Ivana Crnovcic

The Actinomycin Biosynthetic Gene Cluster of Streptomyces chrysomallus: a Genetic Hall of Mirrors for Synthesis of a Molecule with Mirror Symmetry

A gene cluster was identified which contains genes involved in the biosynthesis of actinomycin encompassing 50 kb of contiguous DNA on the chromosome of Streptomyces chrysomallus. It contains 28 genes with biosynthetic functions and is bordered on both sides by IS elements. Unprecedentedly, the cluster consists of two large inverted repeats of 11 and 13 genes, respectively, with four nonribosomal peptide synthetase genes in the middle. Nine genes in each repeat have counterparts in the other, in the same arrangement but in the opposite orientation, suggesting an inverse duplication of one of the arms during the evolution of the gene cluster. All of the genes appear to be organized into operons, each corresponding to a functional section of actinomycin biosynthesis, such as peptide assembly, regulation, resistance, and biosynthesis of the precursor of the actinomycin chromophore 4-methyl-3-hydroxyanthranilic acid (4-MHA). For 4-MHA synthesis, functional analysis revealed genes that encode pathway-specific isoforms of tryptophan dioxygenase, kynurenine formamidase, and hydroxykynureninase, which are distinct from the corresponding enzyme activities of cellular tryptophan catabolism in their regulation and in part in their substrate specificity. Phylogenetic analysis indicates that the pathway-specific tryptophan metabolism in Streptomyces most probably evolved divergently from the normal pathway of tryptophan catabolism to provide an extra or independent supply of building blocks for the synthesis of tryptophan-derived secondary metabolites.

Aromatic C-Methyltransferases with Antipodal Stereoselectivity for Structurally Diverse Phenolic Amino Acids Catalyze the Methylation Step in the Biosynthesis of the Actinomycin Chromophore

The actinomycin biosynthetic gene cluster of Streptomyces chrysomallus harbors two paralogous genes, acmI and acmL, encoding methyltransferases. To unveil their suspected role in the formation of 3-hydroxy-4-methyl-anthranilic acid (4-MHA), the building block of the actinomycin chromophore, each gene was expressed in Escherichia coli. Testing the resulting ∼40 kDa His(6)-tagged proteins with compounds of biogenetic relevance as substrates and S-adenosyl-l-methionine revealed that each exclusively methylated 3-hydroxykynurenine (3-HK) with formation of 3-hydroxy-4-methylkynurenine (4-MHK) identified by its in vitro conversion to 4-MHA with hydroxykynureninase. AcmI and AcmL methylate also hydroxyphenyl-amino propanoic acids such as p-tyrosine, m-tyrosine, or 3,4-dihydroxy-l-phenylalanine (DOPA) but at a lower rate than 3-HK. The presence of the α-amino group was necessary for substrate recognition. Phenolic acids with shorter chains such as 4-hydoxyphenyl-l-glycine (HPG), 3-hydroxybenzoic acid (3-HB), or 3-hydroxyanthranilic acid (3-HA) gave no product. Both enzymes were stereospecific for the optical configuration at α-C with unprecedented antipodal selectivity for the d-enantiomer of 3-HK and the l-enantiomer of p-tyrosine or m-tyrosine. AcmI and AcmL show sequence similarity to various C- and O-methyltransferases from bacteria. Phylogenetic analysis places them into the clade of C-methyltransferases comprising among others orthologues involved in 4-MHA formation of other biosynthesis systems and methyltransferases putatively involved in the C-methylation of tyrosine. Remarkably, computational remodelling of AcmI and AcmL structures revealed significant similarity with the 3-D structures of type 1 O-methyltransferases from plants such as caffeic acid O-methyltransferase (COMT) and other phenylpropanoid methyltransferases. The relevance of 3-HK or 3-HA methylation in the actinomycin biosynthesis pathways of different actinomycetes is discussed.

