Bertolt Gust

PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin

Streptomycetes are high G+C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and λ-Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriTRK2 for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriTRK2 to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate.

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.

Expression of Cre recombinase during transient phage infection permits efficient marker removal in Streptomyces

We report a system for the efficient removal of a marker flanked by two loxP sites in Streptomyces coelicolor, using a derivative of the temperate phage φC31 that expresses Cre recombinase during a transient infection. As the test case for this recombinant phage (called Cre-phage), we present the construction of an in-frame deletion of a gene, pglW, required for phage growth limitation or Pgl in S.coelicolor. Cre-phage was also used for marker deletion in other strains of S.coelicolor.

A gene cluster for prenylated naphthoquinone and prenylated phenazine biosynthesis in Streptomyces cinnamonensis DSM 1042

Streptomyces cinnamonensis DSM 1042 produces two classes of secondary metabolites of mixed isoprenoid/nonisoprenoid origin: the polyketide–isoprenoid compound furanonaphthoquinone I (FNQ I) and several prenylated phenazines, predominantly endophenazine A. We now report the cloning and sequence analysis of a 55 kb gene cluster required for the biosynthesis of these compounds. Several inactivation experiments confirmed the involvement of this gene cluster in the biosynthesis of FNQ I and endophenazine A. The six identified genes for endophenazine biosynthesis showed close similarity to phenazine biosynthetic genes from Pseudomonas. Of the 28 open reading frames identified in the adjacent FNQ I cluster, 13 showed close similarity to genes contained in the cluster for furaquinocin—a structurally similar metabolite from another Streptomyces strain. These genes included a type III polyketide synthase sequence, a momA‐like monooxygenase gene, and two cloQ‐like prenyltransferase genes designated fnq26 and fnq28. Inactivation experiments confirmed the involvement of fnq26 in FNQ I biosynthesis, whereas no change in secondary‐metabolite formation was observed after fnq28 inactivation. The FNQ I cluster contains a contiguous group of five genes, which together encode all the enzymatic functions required for the recycling of S‐adenosylhomocysteine (SAH) to S‐adenosylmethionine (SAM). Two SAM‐dependent methyltransferases are encoded within the cluster. Inactivation experiments showed that fnq9 is responsible for the 7‐O‐methylation and fnq27 for the 6‐C‐methylation reaction in FNQ I biosynthesis.

Aromatic Prenylation in Phenazine Biosynthesis DIHYDROPHENAZINE-1-CARBOXYLATE DIMETHYLALLYLTRANSFERASE FROM STREPTOMYCES ANULATUS

The bacterium Streptomyces anulatus 9663, isolated from the intestine of different arthropods, produces prenylated derivatives of phenazine 1-carboxylic acid. From this organism, we have identified the prenyltransferase gene ppzP. ppzP resides in a gene cluster containing orthologs of all genes known to be involved in phenazine 1-carboxylic acid biosynthesis in Pseudomonas strains as well as genes for the six enzymes required to generate dimethylallyl diphosphate via the mevalonate pathway. This is the first complete gene cluster of a phenazine natural compound from streptomycetes. Heterologous expression of this cluster in Streptomyces coelicolor M512 resulted in the formation of prenylated derivatives of phenazine 1-carboxylic acid. After inactivation of ppzP, only nonprenylated phenazine 1-carboxylic acid was formed. Cloning, overexpression, and purification of PpzP resulted in a 37-kDa soluble protein, which was identified as a 5,10-dihydrophenazine 1-carboxylate dimethylallyltransferase, forming a C–C bond between C-1 of the isoprenoid substrate and C-9 of the aromatic substrate. In contrast to many other prenyltransferases, the reaction of PpzP is independent of the presence of magnesium or other divalent cations. The Km value for dimethylallyl diphosphate was determined as 116 μm. For dihydro-PCA, half-maximal velocity was observed at 35 μm. Kcat was calculated as 0.435 s-1. PpzP shows obvious sequence similarity to a recently discovered family of prenyltransferases with aromatic substrates, the ABBA prenyltransferases. The present finding extends the substrate range of this family, previously limited to phenolic compounds, to include also phenazine derivatives.

