Leonard Kaysser

Cytochrome P450 alkane hydroxylases of the CYP153 family are common in alkane-degrading eubacteria lacking integral membrane alkane hydroxylases.

ABSTRACT Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.

Identification and Manipulation of the Caprazamycin Gene Cluster Lead to New Simplified Liponucleoside Antibiotics and Give Insights into the Biosynthetic Pathway

Caprazamycins are potent anti-mycobacterial liponucleoside antibiotics isolated from Streptomyces sp. MK730-62F2 and belong to the translocase I inhibitor family. Their complex structure is derived from 5′-(β-O-aminoribosyl)-glycyluridine and comprises a unique N-methyldiazepanone ring. The biosynthetic gene cluster has been identified, cloned, and sequenced, representing the first gene cluster of a translocase I inhibitor. Sequence analysis revealed the presence of 23 open reading frames putatively involved in export, resistance, regulation, and biosynthesis of the caprazamycins. Heterologous expression of the gene cluster in Streptomyces coelicolor M512 led to the production of non-glycosylated bioactive caprazamycin derivatives. A set of gene deletions validated the boundaries of the cluster and inactivation of cpz21 resulted in the accumulation of novel simplified liponucleoside antibiotics that lack the 3-methylglutaryl moiety. Therefore, Cpz21 is assigned to act as an acyltransferase in caprazamycin biosynthesis. In vivo and in silico analysis of the caprazamycin biosynthetic gene cluster allows a first proposal of the biosynthetic pathway and provides insights into the biosynthesis of related uridyl-antibiotics.

One‐Pot Enzymatic Synthesis of Merochlorin A and B

The polycycles merochlorin A and B are complex halogenated meroterpenoid natural products with significant antibacterial activities and are produced by the marine bacterium Streptomyces sp. strain CNH-189. Heterologously produced enzymes and chemical synthesis are employed herein to fully reconstitute the merochlorin biosynthesis in vitro. The interplay of a dedicated type III polyketide synthase, a prenyl diphosphate synthase, and an aromatic prenyltransferase allow formation of a highly unusual aromatic polyketide-terpene hybrid intermediate which features an unprecedented branched sesquiterpene moiety from isosesquilavandulyl diphosphate. As supported by in vivo experiments, this precursor is furthermore chlorinated and cyclized to merochlorin A and isomeric merochlorin B by a single vanadium-dependent haloperoxidase, thus completing the remarkably efficient pathway.

Analysis of the Liposidomycin Gene Cluster Leads to the Identification of New Caprazamycin Derivatives

