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.

     

Application of factorial design to the optimization of medium composition in batch cultures of Streptomyces lividans TK21 producing a hybrid antibiotic

Summary The composition of the liquid medium employed to obtain a hybrid antibiotic in batch cultures of a recombinant strain of Streptomyces lividans TK21 has been studied. Starch and glutamic acid are the most appropriate carbon and nitrogen sources to support respectively cell growth and antibiotic production. A central composite experimental design has been employed to derive a statistical model of the effect of phosphate and glutamic acid on growth and antibiotic production, and an initial concentration of 10 mM phosphate and 52.8 mM glutamic acid have been found optimal to maximize the final antibiotic concentration in batch cultures.     

Cloning and analysis of DNA sequences from Streptomyces hygroscopicus encoding geldanamycin biosynthesis

A gene library constructed from large (20 kb) fragments of total DNA from the geldananmycin-producing strain Streptomyces hygroscopicus 3602 cloned in the plasmid vector pIJ61 were used to transform S. lividans TK24. Three transformants of about 800 tested were found to have acquired the ability to produce an antibiotic lethal to a geldanamycin-sensitive strain of Bacillus subtilis. The plasmids isolated from these transformants, pIA101, pIA102 and pIA103, each contained an insert of 15 kb. A 4.5 kb DNA fragment from the insert in pIA102 hybridised to DNA from S. hygroscopicus 3602 and to DNA encoding part of the erythromycin polyketide synthase but not to S. lividans TK24 DNA. The integration-defective phage vector C31 KC515 containing this 4.5 kb fragment was able to lysogenise S. hygroscopicus 3602 to produce lysogens defective in geldanamycin production. Loss of the prophage restored the ability to produce geldanamycin. Extracts of fermentation broth cultures of S. lividans containing pIA101, pIA102 and pIA102 and pIA103 analysed by thin-layer chromatography (TLC) contained compounds identical or very similar to purified geldanamycin, which were not present in S. lividans. These compounds showed a mass spectrum indistinguishable from geldanamycin. The evidence suggests that the clones contain DNA sequences encoding functions required for geldanamycin biosynthesis including components of the polyketide synthase.     

Improvement of A21978C production in Streptomyces roseosporus by reporter-guided rpsL mutation selection

Aims:  Daptomycin, one of the A21978C factors produced by Streptomyces roseosporus, is an acidic cyclic lipopeptide antibiotic with potent activity against a variety of Gram‐positive pathogens. To increase the titre of this extensively used and clinically important antibiotic, we applied a reported‐guided rpsL mutation selection system to generate strains producing high levels of A21978C. Methods and Results:  In the reporter design, dptE was chosen as the overexpressing target, and neo‐encoding neomycin phosphotransferase as the reporter. Using this reporter‐guided selection system, 20% of the selected, streptomycin‐resistant mutants produced greater amounts of A21978C than the starting strain. The selection system increased the screening efficiency about 10‐fold with a frequency of 1·7% A21978C overproducing strains among str^r mutants. A21978C production was increased approximately 2·2‐fold in the rpsL K43N mutant. Conclusions:  The combination of ribosome engineering and reporter‐guided mutant selection generated an A21978C overproducing strain that produced about twice as much A21978C as the parental strain. Significance and Impact of the Study:  The strategies presented here, which integrated the advantages of both ribosome engineering and reporter‐guided mutation selection, could be applied to other bacteria to improve their yield of secondary metabolites.     

Proteomics-driven identification of putative AfsR2-target proteins stimulating antibiotic biosynthesis inStreptomyces lividans

AfsR2, originally identified fromStreptomyces lividans, is a global regulatory protein which stimulates antibiotic biosynthesis. Through its stable chromosomal integration, the high level of gene expression ofafsR2 significantly induced antibiotic production as well as the sporulation ofS. lividans, implying the presence of yet-uncharacterized AfsR2-target proteins. To identify and evaluate the putative AfsR2-target proteins involved in antibiotic regulation, the proteomics-driven approach was applied to the wild-typeS. lividans and theafsR2-integrated actinorhodin overproducing strain. The 2D gel-electrophoresis gave approximately 340 protein spots showing different protein expression patterns between these twoS. lividans strains. Further MALDI-TOF analysis revealed several AfsR2-target proteins, including glyceraldehyde-3-phosphate dehydrogenase, putative phosphate transport system regulator, guanosine pentaphosphate synthetase/polyribonucleotide nucleotidyltransferase, and superoxide dismutase, which suggests that the AfsR2 should be a pleiotropic regulatory protein which controls differential expressions of various kinds of genes inStreptomyces species.     

