Lividans TK24 Research Articles

Characterization of the Polyoxin Biosynthetic Gene Cluster from Streptomyces cacaoi and Engineered Production of Polyoxin H

A gene cluster (pol) essential for the biosynthesis of polyoxin, a nucleoside antibiotic widely used for the control of phytopathogenic fungi, was cloned from Streptomyces cacaoi. A 46,066-bp region was sequenced, and 20 of 39 of the putative open reading frames were defined as necessary for polyoxin biosynthesis as evidenced by its production in a heterologous host, Streptomyces lividans TK24. The role of PolO and PolA in polyoxin synthesis was demonstrated by in vivo experiments, and their functions were unambiguously characterized as O-carbamoyltransferase and UMP-enolpyruvyltransferase, respectively, by in vitro experiments, which enabled the production of a modified compound differing slightly from that proposed earlier. These studies should provide a solid foundation for the elucidation of the molecular mechanisms for polyoxin biosynthesis, and set the stage for combinatorial biosynthesis using genes encoding different pathways for nucleoside antibiotics.

Functional Expression of SAV3818, a Putative TetR-Family Transcriptional Regulatory Gene from Streptomyces avermitilis, Stimulates Antibiotic Production in Streptomyces Species

Avermectin and its analogs are major commercial antiparasitic agents in the fields of animal health, agriculture, and human infections. Previously, comparative transcriptome analysis between the low-producer S. avermitilis ATCC31267 and the high-producer S. avermitilis ATCC31780 using a S. avermitilis whole genome chip revealed that 50 genes were overexpressed at least two-fold higher in S. avermitilis ATCC31780. To verify the biological significance of some of the transcriptomics-guided targets, five putative regulatory genes were individually cloned under the strong-andconstitutive promoter of the Streptomyces expression vector pSE34, followed by the transformation into the lowproducer S. avermitilis ATCC31267. Among the putative genes tested, three regulatory genes including SAV213, SAV3818, and SAV4023 exhibited stimulatory effects on avermectin production in S. avermitilis ATCC31267. Moreover, overexpression of SAV3818 also stimulated actinorhodin production in both S. coelicolor M145 and S. lividans TK21, implying that the SAV3818, a putative TetR-family transcriptional regulator, could be a global upregulator acting in antibiotic production in Streptomyces species.

Isolation and Identification of α,α-Trehalose and Glycerol from an Arctic Psychrotolerant Streptomyces sp. SB9 and their Possible Role in the Strain's Survival

Streptomyces sp. strain SB9 was isolated from perm frost soil samples in Spitsbergen, Arctic Ocean; it grows in a temperature range between 4°C and 28°C. During the survey of biologically active metabolites biosynthesized by this strain, significant amounts of α,α-trehalose (1) and glycerol (2) were detected. The compounds were isolated from the mycelium, were chromatographically separated, and the structures were elucidated on the basis of MS and NMR measurements. A possible role of trehalose in cold adaptation of the strain was examined. It was determined that the mycelium of the strain cultivated at 4°C accumulated 5-fold higher amounts of trehalose in comparison with the cells cultivated at 28°C. The mesofilic reference strains, Streptomyces spectabilis NRRL 2494 and Streptomyces lividans TK64, accumulated 100-fold less trahalose than the psychrotolerant Streptomyces sp. SB9. High amounts of trehalose in the cells could be a reason for adaptation of the strain to life at Arctic conditions.

An ABC transporter encoding gene lndW confers resistance to landomycin E

Streptomyces globisporus 1912 produces a polyketide antibiotic landomycin E (LaE), which possesses anticancer activity. A 1.8 kb DNA fragment at the end of landomycin E biosynthetic gene cluster was sequenced. DNA sequence analysis of this fragment identified one complete open reading frame, designated lndW. The deduced sequence of lndW gene product revealed significant similarity to the ATP-binding domains of the ABC (ATP-binding protein cassette) superfamily of transport-related proteins. Knockout of lndW had no significant effect on resistance to LaE and its production. The expression of lndW in S. globisporus 1912 was proven via transcriptional fusion of lndW promoter to EGFP (enhanced green fluorescent protein). Overexpression of lndW in S. lividans TK24 conferred resistance to LaE. The mechanism of lndW function in LaE biosynthesis is discussed.

