The oxidoreductases LivQ and NeoQ are responsible for the different 6'-modifications in the aminoglycosides lividomycin and neomycin.

AIMS: The 2-deoxystreptamine-containing aminoglycoside antibiotics (AGAs) constitute the largest subgroup of the aminoglycosides. Neomycin (NEO) and lividomycin (LIV) are both representatives of the pseudo-tetrasaccharide group among the NEO-type AGAs. While NEO contains a 6'-NH(2) group, the 6'-position remains unmodified in LIV. The aim of the study was to characterize the substrate specificities of the enzymes involved in the C-6'- and C-6‴-modification in order to explain the different amination patterns. METHODS AND RESULTS: We overproduced and purified the enzymes NeoQ (bifunctional 6'- and 6‴-oxidoreductase) and NeoB (bifunctional 6'- and-6‴-aminotransferase), which had been analysed before (Huang et al. 2007), and compared the enzymatic properties with the corresponding enzymes LivQ (postulated 6‴-oxidoreductase, 72% identity to NeoQ) and LivB (postulated 6‴-aminotransferase, 71% identity to NeoB) from the LIV pathway. By applying a newly established photometric assay, we proved that LivQ oxidized only pseudotetrasaccharidic substrates at the 6‴-position. In contrast, NeoQ accepted also the pseudodisaccharidic paromamine as a substrate and oxidized the 6'- and 6‴-positions on two different precursors of NEO. The aminotransferases LivB and NeoB both transfer NH(2) groups to the 6'-position in the precursor 6'-oxo-paromamine and to the 6‴-position of 6‴-oxo-neomycin C. CONCLUSIONS: The difference in the modification pattern of NEO and LIV at their 6'-positions is based only on the difference in the substrate specificities of the oxidoreductases LivQ and NeoQ, respectively. The aminotransferases LivB and NeoB share identical biochemical properties, and both are capable to transaminate the 6' and also the 6‴-position of the tested AGAs. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data provide information to understand the structural variations in aminoglycosides and may be helpful to interpret variations in other natural product bisoynthesis pathways.

Biotechnology and molecular biology of the alpha-glucosidase inhibitor acarbose.

The alpha-glucosidase inhibitor acarbose, O-[4,6-dideoxy-4[1 s-(1,4,6/5)-4,5,6-trihydroxy-3-hydroxymethyl-2-cyclohexen-1-yl]-amino-alpha-D-glucopyranosyl]-(1-->4)- O-alpha-D-glucopyranosyl-(1-->4)-D-glucopyranose, is produced in large-scale fermentation by the use of strains derived from Actinoplanes sp. SE50. It has been used since 1990 in many countries in the therapy of diabetes type II, in order to enable patients to better control blood sugar contents while living with starch-containing diets. Thus, it is one of the latest successful products of bacterial secondary metabolism to be introduced into the pharmaceutical world market. Cultures of Actinoplanes sp. also produce various other acarbose-like components, of which component C is hard to separate during downstream processing, which is one of the most modern work-up processes developed to date. The physiology, genetics and enzymology of acarbose biosynthesis and metabolism in the producer have been studied to some extent, leading to the proposal of a new pathway and metabolic cycle, the "carbophore". These data could give clues for further biotechnological developments, such as the suppression of side-products, enzymological or biocombinatorial production of new metabolites and the engineering of production rates via genetic regulation in future.

Synthesis of the milk oligosaccharide 2'-fucosyllactose using recombinant bacterial enzymes.

The enzymatic synthesis of GDP-beta-L-fucose and its enzymatic transfer reaction using recombinant enzymes from bacterial sources was examined. The GDP-D-mannose 4,6-dehydratase and the GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase from Escherichia coli K-12, respectively, were used to catalyse the conversion of GDP-alpha-D-mannose to GDP-beta-L-fucose with 78% yield. For the transfer of the L-fucose to an acceptor, we cloned and overproduced the alpha-(1-->2)-fucosyltransferase (FucT2) protein from Helicobacter pylori. We were able to synthesise 2'-fucosyllactose using the overproduced FucT2 enzyme, enzymatically synthesised GDP-L-fucose and lactose. The isolation of 2'-fucosyllactose was accomplished by anion-exchange chromatography and gel filtration to give 65% yield.

Expression and identification of the RfbE protein from Vibrio cholerae O1 and its use for the enzymatic synthesis of GDP-D-perosamine.

