Carboxyphosphonoenolpyruvate phosphonomutase, a novel enzyme catalyzing C-P bond formation.

An enzyme catalyzing the formation of an unusual C-P bond that is involved in the biosynthesis of the antibiotic bialaphos (BA) was isolated from the cell extract of a mutant (NP71) of Streptomyces hygroscopicus SF1293. This enzyme, carboxyphosphonoenolpyruvate (CPEP) phosphonomutase, was first identified as a protein lacking in a mutant (NP213) defective in one of the steps in the pathway to BA. The first 30 residues of the amino terminus of this protein were identical to those predicted by the nucleotide sequence of the gene that restored BA production to NP213. The substrate of the enzyme, a P-carboxylated derivative of phosphoenolpyruvate named CPEP, was also isolated from the broth filtrate of NP213 as a new biosynthetic intermediate of BA. CPEP phosphonomutase catalyzes the rearrangement of the carboxyphosphono group of CPEP to form the C-P bond of phosphinopyruvate.     

Heterologous production of daptomycin in Streptomyces lividans

Daptomycin and the A21978C antibiotic complex are lipopeptides produced by Streptomyces roseosporus and also in recombinant Streptomyces lividans TK23 and TK64 strains, when a 128 kbp region of cloned S. roseosporus DNA containing the daptomycin gene cluster is inserted site-specifically in the ϕC31 attB site. A21978C fermentation yields were initially much lower in S. lividans than in S. roseosporus, and detection was complicated by the production of host metabolites. However A21978C production in S. lividans was improved by deletion of genes encoding the production of actinorhodin and by medium optimization to control the chemical form of the calcium dependent antibiotic (CDA). This latter compound has not previously been chemically characterized as a S. lividans product. Adding phosphate to a defined fermentation medium resulted in formation of only the phosphorylated forms of CDA, which were well separated from A21978C on chromatographic analysis. Adjusting the level of phosphate in the medium led to an improvement in A21978C yield from 20 to 55 mg/l.     

Cloning and characterization of a gene of Streptomyces griseus that increases production of extracellular enzymes in several species of Streptomyces.

Summary A 7.2 kbBglII restriction fragment, which increases the production of several extracellular enzymes, including alkaline phosphatase, amylase, protease, lipase and -galactosidase, was cloned inStreptomyces lividans from the DNA ofS. griseus ATCC 10137. This gene (namedsaf) showed a positive gene dosage effect on production of extracellular enzymes. When thesaf gene was introduced into cells in high copy numbers it delayed the formation of pigments and spores inS. lividans and also retarded actinorhodin production inStreptomyces coelicolor. Thesaf gene hybridized with specific bands in the DNA of severalStreptomyces strains tested. A 1 kb fragment containing thesaf gene was sequenced and contains an open reading frame (ORF) of 306 nucleotides which encodes a polypeptide of M_r 10 500. This ORF is contained within a fragment of 432 by which retained activity inStreptomyces. A fragment with promoter activity is present upstream of thesaf reading frame. The predicted Saf polypeptide has a strong positive charge, and does not show a typical amino acid composition for a membrane protein, and contains a DNA-binding domain similar to those found in several regulatory proteins.     

Comparison of extracellular Escherichia coli AppA phytases expressed in Streptomyces lividans and Pichia pastoris

A phytase gene (appA) from Escherichia coli was cloned into Streptomyces lividans and expressed as an extracellular protein which was then compared with the same enzyme expressed in Pichia pastoris. The phytase expressed in S. lividans was not glycosylated and had a molecular mass of 45 kDa. Compared with the glycosylated phytase expressed in P. pastoris, this non-glycosylated phytase was 25–50% less active (p<0.05) at pH 2 to 3.5 or at 45 and 55 °C, but 50% more active (p<0.05) at 75 °C. The thermo-tolerance of the non-glycosylated phytase was 26 and 48% higher (p<0.05) than that of the glycosylated phytase at 45 and 55 °C, but was 80 and 94% lower (p<0.05) at 65 ° and 75 °C, respectively.     

Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae

Summary A gene conferring high-level resistance to tylosin in Streptomyces lividans and Streptomyces griseofuscus was cloned from a tylosin-producing strain of Streptomyces fradiae. The tylosin-resistance (Tyl^r) gene (tlrA) was isolated on five overlapping DNA fragments which contained a common 2.6 Kb KpnI fragment. The KpnI fragment contained all of the information required for the expression of the Tyl^r phenotype in S. lividans and S. griseofuscus. Southern hybridization indicated that the sequence conferring tylosin resistance was present on the same 5 kb SalI fragment in genomic DNA from S. fradiae and several tylosin-sensitive (Tyl^s) mutants. The cloned tlrA gene failed to restore tylosin resistance in two Tyl^s mutants derived by protoplast formation and regeneration, and it restored partial resistance in a Tyl^s mutant obtained by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The tlrA gene conferred resistance to tylosin, carbomycin, niddamycin, vernamycin-B and, to some degree, lincomycin in S. griseofuscus, but it had no effect on sensitivity to streptomycin or spectinomycin, suggesting that the cloned gene is an MLS (macrolide, lincosamide, streptogramin-B)-resistance gene. Twenty-eight kb of S. fradiae DNA surrounding the tlrA gene was isolated from a genomic library in bacteriophage Charon 4. Introduction of these DNA sequence into S. fradiae mutants blocked at different steps in tylosin biosynthesis failed to restore tylosin production, suggesting that the cloned Tyl^r gene is not closely linked to tylosin biosynthetic genes.     

The terminal inverted repeats of the linear plasmid SCP1 of Streptomyces coelicolor A3(2) possess a truncated copy of the transposon Tn 4811 of Streptomyces lividans 66

Streptomyces coelicolor A3(2), the best genetically studied streptomycete and Streptomyces lividans 66 are very closely related strains. This is further emphasized by our finding that a truncated copy of Tn4811 of S. lividans is present in the terminal inverted repeats of the S. coelicolor giant linear plasmid SCP1. The copy of Tn4811 in SCP1 lacks the first 1276 bp and shows only minor changes in the nucleotide sequence of the remaining 4.12 kb. Tn4811 exists in both ends of SCP1.     

Excision of pIJ408 from the chromosome of Streptomyces glaucescens and its transfer into Streptomyces lividans

Summary Streptomyces glaucescens GLA000 contains the integrated 15 kb DNA element pIJ408 which, during mating of the parent strain with S. lividans, can be transferred into recipient cells. In S. lividans cells, pIJ408 was found in an autonomously replicating form and in a chromosomally integrated state. In the majority of the S. lividans transconjugants studied, a deletion derivative pIJ408. 1 (12.4 kb) occurred. The deletion form was found in some strains only as a free plasmid, in others it was also chromosomally integrated. The integration region of pIJ408 was subcloned and precisely mapped by hybridization, restriction and sequencing analyses. The DNA junction fragments of the integrated plasmid in S. glaucescens, as well as the DNA fragment containing the attachment site of the S. lividans chromosome, were also cloned, submitted to detailed restriction analysis and sequenced. The attachment site of pIJ408 (attP) and the junctions of its integrated form with the chromosomal DNA in S. glaucescens (attL and attR) contain an identical 43 bp sequence. The chromosomal attachment site in S. lividans (attB) differs from the S. glaucescens att sequence by a single base substitution. The similarities between attachment sites of SLP1, pMEA100, pSAM2 and pIJ408 are discussed.     