Construction of a Streptomyces sp.–Escherichia coli conjugative shuttle vector and its application for recombinant biosynthesis of poly(3-hydroxyalkanoic acid)

pGTR760 and pGTR761, two new shuttle vectors, withmultiple cloning sites and capable of conjugal transfer from E. coli to Streptomyces sp. were constructed. The poly-3-hydroxybutyrate (PHB) biosynthetic polycistron from Ralstonia eutropha was cloned into the pGTR760 vector to derive the pCAB_Re plasmid. The pCAB_Re plasmid was conjugally transferred from E. coli S17-1 to Streptomyces lividans TK64. Fluorescence microscopy of the recombinant and the untransformed S. lividans TK64 revealed presence of polyhydroxyalkanoates (PHAs) in both cell types. GC/GC-MS analysis revealed the accumulated polymer to be polyhydroxyoctanoate (PHO). While the untransformed S. lividans cells accumulate 3.5% PHO of cell dry wt, the recombinant cells accumulate 8% PHO of the cell dry wt. The transformation of S. lividans, however, resulted in slower growth rate, delayed sporulation and impaired pigment formation. Scanning electron microscope analysis revealed broken mycelia probably due to release of accumulated PHO granules from the cells.     

Regulatory and biosynthetic effects of the <Emphasis Type="Italic">bkd</Emphasis> gene clusters on the production of daptomycin and its analogs A21978C<Subscript>1–3</Subscript>

Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus in an acidic peptide complex A21978C. In this complex, A21978C_1–3 is most abundant and contains branched-chain fatty acyl groups, while daptomycin has a straight decanoic acyl group. The branched-chain α-keto acid dehydrogenase complex (BCDH complex), encoded by bkd gene clusters in Streptomyces, is responsible for the early step of converting branched-chain amino acids into branched-chain fatty acids. In a daptomycin industrial producer S. roseosporus L30, two alleles of bkd gene clusters, bkdA1B1C1/bkdA2B2C2, and a regulatory gene bkdR located upstream of bkdA2B2C2 are identified. We show that BkdR positively regulated bkdA2B2C2 expression and was negatively auto-regulated, but is not directly involved in regulation of daptomycin gene cluster expression. However, BkdR is required for both daptomycin and A21978C_1–3 production. Furthermore, deletion of bkdA2B2C2 only led to partial reduction of A21978C_1–3 production, while the ΔbkdA1B1C1 mutant shows very weak production of A21978C_1–3, and the double bkd mutant has a similar production profile as the single ΔbkdA1B1C1 mutant, suggesting that bkdA1B1C1 gene cluster plays a dominant role in branched-chain fatty acid biosynthesis. So we reveal a unique regulatory function of BkdR and genetic engineered a bkd null strain for daptomycin production with reduced impurities.     

An efficient blue-white screening system for markerless deletions and stable integrations in Streptomyces chromosomes based on the blue pigment indigoidine biosynthetic gene bpsA

We previously developed an efficient deletion system for streptomycetes based on the positive selection of double-crossover events using bpsA, a gene for producing the blue pigment indigoidine. Using this system, we removed interfering secondary metabolite clusters from Streptomyces lividans TK24, resulting in RedStrep strains with dramatically increased heterologous production of mithramycin A (up to 3-g/l culture). This system, however, required a time-consuming step to remove the resistance marker genes. In order to simplify markerless deletions, we prepared a new system based on the plasmid pAMR18A. This plasmid contains a large polylinker with many unique restriction sites flanked by apramycin and kanamycin resistance genes and the bpsA gene for selecting a double-crossover event. The utility of this new markerless deletion system was demonstrated by its deletion of a 21-kb actinorhodin gene cluster from Streptomyces lividans TK24 with 30% efficiency. We used this system to efficiently remove the matA and matB genes in selected RedStrep strains, resulting in biotechnologically improved strains with a highly dispersed growth phenotype involving non-pelleting small and open mycelia. No further increase in mithramycin A production was observed in these new RedStrep strains, however. We also used this system for the markerless insertion of a heterologous mCherry gene, an improved variant of the monomeric red fluorescent protein, under the control of the strong secretory signal sequence of the subtilisin inhibitor protein, into the chromosome of S. lividans TK24. The resulting recombinant strains efficiently secreted mCherry into the growth medium in a yield of 30 mg/l.