Heterologous Expression of SARS-CoV ORF10 and X5 Genes in E. coli and Streptomyces lividans TK24

In previous studies a variety of novel accessory genes has been identified that were interspersed among the structural genes of the SARS-CoV (severe acute respiratory syndrome coronavirus) genome. The predicted unknown proteins (PUPs) encoded by the accessory genes, which are considered to be unique to the SARS-CoV genome, might play important roles in the SARS-CoV infection. Two of these genes, called ORF10 and X5, were synthesized and introduced into E. coli and Streptomyces lividans TK24, respectively. SDS-PAGE and Western blot revealed that the ORF10 and X5 genes have been expressed in the two hosts. This is the first report of heterologous expression of ORF10 and X5 genes in E. coli and S. lividans TK24. This work makes it possible to study the structure and potential functions of proteins encoding by these two genes.

Artificial Reconstruction of Two Cryptic Angucycline Antibiotic Biosynthetic Pathways

Genome-sequencing projects have revealed that Streptomyces bacteria have the genetic potential to produce considerably larger numbers of natural products than can be observed under standard laboratory conditions. Cryptic angucycline-type aromatic polyketide gene clusters are particularly abundant. Sequencing of two such clusters from Streptomyces sp. PGA64 and H021 revealed the presence of several open reading frames that could be involved in processing the basic angucyclic carbon skeleton. The pga gene cluster contains one putative FAD-dependant monooxygenase (pgaE) and a putatively bifunctional monooxygenase/short chain alcohol reductase (pgaM), whereas the cab cluster contains two similar monooxygenases (cabE and cabM) and an independent reductase (cabV). In this study we have reconstructed the biosynthetic pathways for aglycone synthesis by cloning and sequentially expressing the angucycline tailoring genes with genes required for the synthesis of the unmodified angucycline metabolite-UWM6-in Streptomyces lividans TK24. The expression studies unequivocally showed that, after the production of UWM6, the pathways proceed through the action of the similar monooxygenases PgaE and CabE, followed by reactions catalysed by PgaM and CabMV. Analysis of the metabolites produced revealed that addition of pgaE and cabE genes directs both pathways to a known shunt product, rabelomycin, whereas expression of all genes from a given pathway results in the production of the novel angucycline metabolites gaudimycin A and B. However, one of the end products is most probably further modified by endogenous S. lividans TK24 enzymes. These experiments demonstrate that genes that are either inactive or cryptic in their native host can be used as biosynthetic tools to generate new compounds.

Streptomyces coelicolor oxidase (SCO2837p): A new free radical metalloenzyme secreted by Streptomyces coelicolor A3(2)

The SCO2837 open-reading frame is located within the conserved central core region of the Streptomyces coelicolor A3(2) genome, which contains genes required for essential cellular functions. SCO2837 protein (SCO2837p) expressed by Pichia pastoris is a copper metalloenzyme, catalyzing the oxidation of simple alcohols to aldehydes and reduction of dioxygen to hydrogen peroxide. Distinct optical absorption spectra are observed for oxidized and one-electron reduced holoenzyme, and a free radical EPR signal is present in the oxidized apoprotein, characteristic of the Tyr-Cys redox cofactor previously reported for fungal secretory radical copper oxidases, galactose oxidase and glyoxal oxidase, with which it shares weak sequence similarity. SCO2837p was detected in the growth medium of both S. coelicolor and a recombinant expression host ( Streptomyces lividans TK64) by Western blotting, with the expression level dependent on the nature of the carbon source. This represents the first characterized example of a prokaryotic radical copper oxidase.

IncP plasmids are most effective in mediating conjugation between Escherichia coli and streptomycetes

Mobilizable shuttle plasmids containing the origin of transfer (oriT) region of plasmid F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPalpha) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Streptomyces. The conjugative system of the IncPalpha plasmids was shown to be most effective in conjugative transfer, giving peak values of (2.7 +/- 0.2) x 10(-2) S. lividans TK24 exconjugants per recipient cell. To assess whether the mating-pair formation system or the DNA-processing apparatus of the IncPalpha plasmids is crucial in conjugative transfer, an assay with an IncQ-based mobilizable plasmid (RSF1010) specifying its own DNA-processing system was developed. Only the IncPalpha plasmid mobilized the construct to S. lividans indicating that the mating-pair formation system is primarly responsible for the promiscuous transfer of the plasmids between E. coli and Streptomyces. Dynamic of conjugative transfer from E. coli to S. lividans was investigated and exconjugants starting from the first hour of mating were obtained.