The 4-amino-6-deoxy-monosaccharide D-perosamine is an important element in the glycosylation of interesting cell products, such as antibiotics and lipopolysaccharides (LPS) of Gram-positive and Gram-negative bacteria. The biosynthetic pathway of the precursor molecule, GDP-D-perosamine, in Vibrio cholerae O1 starts with an isomerisation of fructose-6-phosphate catalyzed by the bifunctional enzyme phosphomannose isomerase-guanosine diphosphomannose pyrophosphorylase (RfbA; E.C. 2.7.7.22) creating the intermediate mannose-6-phosphate, which is subsequently converted by the phosphomanno-mutase (RfbB; E.C. 5.4.2.8) and further by RfbA to GDP-D-mannose, to GDP-4 keto-6-deoxymannose by a 4,6-dehydratase (RfbD; E.C. 4.2.1.47) and finally to GDP-D-perosamine by an aminotransferase (RfbE; E.C. not yet classified). We cloned the rfbD and the rfbE genes of V. cholerae O1 in Escherichia coli expression vectors. Both biosynthetic enzymes were overproduced in E. coli BL21 (DE3) and their activities were analyzed. The enzymatic conversion from GDP-D-mannose to GDP-D-perosamine was optimized and the final product, GDP-D-perosamine, was purified and identified by nuclear magnetic resonance, mass spectrometry, and chromatography. The catalytically active form of the GDP-4-keto-6-deoxy-D-mannose-4-aminotransferase seems to be a tetramer of 170 kDa. The His-tag RfbE fusion protein has a Km of 0.06 mM and a Vmax value of 38 nkat/mg protein for the substrate GDP-4-keto-6-deoxy-D-mannose. The Km and Vmax values for the cosubstrate L-glutamate were 0.1 mM and 42 nkat/mg protein, respectively. The intention of this work is to establish a basis for both the in vitro production of GDP-D-perosamine and for an in vivo perosaminylation system in a suitable bacterial host, preferably E. coli.

Identification, cloning, expression, and characterization of the extracellular acarbose-modifying glycosyltransferase, AcbD, from Actinoplanes sp. strain SE50.

An extracellular enzyme activity in the culture supernatant of the acarbose producer Actinoplanes sp. strain SE50 catalyzes the transfer of the acarviosyl moiety of acarbose to malto-oligosaccharides. This acarviosyl transferase (ATase) is encoded by a gene, acbD, in the putative biosynthetic gene cluster for the alpha-glucosidase inhibitor acarbose. The acbD gene was cloned and heterologously produced in Streptomyces lividans TK23. The recombinant protein was analyzed by enzyme assays. The AcbD protein (724 amino acids) displays all of the features of extracellular alpha-glucosidases and/or transglycosylases of the alpha-amylase family and exhibits the highest similarities to several cyclodextrin glucanotransferases (CGTases). However, AcbD had neither alpha-amylase nor CGTase activity. The AcbD protein was purified to homogeneity, and it was identified by partial protein sequencing of tryptic peptides. AcbD had an apparent molecular mass of 76 kDa and an isoelectric point of 5.0 and required Ca(2+) ions for activity. The enzyme displayed maximal activity at 30 degrees C and between pH 6.2 and 6.9. The K(m) values of the ATase for acarbose (donor substrate) and maltose (acceptor substrate) are 0.65 and 0.96 mM, respectively. A wide range of additional donor and acceptor substrates were determined for the enzyme. Acceptors revealed a structural requirement for glucose-analogous structures conserving only the overall stereochemistry, except for the anomeric C atom, and the hydroxyl groups at positions 2, 3, and 4 of D-glucose. We discuss here the function of the enzyme in the extracellular formation of the series of acarbose-homologous compounds produced by Actinoplanes sp. strain SE50.

Analysis of genes involved in 6-deoxyhexose biosynthesis and transfer in Saccharopolyspora erythraea.

