtained which catalyze the formation of L-dimethylallyltryptophan from dimethylallylpyrophosphate and L-tryptophan. The enzyme catalyzing this reaction has been partially characterized. It is absent from young mycelia, but begins to appear at the end of the growth phase, preceding alkaloid formation by about one day.

The AcbC protein from Actinoplanes species is a C7-cyclitol synthase related to 3-dehydroquinate synthases and is involved in the biosynthesis of the alpha-glucosidase inhibitor acarbose.

The putative biosynthetic gene cluster for the α-glucosidase inhibitor acarbose was identified in the producerActinoplanes sp. 50/110 by cloning a DNA segment containing the conserved gene for dTDP-d-glucose 4,6-dehydratase,acbB. The two flanking genes were acbA(dTDP-d-glucose synthase) and acbC, encoding a protein with significant similarity to 3-dehydroquinate synthases (AroB proteins). The acbC gene was overexpressed heterologously in Streptomyces lividans 66, and the product was shown to be a C7-cyclitol synthase using sedo-heptulose 7-phosphate, but not ido-heptulose 7-phosphate, as its substrate. The cyclization product, 2-epi-5-epi-valiolone ((2S,3S,4S,5R)-5-(hydroxymethyl)cyclohexanon-2,3,4,5-tetrol), is a precursor of the valienamine moiety of acarbose. A possible five-step reaction mechanism is proposed for the cyclization reaction catalyzed by AcbC based on the recent analysis of the three-dimensional structure of a eukaryotic 3-dehydroquinate synthase domain (Carpenter, E. P., Hawkins, A. R., Frost, J. W., and Brown, K. A. (1998) Nature 394, 299–302).

Lessons from the rifamycin biosynthetic gene cluster

There is currently intense interest in unravelling the modus operandi of type I modular polyketide synthases in order to lay the ground work for their use in the combinatorial biosynthesis of new bioactive molecules. Much of our knowledge is derived from studies on 6-deoxyerythronolide B (DEBS), the enzyme assembling the polyketide backbone of erythromycin. Work on the rifamycin polyketide synthase has revealed a number of features that differ from those seen with DEBS.