Developmental-Stage-Specific Assembly of ParB Complexes in Streptomyces coelicolor Hyphae

In Streptomyces coelicolor ParB is required for accurate chromosome partitioning during sporulation. Using a functional ParB-enhanced green fluorescent protein fusion, we observed bright tip-associated foci and other weaker, irregular foci in S. coelicolor vegetative hyphae. In contrast, in aerial hyphae regularly spaced bright foci accompanied sporulation-associated chromosome condensation and septation.

A soluble, magnesium-independent prenyltransferase catalyzes reverse and regular C-prenylations and O-prenylations of aromatic substrates

Fnq26 from Streptomyces cinnamonensis DSM 1042 is a new member of the recently identified CloQ/Orf2 class of prenyltransferases. The enzyme was overexpressed in E. coli and purified to apparent homogeneity, resulting in a soluble, monomeric protein of 33.2 kDa. The catalytic activity of Fnq26 is independent of the presence of Mg2+ or other divalent metal ions. With flaviolin (2,5,7‐trihydroxy‐1,4‐naphthoquinone) as substrate, Fnq26 catalyzes the formation of a carbon–carbon‐bond between C‐3 (rather than C‐1) of geranyl diphosphate and C‐3 of flaviolin, i.e. an unusual “reverse” prenylation. With 1,3‐dihydroxynaphthalene and 4‐hydroxybenzoate as substrates Fnq26 catalyzes O‐prenylations.

Phage P1-Derived Artificial Chromosomes Facilitate Heterologous Expression of the FK506 Gene Cluster

We describe a procedure for the conjugative transfer of phage P1-derived Artificial Chromosome (PAC) library clones containing large natural product gene clusters (≥70 kilobases) to Streptomyces coelicolor strains that have been engineered for improved heterologous production of natural products. This approach is demonstrated using the gene cluster for FK506 (tacrolimus), a clinically important immunosuppressant of high commercial value. The entire 83.5 kb FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 present in one 130 kb PAC clone was introduced into four different S. coelicolor derivatives and all produced FK506 and smaller amounts of the related compound FK520. FK506 yields were increased by approximately five-fold (from 1.2 mg L-1 to 5.5 mg L-1) in S. coelicolor M1146 containing the FK506 PAC upon over-expression of the FK506 LuxR regulatory gene fkbN. The PAC-based gene cluster conjugation methodology described here provides a tractable means to evaluate and manipulate FK506 biosynthesis and is readily applicable to other large gene clusters encoding natural products of interest to medicine, agriculture and biotechnology.

Reducing the variability of antibiotic production in Streptomyces by cultivation in 24-square deepwell plates.

Highly reproducible production values of the aminocoumarin antibiotic novobiocin were achieved by cultivation of a heterologous Streptomyces producer strain in commercially available square deepwell plates consisting of 24 wells of 3 ml culture volume each. Between parallel cultivation batches in the deepwell plates, novobiocin accumulation showed standard deviations of 4-9%, compared to 39% in baffled Erlenmeyer flasks. Mycelia used as inoculum could be frozen in the presence of 20% peptone and stored at -70 degrees C, allowing repeated cultivations from the same batch of inoculum over extended periods of time. Originally, novobiocin titers in the deepwell plate (5-12 mg l(-1)) were lower than in Erlenmeyer flasks (24 mg l(-1)). Optimization of the inoculation procedure as well as addition of a siloxylated ethylene oxide/propylene oxide copolymer, acting as oxygen carrier, to the production medium increased novobiocin production to 54 mg l(-1). The additional overexpression of the pathway-specific positive regulator gene novG increased novobiocin production to 163 mg l(-1). Harvesting the precultures in a defined section of growth phase greatly reduced variability between different batches of inoculum. The use of deepwell plates may considerably reduce the workload and cost of investigations of antibiotic biosynthesis in streptomycetes and other microorganisms due to the high reproducibility and the low requirement for shaker space and culture medium.

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.