Glycosylation represents an attractive target for protein engineering of novel antibiotics, because specific attachment of one or more deoxysugars is required for the bioactivity of many antibiotic and antitumour polyketides. However, proper assessment of the potential of these enzymes for such combinatorial biosynthesis requires both more precise information on the enzymology of the pathways and also improved Escherichia coli-actinomycete shuttle vectors. New replicative vectors have been constructed and used to express independently the dnmU gene of Streptomyces peucetius and the eryBVII gene of Saccharopolyspora erythraea in an eryBVII deletion mutant of Sac. erythraea. Production of erythromycin A was obtained in both cases, showing that both proteins serve analogous functions in the biosynthetic pathways to dTDP-L-daunosamine and dTDP-L-mycarose, respectively. Over-expression of both proteins was also obtained in S. lividans, paving the way for protein purification and in vitro monitoring of enzyme activity. In a further set of experiments, the putative desosaminyltransferase of Sac. erythraea, EryCIII, was expressed in the picromycin producer Streptomyces sp. 20032, which also synthesises dTDP-D-desosamine. The substrate 3-alpha-mycarosylerythronolide B used for hybrid biosynthesis was found to be glycosylated to produce erythromycin D only when recombinant EryCIII was present, directly confirming the enzymatic role of EryCIII. This convenient plasmid expression system can be readily adapted to study the directed evolution of recombinant glycosyltransferases.

Preparative synthesis of GDP-beta-L-fucose by recombinant enzymes from enterobacterial sources.

The 6-deoxyhexose L-fucose is an important and characteristic element in glycoconjugates of bacteria (e.g., lipopolysaccharides), plants (e.g., xyloglucans) and animals (e.g., glycolipids, glycoproteins, and oligosaccharides). The biosynthetic pathway of GDP-L-fucose starts with a dehydration of GDP-D-mannose catalyzed by GDP-D-mannose 4,6-dehydratase (Gmd) creating GDP-4-keto-6-deoxymannose which is subsequently converted by the GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase (WcaG; GDP-beta-L-fucose synthetase) to GDP-beta-L-fucose. Both biosynthetic genes gmd and wcaG were cloned from Escherichia coli K12 and the enzymes overexpressed under control of the T7 promoter in the expression vectors pET11a and pET16b, yielding both native and N-terminal His-tag fusion proteins, respectively. The activities of the Gmd and WcaG were analyzed. The enzymatic conversion from GDP-D-mannose to GDP-beta-L-fucose was optimized and the final product was purified. The formation of GDP-beta-L-fucose by the recombinant enzymes was verified by HPLC and NMR analyses. The His-tag fusion variants of the Gmd and WcaG proteins were purified to near homogeneity. The His-tag Gmd recombinant enzyme was inactive, whereas His-tag WcaG showed very similar enzymatic properties relative to the native GDP-beta-L-fucose synthetase. With the purified His-tag WcaG Km and Vmax values, respectively, of 40 microM and 23 nkat/mg protein for the substrate GDP-4-keto-6-deoxy-D-mannose and of 21 microM and 10 nkat/mg protein for the cosubstrate NADPH were obtained; a pH optimum of 7.5 was determined and the enzyme was stimulated to equal extend by the divalent cations Mg2+ and Ca2+. The Gmd enzyme showed a strong feedback inhibition by GDP-beta-L-fucose.

Analysis and regulation of the secY gene(1) from Streptomyces griseus N2-3-11 and interaction of the SecY protein with the SecA protein.

The chromosomal region encoding the secY gene of Streptomyces griseus N2-3-11 was cloned and analyzed. The secY gene encodes a polypeptide of 438 aa with a molecular mass of 47.5 kDa. The transcriptional start point of the secY gene was determined. Northern blot analysis revealed a growth phase-dependent secY expression supporting our previous findings for secA gene expression in S. griseus. Overproduction of the SecY protein was obtained when using Streptomyces lividans TK23 as host. The interaction of the SecY proteins of S. griseus, S. lividans, and Escherichia coli, respectively, with the purified SecA protein of S. griseus was demonstrated for the first time by using ligand affinity blot assays.

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 alpha-glucosidase inhibitor acarbose was identified in the producer Actinoplanes 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).

The genes lmbB1 and lmbB2 of Streptomyces lincolnensis encode enzymes involved in the conversion of L-tyrosine to propylproline during the biosynthesis of the antibiotic lincomycin A.