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.

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 phiC31 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.

Oxidative activities of heterologously expressed CYP107B1 and CYP105D1 in whole-cell biotransformation using Streptomyces lividans TK24

Cytochrome P450 enzymes are a major class of biocatalysts related to the oxidative metabolism of many drugs, assisted by electron transfer partners. The functional expression of the P450 gene in a heterologous host will lead to efficient biotransformation and biodegradation, which are useful in pharmaceutical improvement or environmental cleanup. The soluble cytochrome P450 monooxygenase systems CYP105D1 and CYP107B1 involved in the biotransformation of some xenobiotics, such as secondary metabolites or environmental pollutants, were expressed in Streptomyces lividans TK24 with the Streptomyces expression vector pIJ6021. In whole-cell biotransformation assay using these recombinant strains, the oxidative dealkylation of 7-ethoxycoumarin was detected without any foreign redox partners in the case of CYP107B1, while the activity of CYP105D1 was not monitored until this gene was coexpressed with the ferredoxin gene located downstream of the CYP105D1 gene, and the ferredoxin reductase gene SCF 15.02 from Streptomyces coelicolor A3(II). This result suggests that CYP107B1 is capable of utilizing an endogenous electron transfer partner from the host but not CYP105D1, and that CYP105D1 is complemented by some redox partner imported from closely related strains.

Inactivation of the 20S proteasome in Streptomyces lividans and its influence on the production of heterologous proteins

Proteasomes are self-compartmentalizing proteases first discovered in eukaryotes but also occurring in archaea and in bacteria belonging to the order Actinomycetales. In bacteria, proteasomes have so far no known function. In order to evaluate the influence of the 20S proteasome on the production of heterologous proteins by Streptomyces lividans TK24, the production of a number of heterologous proteins, including soluble human tumour necrosis factor receptor II (shuTNFRII) and salmon calcitonin (sCT), was compared with the wild-type TK24, a proteasome-deficient mutant designated PRO41 and a strain complemented for the disrupted proteasome genes (strain PRO41R). S. lividans cells lacking intact proteasome genes are phenotypically indistinguishable from the wild-type or the complemented strain containing functional proteasomes. Using the expression and secretion signals of the subtilisin inhibitor of Streptomyces venezuelae CBS762.70 (Vsi) for shuTNFRII and those of tyrosinase of Streptomyces antibioticus (MelC1) for the production of sCT, both proteins were secreted in significantly higher amounts in the strain PRO41 than in the wild-type S. lividans TK24 or the complemented strain PRO41R. However, the secretion of other heterologous proteins such as shuTNFRI was not enhanced in the proteasome-deficient strain. This suggests that S. lividans TK24 can degrade some heterologous proteins in a proteasome-dependent fashion. The proteasome-deficient strain may therefore be useful for the efficient production of these heterologous proteins.

Cloning and sequencing of an exoglucanase gene from Streptomyces sp. M23, and its expression in Streptomyces lividans TK-24

A gene encoding exoglucanase (CBHII) of Streptomyces sp. M 23 was cloned and sequenced. The cbhII gene consisted of 1359 bp capable of encoding a polypeptide of 453 amino acids with a calculated molecular mass of 45,175 Da. The deduced amino acid sequence showed homology with those of cellulases belonging to family 6 of the glycosyl hydrolases. The cbhII gene was subcloned into the plasmid pSEV1 and expressed in Streptomyces lividans TK-24. The transformed cells were able to secrete the enzyme efficiently in an active form. The CBHII expressed by S. lividans TK-24 was purified to homogeneity by SDS-polyacrylamide gel and characterized. The recombinant CBHII was stable up to 50 degrees C and more than 30% of the original activity remained after heating at 100 degrees C for 10 min. The amino-terminal amino acid sequence of the recombinant CBHII was identified as GPAAPTARVD. These results agreed well with the properties of the authentic CBHII.