Site-Specific Integration of the Double-Mutation Glucose Isomerase (GIG138PG247D) Gene in Streptomyces lividans and Its Stable Expression

A recombinant expression plasmid pYH12, containing the double-mutation glucose isomerase (GIG138PG247D, GI2) coding gene and its natural regulatory sequence, was constructed for site-specific integration in Streptomyces. The resulting plasmid was introduced into Streptomyces lividans TK54 by protoplast transformation and two apramycin-resistance (Am^R) transformants, designated GY2 and BY7, respectively, were obtained further based on enzyme assays. These results for polymerase chain reaction (PCR), Dot blot, and recovery of cloned fragments from the transformant chromosome indicated that the GI2 gene was integrated into the S. lividans chromosome by site-specific recombination, and which was further verified by Southern blot. We found that the free form of plasmid pYH12 co-existing with the integrated form was present in S. lividans. SDS-PAGE analysis showed that the GI2 gene was expressed in S. lividans. The intracellular GI2 specific activity was 1.15 U/mg. The stability of integrants demonstrated that the cloned GI2 gene was stably integrated and expressed even in the absence of selective pressure. Received: 28 March 2001 / Accepted: 14 May 2001     

Cloning of a Microbispora bispora cellobiohydrolase gene in Streptomyces lividans

The cellobiohydrolase II (CBHII) of Microbispora bispora, originally cloned in Escherichia coli, was subcloned into Streptomyces lividans using shuttle vectors pSKN 01 and pSKN 02. The enzyme was secreted from Streptomyces, whereas it was intracellular in E. coli. The yields of CBHII produced by S. lividans transformants were 15–20-fold higher than those produced by E. coli transformants. The optimal pH of M. bispora native cellobiohydrolase and the cloned enzyme from S. lividans is 6.5. The thermal and pH stability of CHBII produced in M. bispora, E. coli and S. lividans were compared. Enzyme produced in E. coli was inactivated more rapidly (k = 0.252 min^–1 at 90° C; 90% inactivation after 10 min vs. 0.119 min^–1 for the others). CBHII was monitored following electrophoretic separation by reaction with a monoclonal antibody. The apparent molecular mass of the protein produced from the S. lividans clone was 93 kDa, the same as that of the native enzyme, but that of the enzyme produced in E. coli was smaller (82 kDa). Correspondence to: P. Hu     

Unraveling the essential role in conjugation of the Tra protein of Streptomyces lividans plasmid pIJ101

Plasmid pIJ101 from Streptomyces lividans encodes a single gene, tra, that is essential for both plasmid transfer and mobilization of chromosomes during mating. The tra gene product (Tra) is a membrane protein, a portion of which shows similarity to transfer proteins of other streptomycete plasmids as well as additional bacterial chromosome partitioning proteins. This paper reviews past and present work that has focused on elucidating the precise role of the Tra protein of pIJ101 in conjugation in Streptomyces.     

Molecular Cloning of Streptomyces Genes Encoding Vanillate Demethylase

The vanillate demethylase genes from Streptomyces sp. NL15-2K were cloned and sequenced. The vanA and vanB gene homologs, which encode the terminal oxygenase subunit (VanA) and the ferredoxin-type reductase subunit (VanB) of the enzyme respectively, were found in the sequenced 7.5-kb DNA region. Expression of the vanAB genes in Streptomyces lividans 1326 resulted in in vivo demethylation of veratric acid to vanillic acid.     

Methylation of 16S ribosomal RNA and resistance to aminoglycoside antibiotics in clones of Streptomyces lividans carrying DNA from Streptomyces tenjimariensis

Summary A single gene from Streptomyces tenjimariensis, conferring resistance to kanamycin, apramycin and sisomicin, has been cloned in Streptomyces lividans. The mechanism of resistance involves methylation of 16S RNA in the 30S ribosomal subunit.     