The genes lmbA,B1,B2 in the lincomycin A production gene cluster of Streptomyces lincolnensis were shown to form a common transcription unit with the promoter located directly upstream of lmbA. The proteins LmbB1 (mol. mass, 18 kDa) and LmbB2 (mol. mass 34 kDa), when over-produced together in Escherichia coli, brought about enzyme activities for the specific conversion of both L-tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA) to a yellow-colored product. The LmbB1 protein alone catalyzed the conversion of L-DOPA, but not of L-tyrosine. The purified LmbB1 protein showed a Km for L-DOPA of 258.3 microM. The L-tyrosine converting activity could not been demonstrated in vitro. The preliminary interpretation of these data suggests that the protein LmbB1 is an L-DOPA extradiol-cleaving 2,3-dioxygenase and that the protein LmbB2, either alone or in accord with LmbB1, represents an L-tyrosine 3-hydroxylase. This sequence of putative oxidation reactions on L-tyrosine seems to represent a new pathway different from the ones catalyzed by mammalian L-tyrosine hydroxylases or the wide-spread tyrosinases. The protein LmbA seemed not to be involved in this process. The labile, yellow-colored product from L-DOPA could not be converted to a picolinic acid derivative [3-(2-carboxy-5-pyridyl)alanine] in the presence of ammonia. Therefore, it probably is not a derivative of a cis, cis-3-hydroxymuconic acid semialdehyde; instead, its speculative structure represents a heterocyclic precursor of the propylhygric acid moiety of lincomycin A.

Cloning and transcriptional analysis of the rplKA-or f31-rplJL gene cluster of Streptomyces griseus.

A 5018-bp DNA fragment of the rpl/rpo BC gene cluster (here called the rif cluster) of Streptomyces griseus N2-3-11 was analysed by DNA sequencing and transcription studies. By sequence comparison of the deduced proteins, five genes and part of an open reading frame (orf) were identified. The genes encoding the ribosomal (r-) proteins L1 (rplA), L7/12 (rplJ), L10 (rplK) and L11 (rplL), a protein of known function (orf31), and the N-terminus of the beta subunit of RNA polymerase (rpoB), are organized in three operons, rplKA, rplJL and rpoB(C), and the monocistronic transcription unit orf31. The promoters of these transcription units, rplKp, orf31p, rplJp, and rpoBp, were identified and the growth-phase dependence of the transcription of these operons was analysed. Binding sites for the ribosomal proteins L1 and L10 were identified by sequence comparison, suggesting that the r-proteins RplA and RplJ are involved in feedback regulation of their respective operons by binding to specific RNA-binding sites present in both the mRNA and the 23S rRNA, as has been described for other bacteria. The analyses of the rpoBp promoter by means of promoter-probe plasmids suggested a possible attenuator-based regulatory mechanism for the transcription of the rpoB(C) operon.

Crystal structure of L-arginine:inosamine-phosphate amidinotransferase StrB1 from Streptomyces griseus: an enzyme involved in streptomycin biosynthesis.

Inosamine-phosphate amidinotransferases catalyze two nonconsecutive transamidination reactions in the biosynthesis of the streptomycin family of antibiotics. L-Arginine:inosamine-phosphate amidinotransferase StrB1 from Streptomyces griseus (StrB1) was cloned as an N-terminal hexa-histidine fusion protein, purified by affinity chromatography, and crystallized, and its crystal structure was solved by Patterson search methods at 3.1 A resolution. The structure is composed of five betabeta alphabeta-modules which are arranged circularly into a pseudo-5-fold symmetric particle. The three-dimensional structure is closely related to the structure of human L-arginine:glycine amidinotransferase (AT), but five loops (the 40-, 170-, 220-, 250-, and 270-loop) are organized very differently. The major changes are found in loops around the active site which open the narrow active site channel of AT to form an open and solvent-exposed cavity. In particular, module II of StrB1 is AT-like but lacks a 10-residue alpha-helix in the 170-loop. The concomitant reorganization of neighboring surface loops that surround the active site, i.e., the 40-loop and the 270-loop, results in an arrangement of loops which allows an unrestricted access of substrates to the cavity. However, the residues which are involved in substrate binding and catalysis are conserved in AT and StrB1 and are at equivalent topological positions, suggesting a similar reaction mechanism among amidinotransferases. The binding site for L-arginine had been deduced from its complex with AT. Molecular modeling revealed a possible binding mode for the second substrate scyllo-inosamine 4-phosphate.

The StrQ protein encoded in the gene cluster for 5'-hydroxystreptomycin of Streptomyces glaucescens GLA.0 is a alpha-D-glucose-1-phosphate cytidylyltransferase (CDP-D-glucose synthase).