High-level expression of a novel amine-synthesizing enzyme, N-substituted formamide deformylase, in Streptomyces with a strong protein expression system

N-substituted formamide deformylase (NfdA) from Arthrobacter pascens F164 is a novel deformylase involved in the metabolism of isonitriles. The enzyme catalyzes the deformylation of an N-substituted formamide, which is produced from the corresponding isonitrile, to yield the corresponding amine and formate. The nfdA gene from A. pascens F164 was cloned into different types of expression vectors for Escherichia coli and Streptomyces strains. Expression in E. coli resulted in the accumulation of an insoluble protein. However, Streptomyces strains transformed with a P nitA -NitR system, which we very recently developed as a regulatory gene expression system for streptomycetes, allowed the heterologous overproduction of NfdA in an active form. When Streptomyces lividans TK24 transformed with pSH19- nfdA was cultured under the optimum conditions, the NfdA activity of the cell-free extract amounted to 8.5 U/mg, which was 29-fold higher than that of A. pascens F164. The enzyme also comprised ≈20% of the total extractable cellular protein. The recombinant enzyme was purified to homogeneity and characterized. The expression system established here will allow structural analysis and mutagenesis studies of NfdA.

Identification and Characterization of Genes fromStreptomycessp. Strain K30 Responsible for Clear Zone Formation on Natural Rubber Latex and Poly(cis-1,4-isoprene) Rubber Degradation

Streptomyces sp. strain K30 was isolated from soil next to a city high way in Münster (Germany) according to its ability to degrade natural and synthetic poly(cis-1,4-isoprene) rubber and to form clear zones on natural rubber latex agar plates. The clear zone forming phenotype was used to clone the responsible gene by phenotypic complementation of a clear zone negative mutant. An open reading frame (lcp) of 1,191 bp was identified, which was preceded by a putative signal sequence and restored the capability to form clear zones on natural rubber latex in the mutant. The putative translation product exhibited strong homologies (50% aa identity) to a putative secreted protein from Streptomyces coelicolor strain A3(2), another clear zone forming strain. Heterologous expression of lcp of Streptomyces sp. strain K30 in Streptomyces lividans strain TK23 enabled the latter to form clear zones on latex-overlay agar plates and to accumulate a degradation product of about 12 kDa containing aldehyde groups. Two ORFs putatively encoding a heterodimeric molybdenum hydroxylase (oxiAB) were identified downstream of lcp in Streptomyces sp. strain K30 strain which exerted a positive effect on clear zone formation and enabled the strain to oxidize the resulting aldehydes. Heterologous expression of a fragment harboring lcp plus oxiAB in S. lividans TK23 resulted in accumulation of aldehydes only in the presence of 10 mM tungstate. Determination of protein content during cultivation on poly(cis-1,4-isoprene) revealed an increase of the cellular protein, and gel permeation chromatography analysis indicated a shift of the molecular weight distribution of the rubber to lower values in the transgenic S. lividans strains and in the wild type, thus confirming utilization and degradation of rubber. Therefore, for the first time, genes responsible for clear zone formation on natural rubber latex and synthetic cis-1,4-polyisoprene degradation in Gram-positive bacteria were identified and characterized.

Neocarzinostatin naphthoate synthase: an unique iterative type I PKS from neocarzinostatin producer Streptomyces carzinostaticus

Enediyne antibiotics are known for their potent antitumor activities. One such enediyne, neocarzinostatin (NCS), consists of a 1:1 complex of non-peptide chromophore ( 1a), and peptide apoprotein. The structurally diverse non-peptide chromophore is responsible for its biological activity. One of its structural components, the naphthoic acid moiety (2,7-dihydroxy-5-methyl-1-naphthoic acid, 1d) is synthesized by a polyketide synthase (PKS) pathway through condensing six intact acetate units. The 5.45 kb iterative type I PKS, neocarzinostatin naphthoate synthase (NNS), responsible for naphthoic acid moiety biosynthesis, shares sequence homology with 6-methyl salicylic acid synthase of fungi and orsellinic acid synthases (AviM and CalO5) of Streptomyces origin. Cultures of S. lividans TK24 and S. coelicolor YU105 containing plasmids with NNS were able to produce 2-hydroxy-5-methyl-1-naphthoic acid ( 2a), a key intermediate of naphthoic acid moiety in NCS. In addition to 2a, a novel product, 2-hydroxy-5-hydroxymethyl-1-naphthoic acid ( 2d) was isolated. This is the first report of a bacterial iterative type I PKS from an enediyne producer which enables the biosynthesis of bicyclic aromatic compounds.

BlaB, a protein involved in the regulation of Streptomyces cacaoi beta-lactamases, is a penicillin-binding protein.