Strain Prioritization and Genome Mining for Enediyne Natural Products

ABSTRACT The enediyne family of natural products has had a profound impact on modern chemistry, biology, and medicine, and yet only 11 enediynes have been structurally characterized to date. Here we report a genome survey of 3,400 actinomycetes, identifying 81 strains that harbor genes encoding the enediyne polyketide synthase cassettes that could be grouped into 28 distinct clades based on phylogenetic analysis. Genome sequencing of 31 representative strains confirmed that each clade harbors a distinct enediyne biosynthetic gene cluster. A genome neighborhood network allows prediction of new structural features and biosynthetic insights that could be exploited for enediyne discovery. We confirmed one clade as new C-1027 producers, with a significantly higher C-1027 titer than the original producer, and discovered a new family of enediyne natural products, the tiancimycins (TNMs), that exhibit potent cytotoxicity against a broad spectrum of cancer cell lines. Our results demonstrate the feasibility of rapid discovery of new enediynes from a large strain collection. IMPORTANCE Recent advances in microbial genomics clearly revealed that the biosynthetic potential of soil actinomycetes to produce enediynes is underappreciated. A great challenge is to develop innovative methods to discover new enediynes and produce them in sufficient quantities for chemical, biological, and clinical investigations. This work demonstrated the feasibility of rapid discovery of new enediynes from a large strain collection. The new C-1027 producers, with a significantly higher C-1027 titer than the original producer, will impact the practical supply of this important drug lead. The TNMs, with their extremely potent cytotoxicity against various cancer cells and their rapid and complete cancer cell killing characteristics, in comparison with the payloads used in FDA-approved antibody-drug conjugates (ADCs), are poised to be exploited as payload candidates for the next generation of anticancer ADCs. Follow-up studies on the other identified hits promise the discovery of new enediynes, radically expanding the chemical space for the enediyne family. Recent advances in microbial genomics clearly revealed that the biosynthetic potential of soil actinomycetes to produce enediynes is underappreciated. A great challenge is to develop innovative methods to discover new enediynes and produce them in sufficient quantities for chemical, biological, and clinical investigations. This work demonstrated the feasibility of rapid discovery of new enediynes from a large strain collection. The new C-1027 producers, with a significantly higher C-1027 titer than the original producer, will impact the practical supply of this important drug lead. The TNMs, with their extremely potent cytotoxicity against various cancer cells and their rapid and complete cancer cell killing characteristics, in comparison with the payloads used in FDA-approved antibody-drug conjugates (ADCs), are poised to be exploited as payload candidates for the next generation of anticancer ADCs. Follow-up studies on the other identified hits promise the discovery of new enediynes, radically expanding the chemical space for the enediyne family.

Occurrence and biosynthesis of C-demethylactinomycins in actinomycin-producing Streptomyces chrysomallus and Streptomyces parvulus

Streptomyces chrysomallus and Streptomyces parvulus produce novel C-demethylactinomycins besides their normal actinomycins when fed with 3-hydroxyanthranilic acid (3-HA). The 3-HA is incorporated into pentapeptide lactone precursors in competition with the regular precursor 4-methyl-3-hydroxyanthranilic acid (4-MHA). The resultant 3-HA pentapeptide lactones can condense with each other, as well as with the continuously formed 4-MHA pentapeptide lactones giving C-demethylactinomycins lacking one or both methyl groups in their phenoxazinone chromophores. In case of C-demethylactinomyins lacking one methyl group, the condensation was shown to be regiospecific directing the 3-HA portion almost exclusively to the α-side of the phenoxazinone chromophore. As 3-HA is a weaker substrate for the 4-MHA-incorporating enzyme actinomycin synthetase I than 4-MHA, C-demethylactinomycins never exceeded 7–8% of total actinomycin formed. Surprisingly, C-demethylactinomycins (up to 0.8%) were also found in the actinomycin mixtures of unsupplemented streptomycete cultures after longer cultivation times, indicating the natural presence of 3-HA. Feeding with 3-hydroxykynurenine (3-HK) induced also formation of C-demethylactinomycins indicating that 3-HK is source of 3-HA. Analysis of tryptophan metabolites in the intracellular pools of the streptomycetes using 5-3H-tryptophan as radiotracer revealed formation of 4-MHA, but not of 3-HA. This indicates that intracellular 3-HK is almost exclusively converted to 3-hydroxy-4-methylkynurenine (4-MHK), which has been identified previously as direct precursor of 4-MHA. However, small amount of 3-HK leaking out from the 4-MHA pathway can be prematurely converted to 3-HA all along the cultivation of the streptomycetes resulting in the formation of natural C-demethylactinomycins.