The gene strQ was identified as the last gene of a putative transcription unit, strB1FGHPQ, located in the gene cluster for the production of 5'-hydroxy-streptomycin (OH-Sm) in Streptomyces glaucescens GLA.0. [In contrast, the corresponding operon in the str/sts-gene cluster of the Sm-producer Streptomyces griseus, strB1FGHIK, differs in the two distal genes; Mansouri, K. & Piepersberg, W. (1991) Mol. Gen. Genet. 228, 459-469]. The deduced StrQ protein exhibited similarities to members of the enzyme family of hexose-1-phosphate nucleotidylyltransferases (NDP-hexose synthases or pyrophosphorylases), with the strongest similarity to the subfamily of alpha-D-glucose-1-phosphate cytidylyltransferases (CDP-D-glucose synthases). The StrQ protein was heterologously expressed in Escherichia coli. The purified protein revealed an enzyme activity of that of a CDP-D-glucose synthase and a substrate specificity restricted to CTP and alpha-D-glucose 1-phosphate. The K(m) and Vmax values determined for CTP are 44 microM and 920 microM and for alpha-D-glucose 1-phosphate 195 microM and 1.06 mM, respectively. The CDP-D-glucose synthase activity was also detected in cells of S. glaucescens under the conditions of antibiotic production, but was absent from cells of the streptomycin producer S. griseus N2-3-11. Also, the genomes of several strains of S. griseus did not seem to possess strQ-related genes. In contrast, hybridisation experiments indicated that genes homologous to strQ were probably present in various other actinomycetes producing aminoglycosides. A possible function of the StrQ protein in the OH-Sm biosynthetic pathway of GLA.0 is discussed.

Protein secretion in Streptomyces griseus N2-3-11: characterization of the secA gene and its growth phase-dependent expression.

The chromosomal region encoding the secA gene of Streptomyces griseus N2-3-11 was cloned and analyzed. The secA gene encodes a polypeptide of 939 aa with a molecular mass of 105 kDa. The growth defect of temperature sensitive Escherichia coli secA mutants was not restored by the S. griseus SecA. The secA promoter was analyzed and the transcriptional start point of the gene was determined. Northern blot and Western blot analyses revealed a growth phase dependent secA expression. The integration of an additional copy of the S. griseus secA gene into the genome of S. lividans TK23 had no visible effect on the efficiency of protein secretion.

Identification of stsC, the gene encoding the L-glutamine:scyllo-inosose aminotransferase from streptomycin-producing Streptomycetes.

Eight new genes, strO-stsABCDEFG, were identified by sequencing DNA in the gene cluster that encodes proteins for streptomycin production of Streptomyces griseus N2-3-11. The StsA (calculated molecular mass 43.5 kDa) and StsC (45.5 kDa) proteins - together with another gene product, StrS (39.8 kDa), encoded in another operon of the same gene cluster - show significant sequence identity and are members of a new class of pyridoxal-phosphate-dependent aminotransferases that have been observed mainly in the biosynthetic pathways for secondary metabolites. The aminotransferase activity was demonstrated for the first time by identification of the overproduced and purified StsC protein as the L-glutamine:scyllo-inosose aminotransferase, which catalyzes the first amino transfer in the biosynthesis of the streptidine subunit of streptomycin. The stsC and stsA genes each hybridized specifically to distinct fragments in the genomic DNA of most actinomycetes tested that produce diaminocyclitolaminoglycosides. In contrast, only stsC, but not stsA, hybridized to the DNA of Streptomyces hygroscopicus ssp. glebosus, which produces the monoaminocyclitol antibiotic bluensomycin; this suggests that both genes are specifically used in the first and second steps of the cyclitol transamination reactions. Sequence comparison studies performed with the deduced polypeptides of the genes adjacent to stsC suggest that the enzymes encoded by some of these genes [strO (putative phosphatase gene), stsB (putative oxidoreductase gene), and stsE (putative phosphotransferase gene)] also could be involved in (di-)aminocyclitol synthesis.

The str gene cluster for the biosynthesis of 5'-hydroxystreptomycin in Streptomyces glaucescens GLA.0 (ETH 22794): new operons and evidence for pathway-specific regulation by StrR.