Streptomyces cacaoi beta-lactamase genes are controlled by two regulators named blaA and blaB. Whereas BlaA has been identified as a LysR-type activator, the function of BlaB is still unknown. Its primary structure is similar to that of the serine penicillin-recognizing enzymes (PREs). Indeed, the SXXK and KTG motifs are perfectly conserved in BlaB, whereas the common SXN element found in PREs is replaced by a SDG motif. Site-directed mutations were introduced in these motifs and they all disturb beta-lactamase regulation. A water-soluble form of BlaB was also overexpressed in the Streptomyces lividans TK24 cytoplasm and purified. To elucidate the activity of BlaB, several compounds recognized by PREs were tested. BlaB could be acylated by some of them, and it can therefore be considered as a penicillin-binding protein. BlaB is devoid of beta-lactamase, D-aminopeptidase, DD-carboxypeptidase or thiolesterase activity.

Resistance genes of aminocoumarin producers: two type II topoisomerase genes confer resistance against coumermycin A1 and clorobiocin.

The aminocoumarin resistance genes of the biosynthetic gene clusters of novobiocin, coumermycin A(1), and clorobiocin were investigated. All three clusters contained a gyrB(R) resistance gene, coding for a gyrase B subunit. Unexpectedly, the clorobiocin and the coumermycin A(1) clusters were found to contain an additional, similar gene, named parY(R). Its predicted gene product showed sequence similarity with the B subunit of type II topoisomerases. Expression of gyrB(R) and likewise of parY(R) in Streptomyces lividans TK24 resulted in resistance against novobiocin and coumermycin A(1), suggesting that both gene products are able to function as aminocoumarin-resistant B subunits of gyrase. Southern hybridization experiments showed that the genome of all three antibiotic producers and of Streptomyces coelicolor contained two additional genes which hybridized with either gyrB(R) or parY(R) and which may code for aminocoumarin-sensitive GyrB and ParY proteins. Two putative transporter genes, novA and couR5, were found in the novobiocin and the coumermycin A(1) cluster, respectively. Expression of these genes in S. lividans TK24 resulted in moderate levels of resistance against novobiocin and coumermycin A(1), suggesting that these genes may be involved in antibiotic transport.

Studies on a second and third ring cyclization in anthracycline biosynthesis.

This paper focuses on study of second and third ring cyclization in anthracycline biosynthesis by a heterologous gene expression. Firstly, anthracycline non-producing Streptomyces peucetius mutant, D2 was heterologously complemented to produce daunomycins with plasmids pSgs44 and pSYE66, which contain putative cyclase genes of S. galilaeus and S. nogalater, respectively. A point mutation in the cyclase gene dpsY of D2 has changed glycine to serine resulting inactivation of the enzyme. Secondly, the putative cyclase gene snoaM from S. nogalater, was expressed in a gene cassette in S. lividans TK24 and S. coelicolor CH999 to study the influence of the cyclase gene on auramycinone production and the impact of endogenous genes on production profiles. The results obtained confirms that a cyclase closing the second and third ring of a polyketide is essential in anthracycline biosynthesis.

Molecular characterization and analysis of the biosynthetic gene cluster for the azoxy antibiotic valanimycin.

Streptomyces viridifaciens MG456-hF10 produces the antibiotic valanimycin, a naturally occurring azoxy compound. Valanimycin is known to be derived from valine and serine with the intermediacy of isobutylamine and isobutylhydroxylamine, but little is known about the stages in the pathway leading to the formation of the azoxy group. In previous studies, a cosmid containing S. viridifaciens DNA was isolated that conferred valanimycin production upon Strepto-myces lividans TK24. Subcloning of DNA from the valanimycin-producing cosmid has led to the identi-fication of a 22 kb segment of DNA sufficient to allow valanimycin production in S. lividans TK24. Sequencing of this DNA segment and the surrounding DNA revealed the presence of 20 genes. Gene disruption experiments defined the boundaries of the valanimycin gene cluster, which appears to contain 14 genes. The cluster includes an amino acid decar-boxylase gene (vlmD), a valanimycin resistance gene (vlmF ), at least two regulatory genes (vlmE, vlmI ), two genes encoding a flavin monooxygenase (vlmH, vlmR), a seryl tRNA synthetase gene (vlmL ) and seven genes of unknown function. Overproduction and characterization of VlmD demonstrated that it catalyses the decarboxylation of l-valine. An unusual feature of the valanimycin gene cluster is that four genes involved in branched amino acid biosynthesis are located near its 5' end.