Glucose kinase of Streptomyces coelicolor A3(2): large-scale purification and biochemical analysis

Glucose kinase of Streptomyces coelicolor A3(2) is essential for glucose utilisation and is required for carbon catabolite repression (CCR) exerted through glucose and other carbon sources. The protein belongs to the ROK-family, which comprises bacterial sugar kinases and regulators. To better understand glucose kinase function, we have monitored the cellular activity and demonstrated that the choice of carbon sources did not significantly change the synthesis and activity of the enzyme. The DNA sequence of the Streptomyces lividans glucose kinase gene glkA was determined. The predicted gene product of 317 amino acids was found to be identical to S. coelicolor glucose kinase, suggesting a similar role for this protein in both organisms. A procedure was developed to produce pure histidine-tagged glucose kinase with a yield of approximately 10 mg/l culture. The protein was stable for several weeks and was used to raise polyclonal antibodies. Purified glucose kinase was used to explore protein-protein interaction by surface plasmon resonance. The experiments revealed the existence of a binding activity present in S. coelicolor cell extracts. This indicated that glucose kinase may interact with (an)other factor(s), most likely of protein nature. A possible cross-talk with proteins of the phosphotransferase system, which are involved in carbon catabolite repression in other bacteria, was investigated.     

Directed evolution of <Emphasis Type="Italic">Streptomyces lividans</Emphasis> xylanase B toward enhanced thermal and alkaline pH stability

The thermal and alkaline pH stability of Streptomyces lividans xylanase B was improved greatly by random mutagenesis using DNA shuffling. Positive clones with improved thermal stability in an alkaline buffer were screened on a solid agar plate containing RBB-xylan (blue). Three rounds of directed evolution resulted in the best mutant enzyme 3SlxB6 with a significantly improved stability. The recombinant enzyme exhibited significant thermostability at 70°C for 360 min, while the wild-type lost 50% of its activity after only 3 min. In addition, mutant enzyme 3SlxB6 shows increased stability to treatment with pH 9.0 alkaline buffer. The K _m value of 3SlxB6 was estimated to be similar to that of wild-type enzyme; however k _cat was slightly decreased, leading to a slightly reduced value of k _cat/K _m, compared with wild-type enzyme. DNA sequence analysis revealed that eight amino acid residues were changed in 3SlxB6 and substitutions included V3A, T6S, S23A, Q24P, M31L, S33P, G65A, and N93S. The stabilizing effects of each amino acid residue were investigated by incorporating mutations individually into wild-type enzyme. Our results suggest that DNA shuffling is an effective approach for simultaneous improvement of thermal and alkaline pH stability of Streptomyces lividans xylanase B even without structural information.     

Isolation and genetic structure of IS112, an insertion sequence responsible for the inactivation of the SalI restriction-modification system of Streptomyces albus G.

Summary IS112 is a transposable element identified in Streptomyces albus G by its frequent mutagenic insertion into the genes for the SalI restriction-modification system. IS112 is present in several copies in the genome of S. albus G. Homologous sequences were detected in other Streptomyces strains. Sequence analysis revealed that IS112 has a length of 883 by with a GC content of 67.4%. The copy that was isolated contained imperfect inverted repeats (16/20 match) at its ends and was flanked by a 2 by duplication at the target site, which was located within the gene (salIR) for the Sall endonuclease. A long open reading frame (ORF) encoding a putative polypeptide of 256-253 amino acids spans almost the entire sequence. Significant homology was detected between this polypeptide and that corresponding to ORFB of IS493, an insertion sequence recently isolated from Streptomyces lividans 66.

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Increased heterologous production of the antitumoral polyketide mithramycin A by engineered Streptomyces lividans TK24 strains