Two divergently oriented operons, strXU and strVW, located within the gene cluster for 5'-hydroxystreptomycin (5'-OH-Sm) biosynthesis in Streptomyces glaucescens strain GAL.0 (ETH 22794), were analysed by DNA sequencing and transcription/regulation studies. Three genes, strU and strVW, are conserved in a similar arrangement but in a different location within the str/sts gene cluster of the Sm-producing strain S. griseus N2-3-11. The four putative products resemble NDP-4-ketohexose 3,5-epimerases (StrX, M(r) 20.2 kDa), NAD(P)-dependent oxidoreductases (StrU, 45.6 kDa), and ABC-transporters (StrV, 61.8 kDa; StrW, 63.4 kDa). These genes are apparently involved in the biosynthesis of 5'-OH-Sm because the promoters of both operons are activated in trans by the activator StrR of S. griseus N2-3-11, when cloned in S. lividans 66 TK23. A sequence motif resembling the consensus sequence GTTCGActG(N)11CagTcGAAc for binding of StrR was identified within the intergenic region of strX and strV. Specific binding of StrR to this site was demonstrated by gel retardation assays using purified His*Tag-StrR.

Nucleotide sequences of streptomycete 16S ribosomal DNA: towards a specific identification system for streptomycetes using PCR.

To facilitate the differential identification of the genus Streptomyces, the 16S rRNA genes of 17 actinomycetes were sequenced and screened for the existence of streptomycete-specific signatures. The 16S rDNA of the Streptomyces strains and Amycolatopsis orientalis subsp. lurida exhibited 95-100% similarity, while that of the 16S rDNA of Actinoplanes utahensis showed only 88% similarity to the streptomycete 16S rDNAs. Potential genus-specific sequences were found in regions located around nucleotide positions 120, 800 and 1100. Several sets of primers derived from these characteristic regions were investigated as to their specificity in PCR-mediated amplifications. Most sets allowed selective amplification of the streptomycete rDNA sequences studied. RFLPs in the 16S rDNA permitted all strains to be distinguished.

Molecular characterization of the lincomycin-production gene cluster of Streptomyces lincolnensis 78-11.

The lincomycin (LM)-production gene cluster of the overproducing strain Streptomyces lincolnensis 78-11 was cloned, analysed by hybridization, as well as by DNA sequencing, and compared with the respective genome segments of other lincomycin producers. The lmb/lmr gene cluster is composed of 27 open reading frames with putative biosynthetic or regulatory functions (lmb genes) and three resistance (lmr) genes, two of which, lmrA and lmrC, flank the cluster. A very similar overall organization of the lmb/lmr cluster seems to be conserved in four other LM producers, although the clusters are embedded in non-homologous genomic surroundings. In the wild-type strain (S. lincolnensis NRRL2936), the lmb/lmr-cluster apparently is present only in single copy. However, in the industrial strain S. lincolnensis 78-11 the non-adjacent gene clusters for the production of LM and melanin (melC) both are duplicated on a large (0.45-0.5 Mb) fragment, accompanied by deletion events. This indicates that enhanced gene dosage is one of the factors for the overproduction of LM and demonstrates that large-scale genome rearrangements can be a result of classical strain improvement by mutagenesis. Only a minority of the putative Lmb proteins belong to known protein families. These include members of the gamma-glutamyl transferases (LmbA), amino acid acylases (LmbC), aromatic amino acid aminotransferases (LmbF), imidazoleglycerolphosphate dehydratases (LmbK), dTDP-glucose synthases (LmbO), dTDP-glucose 4,6-dehydratases (LmbM) and (NDP-) ketohexose (or ketocyclitol) aminotransferases (LmbS). In contrast to earlier proposals on the biosynthetic pathway of the C-8 sugar moiety (methylthiolincosaminide), this branch of the LM pathway actually seems to be based on nucleotide-activated sugars as precursors.

Application of random amplified polymorphic DNA (RAPD) assays in identifying conserved regions of actinomycete genomes.

Various arbitrary primers as well as pUC18/19 'reverse' sequencing primers were used for random amplified polymorphic DNA assays. Use of a modified reverse primer led to amplification of one major approx. 1100-bp band from the chromosomal DNA of all actinomycetes tested; however, the band was not found when DNAs from other bacteria were used in comparable experiments. Hybridization experiments showed that these bands all contained similar genomic regions. Subsequent sequencing of four of these fragments showed they each contained the sequence of the 3' end of the 23S rRNA gene, the intergenic region and the start of the 5S rRNA gene.