The liposidomycins (LPMs, Scheme 1) were identified in a culture broth of a Streptomyces strain in 1985 by screening for inhibitors of peptidoglycan synthesis. They show strong and selective activity against the Escherichia coli translocase I MraY (LPM C: IC50 30 ng mL ) but do not inhibit eukaryotic glycoconjugate formation, unlike other translocase I inhibitors such as the tunicamycins. 3] They comprise a 5’-substituted uridine, a 5-amino-5-deoxyribose-2-sulfate and an N-methylated perhydro-1,4-diazepine (Scheme 1). A fatty acid side chain of variable length and conformation distinguishes the different LPMs and is linked to a unique 3-methylglutaryl group. The overall structure resembles only the caprazamycins (CPZs; Scheme 1). The caprazamycins contain a permethylated l-rhamnose but lack the sulfate group at the 2’’-position of the aminoribose. In bacterial secondary metabolism, sulfurylation is rare, and only a few sulfated compounds have been investigated regarding their biosynthesis. Though the LPMs are potent inhibitors of the translocase I in vitro, they exhibit only weak antimicrobial activity. However, changing the medium components and UVradiation of the original producer strain led to the production of LPM derivatives that strongly inhibited the growth of mycobacteria. Interestingly, the change of activity was mainly due to compounds that had lost the sulfate moiety. It seems that the hydrophilic sulfate attached to the 2’’-position of the aminoribose confers poor membrane permeability to the LPMs. On the other hand, a comparison of the sulfurylated LPM A-(I) and non-sulfurylated LPM A-(III) showed LPM A-(I) to be more selective in vitro against bacterial peptidoglycan biosynthesis versus eukaryotic glycoprotein formation by one order of magnitude. Thus, the role of the sulfate group remains an intriguing subject for future investigations. We recently identified the biosynthetic gene cluster for CPZs in Streptomyces sp. MK730-62F2. Due to the structural resemblance between the LPMs and CPZs, we assumed similar enzymes to be involved in the formation of both compounds. Two cosmids out of 1100 could be identified in a genomic library of the LPM producer Streptomyces sp. SN-1061M that were positive for a hypothetical N-methyltransferase similar to Cpz11. Cosmid 3G5 was finally selected for complete shot-gun sequencing. The nucleotide sequence of the gene cluster has been deposited in GenBank under accession no. GU219978. A total of 25 open reading frames, designated lpmA–lpmY, were assigned to the LPM gene cluster presumably encoding for biosynthesis, resistance, transport and regulation (Scheme 2, and Table S1 in the Supporting Information). In order to verify that the identified genes were sufficient for the biosynthesis of LPMs, we prepared the cosmid 3G5 for heterologous expression as described elsewhere. The generated cosmid lipLK01 was introduced into S. coelicolor M512, and culture extracts of the respective mutants were analysed by LCESI-MS/MS. In UV chromatograms of S. coelicolor M512/lipLK01 extracts (Figure 1 C), four prominent peaks at tR = 22.4, 24.7, 26.8 and 30.0 min appeared, which could not be found in extracts of S. coelicolor M512 without the LPM cluster (Figure 1 B). The genuine LPM producer Streptomyces sp. SN-1061M produced similar peaks that represent the LPMs Z, A, B, C, G, K, L and M (Figure 1 A). Mass peaks for known LPMs B, C, H, L, M and X with corresponding retention times were detected in product ion chromatograms of extracts from S. coelicolor M512/lipLK01 (Figure S1). Characteristic fragmentation patterns were observed by collision-induced dissociation of the sulfate group, aminoribose, uracil and fatty acid group, as published for FAB-MS. Similar fragmentations were recently reported for the caprazamycins. The analytical data prove that S. coelicolor M512 containing lipLK01 produces LPMs, and this confirms the identification of the LPM gene cluster. Heterologous production of intact LPMs shows that all the required genes for the formation of LPMs are encoded on cosmid 3G5. A comparison of the annotated genes on this cosmid and the CPZ biosynthetic cluster reveals striking similarities (Scheme 2). This indicates that the biosynthesis of the nonsulfurylated, nonglycosylated liponucleoside core structure, the regulation and the resistance mechanism are analogous for both compounds (Scheme 3, below). Genes directing the attachment and methylation of a deoxysugar, similar to the cpz28–31 genes at the right end of the CPZ cluster, were not identified on cosmid 3G5; this reflects the absence of the rhamnosyl moiety in the LPMs (Scheme 2). Instead, downstream of lpmY, we found an open reading frame, orf12, that encodes for a hypothetical protein with no apparent function in LPM biosynthesis. Consequently, lpmY was defined to be the right border of the LPM cluster. On the left end, upstream of lpmG, a set of genes lpmA–lpmF is proposed to play a role in sulfurylation of the LPMs. Surprisingly, similar genes were identified adjacent to the CPZ gene cluster and have been shown previously by inactivation experiments not to be involved in CPZ formation. Adjacent to lpmA are the genes orf1–11, which are highly homologous to a contiguous stretch of sequence from S. coelicolor (SCO2293–SCO2301). Therefore, the biosyn[a] L. Kaysser, S. Siebenberg, Dr. B. Gust Eberhard-Karls-Universit t T bingen, Pharmazeutische Biologie Auf der Morgenstelle 8, 72076 T bingen (Germany) Fax: (+ 49) 7071-29-5250 E-mail : bertolt.gust@uni-tuebingen.de [b] Dr. B. Kammerer Hochschule Biberach, Institut f r Pharmazeutische Biotechnologie Hubertus-Liebrecht-Strasse 35, 88400 Biberach (Germany) Fax: (+ 49) 7351-582-119 Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.200900637.