Engineered production and evaluation of 6|[prime]|-deoxy-tallysomycin H-1 revealing new insights into the structure|[ndash]|activity relationship of the anticancer drug bleomycin

The bleomycins (BLMs), a family of glycopeptide antibiotics, are currently used clinically in combination with a number of other agents for the treatment of malignant tumors. Other members of the BLM family include tallysomycins (TLMs), phleomycins and zorbamycin (ZBM). We previously cloned and characterized the biosynthetic gene clusters for BLMs, TLMs and ZBM. Applications of combinatorial biosynthesis strategies to the three biosynthetic machineries enabled the engineered production of several BLM analogs with unique structural characteristics and varying DNA cleavage activities, thereby providing an outstanding opportunity to study the structure–activity relationship (SAR) for the BLM family of anticancer drugs. We now report the engineered production of a new BLM–TLM–ZBM hybrid metabolite, named 6′-deoxy-TLM H-1, which consists of the 22-desmethyl-BLM aglycone, the TLM A C-terminal amine and the ZBM disaccharide, by heterologous expression of the zbmGL genes from the ZBM biosynthetic gene cluster in the Streptoalloteichus hindustanus ΔtlmH mutant strain SB8005. Evaluation of the DNA cleavage activities of 6′-deoxy-TLM H-1 as a measurement for its potential anticancer activity, in comparison with TLM H-1 and BLM A2, reveals new insight into the SAR of BLM family of anticancer drugs.

Genome Mining of Micromonospora yangpuensis DSM 45577 as a Producer of an Anthraquinone-Fused Enediyne

A new anthraquinone-fused enediyne, yangpumicin A (YPM A, 1), along with four Bergman cyclization congeners (YPM B-E, 2-5), was isolated from Micromonospora yangpuensis DSM 45577 after mining enediyne biosynthetic gene clusters from public actinobacterial genome databases and prioritizing the hits by an enediyne genome neighborhood network analysis for discovery. YPM A is potent against a broad spectrum of human cancer cell lines. The discovery of 1 provides new opportunities for the functionalization of enediynes to develop new conjugation chemistries for antibody-drug conjugates.

Engineered production of cancer targeting peptide (CTP)-containing C-1027 in Streptomyces globisporus and biological evaluation.

Conjugation of cancer targeting peptides (CTPs) with small molecular therapeutics has emerged as a promising strategy to deliver potent (but typically nonspecific) cytotoxic agents selectively to cancer cells. Here we report the engineered production of a CTP (NGR)-containing C-1027 and evaluation of its activity against selected cancer cell lines. C-1027 is an enediyne chromoprotein produced by Streptomyces globisporus, consisting of an apo-protein (CagA) and an enediyne chromophore (C-1027). NGR is a CTP that targets CD13 in tumor vasculature. S. globisporus SB1026, a recombinant strain engineered to encode CagA with the NGR sequence fused at its C-terminus, directly produces the NGR-containing C-1027 that is equally active as the native C-1027. Our results demonstrate the feasibility to produce CTP-containing enediyne chromoproteins by metabolic pathway engineering and microbial fermentation and will inspire efforts to engineer other CTP-containing drug binding proteins for targeted delivery.