     

Heterologous Expression of Novobiocin and Clorobiocin Biosynthetic Gene Clusters

A method was developed for the heterologous expression of biosynthetic gene clusters in different Streptomyces strains and for the modification of these clusters by single or multiple gene replacements or gene deletions with unprecedented speed and versatility. λ-Red-mediated homologous recombination was used for genetic modification of the gene clusters, and the attachment site and integrase of phage C31 were employed for the integration of these clusters into the heterologous hosts. This method was used to express the gene clusters of the aminocoumarin antibiotics novobiocin and clorobiocin in the well-studied strains Streptomyces coelicolor and Streptomyces lividans, which, in contrast to the natural producers, can be easily genetically manipulated. S. coelicolor M512 derivatives produced the respective antibiotic in yields comparable to those of natural producer strains, whereas S. lividans TK24 derivatives were at least five times less productive. This method could also be used to carry out functional investigations. Shortening of the cosmids' inserts showed which genes are essential for antibiotic production.     

Heterologous production of paromamine in <Emphasis Type="Italic">Streptomyces lividans</Emphasis> TK24 using kanamycin biosynthetic genes from Streptomyces kanamyceticus ATCC12853

The 2-deoxystreptamine and paromamine are two key intermediates in kanamycin biosynthesis. In the present study, pSK-2 and pSK-7 recombinant plasmids were constructed with two combinations of genes: kanABK and kanABKF and kacA respectively from kanamycin producer Streptomyces kanamyceticus ATCC12853. These plasmids were heterologously expressed into Streptomyces lividans TK24 independently and generated two recombinant strains named S. lividans Sk-2/SL and S. lividans SK-7/SL, respectively. ESI/ MS and ESI-LC/MS analysis of the metabolite from S. lividans SK-2/SL showed that the compound had a molecular mass of 163 [M + H]⁺, which corresponds to that of 2-deoxystreptamine. ESI/MS and MS/MS analysis of metabolites from S. lividans SK-7/SL demonstrated the production of paromamine with a molecular mass of 324 [M + H]⁺. In this study, we report the production of paromamine in a heterologous host for the first time. This study will evoke to explore complete biosynthetic pathways of kanamycin and related aminoglycoside antibiotics.     

Production of cephamycin C by <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis> NT4 using solid-state fermentation

Cephamycin C is an extracellular broad spectrum β-lactam antibiotic produced by Streptomyces clavuligerus, S. cattleya and Nocardia lactamdurans. In the present study, different substrates for solid-state fermentation were screened for maximum cephamycin C production by S. clavuligerus NT4. The fermentation parameters such as substrate concentration, moisture content, potassium dihydrogen phosphate, inoculum size and ammonium oxalate were optimized by response surface methodology (RSM). The optimized conditions yielded 21.68 ± 0.76 mg gds⁻¹ of cephamycin C as compared to 10.50 ± 1.04 mg gds⁻¹ before optimization. Effect of various amino acids on cephamycin C production was further studied by using RSM, which resulted in increased yield of 27.41 ± 0.65 mg gds⁻¹.     

Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans

Summary A broad-spectrum mercury resistance locus (mer) from a spontaneous chloramphenicol-sensitive (Cm^s), arginine auxotrophic (Arg^–) mutant of Streptomyces lividan 1326 was isolated on a 6 kb DNA fragment by shotgun cloning into the mercury-sensitive derivative S. lividans TK64 using the vector pIJ702. The mer genes form part of a very large amplifiable DNA sequence present in S. lividans 1326. This element was amplified to about 20 copies per chromosome in the Cm^s Arg^– mutant and was missing from strains like S. lividans TK64, cured for the plasmid SLP3. DNA sequence analysis of a 5 kb region encompassing the whole region required for broad-spectrum mercury resistance revealed six open reading frames (ORFs) transcribed in opposite directions from a common intercistronic region. The protein sequences predicted from the two ORFs transcribed in one direction showed a high degree of similarity to mercuric reductase and organomercurial lyase from other gram-negative and gram-positive sources. Few, if any, similarities were found between the predicted polypeptide sequences of the other four ORFs and other known proteins.     

Comparative Proteomic Analysis of Streptomyces lividans Wild-Type and ppk Mutant Strains Reveals the Importance of Storage Lipids for Antibiotic Biosynthesis

Streptomyces lividans TK24 is a strain that naturally produces antibiotics at low levels, but dramatic overproduction of antibiotics occurs upon interruption of the ppk gene. However, the role of the Ppk enzyme in relation to the regulation of antibiotic biosynthesis remains poorly understood. In order to gain a better understanding of the phenotype of the ppk mutant, the proteomes of the wild-type (wt) and ppk mutant strains, grown for 96 h on R2YE medium limited in phosphate, were analyzed. Intracellular proteins were separated on two-dimensional (2D) gels, spots were quantified, and those showing a 3-fold variation or more were identified by mass spectrometry. The expression of 12 proteins increased and that of 29 decreased in the ppk mutant strain. Our results suggested that storage lipid degradation rather than hexose catabolism was taking place in the mutant. In order to validate this hypothesis, the triacylglycerol contents of the wt and ppk mutant strains of S. lividans as well as that of Streptomyces coelicolor M145, a strain that produces antibiotics at high levels and is closely related to S. lividans, were assessed using electron microscopy and thin-layer chromatography. These studies highlighted the large difference in triacylglycerol contents of the three strains and confirmed the hypothetical link between storage lipid metabolism and antibiotic biosynthesis in Streptomyces.     

Overexpression and biochemical characterization of DagA from <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A3(2): an endo-type β-agarase producing neoagarotetraose and neoagarohexaose

The DagA product of Streptomyces coelicolor is an agarase with a primary translation product (35 kDa) of 309 amino acids, including a 30-amino acid signal peptide. Although dagA expression in Streptomyces lividans under the control of its own set of promoters was previously reported, its enzymatic properties have never been elucidated. To develop an improved expression system for dagA, three types of strong promoters for the Streptomyces host were linked to dagA, and their efficiencies in DagA production were compared in S. lividans TK24. All of the transformants with dagA grew at improved rates and produced larger amounts of DagA in the modified R2YE medium containing 0.5% agar as the sole carbon source. Of the three transformants, the S. lividans TK24/pUWL201-DagA (ermE promoter) produced the highest agarase activity (A ₅₄₀ = 4.24), and even the S. lividans TK24/pHSEV1-DagA (tipA promoter) and S. lividans TK24/pWHM3-DagA (sprT promoter) produced higher agarase activity (A ₅₄₀ = 0.24 and 0.12, respectively) than the control (A ₅₄₀ = 0.01) in the modified R2YE medium. The mature form of DagA protein (32 kDa) was successfully purified by one-step affinity column chromatography by using agarose beads with excellent yield. The purified DagA was found to exhibit maximal agarase activity at 40°C and pH 7.0. The K _m, V _max, and K _cat values for agarose were 2.18 mg/ml (approximately 1.82 × 10⁻⁵ M), 39.06 U/mg of protein, and 9.5 × 10³/s, respectively. Thin layer chromatography (TLC) analysis, matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry, and Fourier transform nuclear magnetic resonance (FT-NMR) spectrometry of the hydrolyzed products of agarose by DagA revealed that DagA is an endo-type β-agarase that degrades agarose into neoagarotetraose and neoagarohexaose.     