Identification of a Napsamycin Biosynthesis Gene Cluster by Genome Mining

Napsamycins are potent inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan biosynthesis, and are classified as uridylpeptide antibiotics. They comprise an N‐methyl diaminobutyric acid, an ureido group, a methionine and two non‐proteinogenic aromatic amino acid residues in a peptide backbone that is linked to a 5′‐amino‐3′‐deoxyuridine by an unusual enamide bond. The napsamycin gene cluster was identified in Streptomyces sp. DSM5940 by using PCR probes from a putative uridylpeptide biosynthetic cluster found in S. roseosporus NRRL15998 by genome mining. Annotation revealed 29 hypothetical genes encoding for resistance, regulation and biosynthesis of the napsamycins. Analysis of the gene cluster indicated that the peptide core structure is assembled by a nonlinear non‐ribosomal peptide synthetase (NRPS)‐like mechanism that involves several discrete single or didomain proteins. Some genes could be assigned, for example, to the synthesis of the N‐methyl diaminobutyric acid, to the generation of m‐tyrosine and to the reduction of the uracil moiety. The heterologous expression of the gene cluster in Streptomyces coelicolor M1154 resulted in the production of napsamycins and mureidomycins as demonstrated by LC‐ESI‐MS and MS/MS analysis. The napsamycin gene cluster provides a molecular basis for the detailed study of the biosynthesis of this class of structurally unusual compounds.

Heterologous expression of the biosynthetic gene clusters of coumermycin A1, clorobiocin and caprazamycins in genetically modified Streptomyces coelicolor strains

The biosynthetic gene clusters of the aminocoumarin antibiotics clorobiocin and coumermycin A(1) and of the liponucleoside antibiotic caprazamycin were stably integrated into the genomes of different host strains derived from Streptomyces coelicolor A3(2). For the heterologous expression of clorobiocin derivatives in a chemically defined medium, inclusion of 0.6% of the siloxylated ethylene oxide/propylene oxide copolymer Q2-5247 into the growth medium proved to result in a 4.8-fold increase of productivity. Presumably, this copolymer acts as an oxygen carrier. The additional inclusion of cobalt chloride (0.2-2 mg l(-1)) dramatically increased the percentage of the desired compound clorobiocin within the total produced clorobiocin derivatives. This is very likely due to a stimulation of a cobalamin-dependent methylation reaction catalyzed by the enzyme CloN6 of clorobiocin biosynthesis. All three investigated host strains (S. coelicolor M512, M1146 and M1154) gave similar production rates of total clorobiocin derivatives (on average, 158 mg l(-1) in the presence of 0.6% Q2-5247 and 0.2 mg l(-1) CoCl(2)). In contrast, heterologous production of caprazamycin derivatives was optimal in strain M1154 (amounts of 152 mg l(-1) on average).

A Multitasking Vanadium-Dependent Chloroperoxidase as an Inspiration for the Chemical Synthesis of the Merochlorins†

The vanadium-dependent chloroperoxidase Mcl24 was discovered to mediate a complex series of unprecedented transformations in the biosynthesis of the merochlorin meroterpenoid antibiotics. In particular, a site-selective naphthol chlorination is followed by an oxidative dearomatization/terpene cyclization sequence to build up the stereochemically complex carbon framework of the merochlorins in one step. Inspired by the enzyme reactivity, a chemical chlorination protocol paralleling the biocatalytic process was developed. These chemical studies led to the identification of previously overlooked merochlorin natural products.

A New Arylsulfate Sulfotransferase Involved in Liponucleoside Antibiotic Biosynthesis in Streptomycetes