Mithramycin A is an antitumor compound used for treatment of several types of cancer including chronic and acute myeloid leukemia, testicular carcinoma, hypercalcemia and Paget’s disease. Selective modifications of this molecule by combinatorial biosynthesis and biocatalysis opened the possibility to produce mithramycin analogues with improved properties that are currently under preclinical development. The mithramycin A biosynthetic gene cluster from Streptomyces argillaceus ATCC12956 was cloned by transformation assisted recombination in Saccharomyces cerevisiae and heterologous expression in Streptomyces lividans TK24 was evaluated. Mithramycin A was efficiently produced by S. lividans TK24 under standard fermentation conditions. To improve the yield of heterologously produced mithramycin A, a collection of derivative strains of S. lividans TK24 were constructed by sequential deletion of known potentially interfering secondary metabolite gene clusters using a protocol based on the positive selection of double crossover events with blue pigment indigoidine-producing gene. Mithramycin A production was evaluated in these S. lividans strains and substantially improved mithramycin A production was observed depending on the deleted gene clusters. A collection of S. lividans strains suitable for heterologous expression of actinomycetes secondary metabolites were generated and efficient production of mithramycin A with yields close to 3 g/L, under the tested fermentation conditions was achieved using these optimized collection of strains.

     

Efficient purification of transglutaminase from recombinant <Emphasis Type="Italic">Streptomyces platensis</Emphasis> at various scales

An efficient system for the fast and efficient purification of transglutaminase from recombinant Streptomyces platensis and expressed in Streptomyces lividans 25-2 is described. Because the purification procedure of this system is flexible, culture broth from laboratory (20 l) and pilot-plant (130 l) fermentations were used to purify the enzyme to electrophoretic homogeneity with high purity (90–95%) and yield (61–77%) within 1 or 2 days.     

The Streptomyces ghanaensis low copy plasmid pSG2 and its use for vector construction

A plasmid, pSG2, was isolated from Streptomyces ghanaensis and characterized by electron microscopy, buoyant density measurement, and restriction enzyme analysis. The length of 13.8 kb, single restriction sites for HindIII, EcoRV and PvuII and the possibility of deleting non-essential regions of the plasmid made pSG2 a suitable basic replicon for vector development. pSG2 has a copy number of about four. Plasmid pSG2 was fused to a pACYC184 derivative modified to harbour a thiostrepton resistance gene. The resulting plasmid, designated pSW1, is a 16.6 kb shuttle plasmid which replicates in Escherichia coli and in several Streptomyces strains, including S. ghanaensis, S. lividans and S. viridochromogenes. Replacement of a Bg/II-fragment of plasmid pSG2 by a fragment encoding thiostrepton resistance resulted in a low copy 12.2 kb Streptomyces plasmid. This plasmid, designated pSW2, is a Streptomyces broad host range plasmid.     

Evaluation of TatABC overproduction on Tat- and Sec-dependent protein secretion in Streptomyces lividans

The majority of bacterial proteins are exported across the cytoplasmic membrane via the Sec pathway, but also the more recently discovered twin-arginine translocation (Tat) route seems to play an important role for protein secretion in Streptomyces lividans in whose genome tatA, tatB and tatC have been identified. In the present work we showed that simultaneous overproduction of TatABC improved the Tat-dependent secretion capacity as could be concluded from the increased amount of secreted xylanase C, an exclusive Tat-dependent substrate. This result demonstrates that next to the availability of energy to drive secretion, also the number of translocases can be rate-limiting for Tat-dependent secretion. On the other hand, tatABC overexpression was found to diminish secretion of the Sec-dependent proteins xylanase B and subtilisin inhibitor in S. lividans. These results reveal cross-talk between both pathways in S. lividans.     

Extracellular production of a sphingomyelinase from <Emphasis Type="Italic">Streptomyces griseocarneus</Emphasis> using <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

The structural gene for sphingomyelinase (SMase) from Streptomyces griseocarneus, was introduced into Streptomyces lividans using a shuttle vector, pUC702, for Escherichia coli/S. lividans. High-level secretory production of SMase was achieved using the promoter, signal sequence and terminator regions of phospholipase D from Streptoverticillium cinnamoneum. The transformant constitutively expressed a high specific activity of SMase extracellularly during batch culture. Maximum SMase activity (555 ± 114 U/mg protein) was with 1.75 M MgCl₂ which was about 50-fold more than that with 10 mM MgCl₂.