Structural Elucidation of Alterporriol B,a Novel Metabolic Pigment Produced by Alternaria porri (Ellis) Ciferri

Restriction-reduced mutants were isolated from Streptomyces rosa subsp. notoensis KA301 and S. tanashiensis strain Kala which produce the benzoisochromanequinone antibiotics nanaomycin and kalafungin, respectively. The mutants of S. rosa, which can be transformed with a multi-copy plasmid and in which the actinophage Pa16 can propagate, were selected. They were transformed with a single-copy plasmid propagated in S. lividans TK24, and with its modified plasmid propagated in the mutant at higher efficiency. The mutants of S. tanashiensis were selected by their capability to be transformed with a multi-copy plasmid. The efficiency of transformation with a single-copy plasmid propagated in S. lividans TK24 was low, but was much increased by heating the protoplasts at 42°C for 15 min prior to the transformation. These mutants derived from both strains probably lack at least one of their restriction systems.     

The bialaphos biosynthetic genes ofStreptomyces hygroscopicus: Molecular cloning and characterization of the gene cluster

Summary We have isolated and studied the organization ofStreptomyces hygroscopicus genes responsible for the biosynthesis of the antibiotic herbicide bialaphos. Bialaphos production genes were cloned from genomic DNA using a plasmid vector (pIJ702). Three plasmids were isolated which restored productivity toS. hygroscopicus mutants blocked at different steps of the biosynthetic pathway. Subcloning experiments using other nonproducing mutants showed that four additional bialaphos production genes were also contained on these plasmids. A gene conferring resistance to bialaphos, which was independently cloned using the plasmid vector pIJ61, and an antibiotic-sensitive host (S. lividans), was also linked to the production genes. Cosmids were isolated which defined the location of these genes in a 16 kb cluster.     

Iso-migrastatin titer improvement in the engineered <Emphasis Type="Italic">Streptomyces lividans</Emphasis> SB11002 strain by optimization of fermentation conditions

The heterologous production of iso-migrastatin (iso-MGS) was successfully demonstrated in an engineered S. lividans SB11002 strain, which was derived from S. lividans K4-114, following introduction of pBS11001, which harbored the entire mgs biosynthetic gene cluster. However, under similar fermentation conditions, the iso-MGS titer in the engineered strain was significantly lower than that in the native producer — Streptomyces platensis NRRL 18993. To circumvent the problem of low iso-MGS titers and to expand the utility of this heterologous system for iso-MGS biosynthesis and engineering, systematic optimization of the fermentation medium was carried out. The effects of major components in the cultivation medium, including carbon, organic and inorganic nitrogen sources, were investigated using a single factor optimization method. As a result, sucrose and yeast extract were determined to be the best carbon and organic nitrogen sources, resulting in optimized iso-MGS production. Conversely, all other inorganic nitrogen sources evaluated produced various levels of inhibition of iso-MGS production. The final optimized R2YE production medium produced iso-MGS with a titer of 86.5 mg/L, about 3.6-fold higher than that in the original R2YE medium, and 1.5 fold higher than that found within the native S. platensis NRRL 18993 producer.     

High yield fermentation and purification of Tendamistat disulphide analogues secreted by Streptomyces lividans

In our studies of structure-function correlation of polypeptides we used Tendamistat (TM), an -amylase-inhibitor of Streptomyces tendae, as a model to investigate the influence of different mutants on the expression and secretion of the protein. In addition, we examined the influence of replacing the two disulphide-bridges that stabilize the two-loop structure of the whole protein. The single mutants C27S, C27T, C45A, the double mutants C11A/C27A, C11A/C27S, C11A/C27T, C11A/C27L, C45/C73A and the fourfold mutant C11A/C27A/C45A/C73A were prepared. The mutated TM gene was expressed in S. lividans TK 24, which secretes the active form of the inhibitor into the culture medium. Compared with the wild-type, the double-mutated TM derivatives show an increase in secreted protein by a factor of two to ten. In contrast, the single-mutated inhibitor analogues show the reverse effect. In order to examine the influence of temperature and culture media on the production of protein derivative we used the most unstable C11A analogue. Our expression studies at 10, 19, 28 and 37° C established 19° C as the optimal temperature for production of the protein derivatives. The correlation between the stability and secretion of TM is discussed in the context of our knowledge of protein translocation in bacteria. Based on these experiments we optimized the fermentation parameters, isolated TM analogous on a large scale, and verified them. Correspondence to: J. W. Engels