Sulfotransferases are involved in a variety of physiological processes and typically use 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as the sulfate donor substrate. In contrast, microbial arylsulfate sulfotransferases (ASSTs) are PAPS-independent and utilize arylsulfates as sulfate donors. Yet, their genuine acceptor substrates are unknown. In this study we demonstrate that Cpz4 from Streptomyces sp. MK730–62F2 is an ASST-type sulfotransferase responsible for the formation of sulfated liponucleoside antibiotics. Gene deletion mutants showed that cpz4 is required for the production of sulfated caprazamycin derivatives. Cloning, overproduction, and purification of Cpz4 resulted in a 58-kDa soluble protein. The enzyme catalyzed the transfer of a sulfate group from p-nitrophenol sulfate (Km 48.1 μm, kcat 0.14 s−1) and methyl umbelliferone sulfate (Km 34.5 μm, kcat 0.15 s−1) onto phenol (Km 25.9 and 29.7 mm, respectively). The Cpz4 reaction proceeds by a ping pong bi-bi mechanism. Several structural analogs of intermediates of the caprazamycin biosynthetic pathway were synthesized and tested as substrates of Cpz4. Des-N-methyl-acyl-caprazol was converted with highest efficiency 100 times faster than phenol. The fatty acyl side chain and the uridyl moiety seem to be important for substrate recognition by Cpz4. Liponucleosides, partially purified from various mutant strains, were readily sulfated by Cpz4 using p-nitrophenol sulfate. No product formation could be observed with PAPS as the donor substrate. Sequence homology of Cpz4 to the previously examined ASSTs is low. However, numerous orthologs are encoded in microbial genomes and represent interesting subjects for future investigations.

Formation and Attachment of the Deoxysugar Moiety and Assembly of the Gene Cluster for Caprazamycin Biosynthesis

ABSTRACT Caprazamycins are antimycobacterials produced by Streptomyces sp. MK730-62F2. Previously, cosmid cpzLK09 was shown to direct the biosynthesis of caprazamycin aglycones, but not of intact caprazamycins. Sequence analysis of cpzLK09 identified 23 genes involved in the formation of the caprazamycin aglycones and the transfer and methylation of the sugar moiety, together with genes for resistance, transport, and regulation. In this study, coexpression of cpzLK09 in Streptomyces coelicolor M512 with pRHAM, containing all the required genes for dTDP-l-rhamnose biosynthesis, led to the production of intact caprazamycins. In vitro studies showed that Cpz31 is responsible for the attachment of the l-rhamnose to the caprazamycin aglycones, generating a rare acylated deoxyhexose. An l-rhamnose gene cluster was identified elsewhere on the Streptomyces sp. MK730-62F2 genome, and its involvement in caprazamycin formation was demonstrated by insertional inactivation of cpzDIII. The l-rhamnose subcluster was assembled with cpzLK09 using Red/ET-mediated recombination. Heterologous expression of the resulting cosmid, cpzEW07, led to the production of caprazamycins, demonstrating that both sets of genes are required for caprazamycin biosynthesis. Knockouts of cpzDI and cpzDV in the l-rhamnose subcluster confirmed that four genes, cpzDII, cpzDIII, cpzDIV, and cpzDVI, are sufficient for the biosynthesis of the deoxysugar moiety. The presented recombineering strategy may provide a useful tool for the assembly of biosynthetic building blocks for heterologous production of microbial compounds.

Diversity of ABBA Prenyltransferases in Marine Streptomyces sp CNQ-509: Promiscuous Enzymes for the Biosynthesis of Mixed Terpenoid Compounds

Terpenoids are arguably the largest and most diverse family of natural products, featuring prominently in e.g. signalling, self-defence, UV-protection and electron transfer. Prenyltransferases are essential players in terpenoid and hybrid isoprenoid biosynthesis that install isoprene units on target molecules and thereby often modulate their bioactivity. In our search for new prenyltransferase biocatalysts we focused on the marine-derived Streptomyces sp. CNQ-509, a particularly rich source of meroterpenoid chemistry. Sequencing and analysis of the genome of Streptomyces sp. CNQ-509 revealed seven putative phenol/phenazine-specific ABBA prenyltransferases, and one putative indole-specific ABBA prenyltransferase. To elucidate the substrate specificity of the ABBA prenyltransferases and to learn about their role in secondary metabolism, CnqP1 –CnqP8 were produced in Escherichia coli and incubated with various aromatic and isoprenoid substrates. Five of the eight prenyltransferases displayed enzymatic activity. The efficient conversion of dihydroxynaphthalene derivatives by CnqP3 (encoded by AA958_24325) and the co-location of AA958_24325 with genes characteristic for the biosynthesis of THN (tetrahydroxynaphthalene)-derived natural products indicates that the enzyme is involved in the formation of debromomarinone or other naphthoquinone-derived meroterpenoids. Moreover, CnqP3 showed high flexibility towards a range of aromatic and isoprenoid substrates and thus represents an interesting new tool for biocatalytic applications.