Activities of recombinant human bleomycin hydrolase on bleomycins and engineered analogues revealing new opportunities to overcome bleomycin-induced pulmonary toxicity

The bleomycins (BLMs) are widely used in combination therapies for the treatment of various cancers. Dose-dependent and cumulative pulmonary toxicity is the major cause of BLM-associated morbidity, limiting the broad uses of BLMs as anticancer drugs. The organ specificity of BLM-induced toxicity has been correlated with the expression of the hBLMH gene, encoding the human bleomycin hydrolase (hBLMH), which is poorly expressed in the lung. hBLMH hydrolyzes BLMs into the biologically inactive deamido BLMs, thereby protecting organs from BLM-induced toxicity. Here we report (i) expression of hBLMH and production and isolation of recombinant human bleomycin hydrolase (rhBLMH) from E. coli, (ii) structural characterization of deamido BLM A2 and B2 isolated from rhBLMH-catalyzed hydrolysis of BLM A2 and B2, and (iii) kinetic characterization of the rhBLMH-catalyzed hydrolysis of BLM A2 and B2, in comparison with five BLM analogues. rhBLMH from E. coli catalyzes rapid and efficient hydrolysis of all BLMs tested, exhibiting a superior catalytic efficiency for BLM B2. These findings reveal new opportunities to overcome BLM-induced pulmonary toxicity in chemotherapies, potentially by exploring BLM B2 as the preferred congener, engineering designer BLMs with optimized activity for rhBLMH, or co-administrating rhBLMH directly into the lung as a potential protein therapeutic.

Genetic interrelations in the actinomycin biosynthetic gene clusters of Streptomyces antibioticus IMRU 3720 and Streptomyces chrysomallus ATCC11523, producers of actinomycin X and actinomycin C.

Sequencing the actinomycin (acm) biosynthetic gene cluster of Streptomyces antibioticus IMRU 3720, which produces actinomycin X (Acm X), revealed 20 genes organized into a highly similar framework as in the bi-armed acm C biosynthetic gene cluster of Streptomyces chrysomallus but without an attached additional extra arm of orthologues as in the latter. Curiously, the extra arm of the S. chrysomallus gene cluster turned out to perfectly match the single arm of the S. antibioticus gene cluster in the same order of orthologues including the the presence of two pseudogenes, scacmM and scacmN, encoding a cytochrome P450 and its ferredoxin, respectively. Orthologues of the latter genes were both missing in the principal arm of the S. chrysomallus acm C gene cluster. All orthologues of the extra arm showed a G +C-contents different from that of their counterparts in the principal arm. Moreover, the similarities of translation products from the extra arm were all higher to the corresponding translation products of orthologue genes from the S. antibioticus acm X gene cluster than to those encoded by the principal arm of their own gene cluster. This suggests that the duplicated structure of the S. chrysomallus acm C biosynthetic gene cluster evolved from previous fusion between two one-armed acm gene clusters each from a different genetic background. However, while scacmM and scacmN in the extra arm of the S. chrysomallus acm C gene cluster are mutated and therefore are non-functional, their orthologues saacmM and saacmN in the S. antibioticus acm C gene cluster show no defects seemingly encoding active enzymes with functions specific for Acm X biosynthesis. Both acm biosynthetic gene clusters lack a kynurenine-3-monooxygenase gene necessary for biosynthesis of 3-hydroxy-4-methylanthranilic acid, the building block of the Acm chromophore, which suggests participation of a genome-encoded relevant monooxygenase during Acm biosynthesis in both S. chrysomallus and S. antibioticus.

Comparative Studies of the Biosynthetic Gene Clusters for Anthraquinone-Fused Enediynes Shedding Light into the Tailoring Steps of Tiancimycin Biosynthesis

Comparative analyses of the four known anthraquinone-fused enediynes biosynthetic gene clusters identified four genes, tnmE6, tnmH, tnmL, and tnmQ, unique to the tnm gene cluster. Larger scale fermentation of both the S. sp. CB03234 wild-type and the Δ tnmH and Δ tnmL mutant strains resulted in the characterization of 20 new tiancimycin (TNM) congeners, including five enediynes. These findings enabled a proposal for the late stage of TNM biosynthesis featuring an intermediate possibly common for all anthraquinone-fused enediynes.