<Emphasis Type="Italic">fabC</Emphasis> of <Emphasis Type="Italic">Streptomyces lydicus</Emphasis> involvement in the biosynthesis of streptolydigin

Streptolydigin, a secondary metabolite produced by Streptomyces lydicus, is a potent inhibitor of bacterial RNA polymerases. It has been suggested that streptolydigin biosynthesis is associated with polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS). Thus, there is great interest in understanding the role of fatty acid biosynthesis in the biosynthesis of streptolydigin. In this paper, we cloned a type II fatty acid synthase (FAS II) gene cluster of fabDHCF from the genome of S. lydicus and constructed the SlyfabCF-disrupted mutant. Sequence analysis showed that SlyfabDHCF is 3.7 kb in length and encodes four separated proteins with conserved motifs and active residues, as shown in the FAS II of other bacteria. The SlyfabCF disruption inhibited streptolydigin biosynthesis and retarded mycelial growth, which were likely caused by the inhibition of fatty acid synthesis. Streptolydigin was not detected in the culture of the mutant strain by liquid chromatography–mass spectrometry. Meanwhile, the streptolol moiety of streptolydigin accumulated in cultures. As encoded by fabCF, acyl carrier protein (ACP) and β-ketoacyl-ACP synthase II are required for streptolydigin biosynthesis and likely involved in the step between PKS and NRPS. Our results provide the first genetic and metabolic evidence that SlyfabCF is shared by fatty acid synthesis and antibiotic streptolydigin synthesis.     

Identification of three Zwittermicin A biosynthesis-related genes from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">kurstaki</Emphasis> strain YBT-1520

Zwittermicin A (ZwA) is a novel, broad-spectrum linear aminopolyol antibiotic produced by some Bacillus cereus and Bacillus thuringiensis. However, only part of its biosynthesis cluster has been identified and characterized from B. cereus UW85. To better understand the biosynthesis cluster of ZwA, a bacterial artificial chromosome (BAC) library of B. thuringiensis subsp. kurstaki strain YBT-1520, a ZwA-producing strain, was constructed. Two BAC clones, 1F8 and 5E2, were obtained by PCR, which overlap the known ZwA biosynthesis cluster of B. cereus UW85. This ZwA biosynthesis cluster is at least 38.6 kb and is located on the chromosome, instead of the plasmid. Partial DNA sequencing revealed both BAC clones carry three new ZwA biosynthesis-related genes, zwa6, zwa5A and zwa5B, which were found at the corresponding location of B. cereus UW85. Putative amino acid sequences of these genes shown that ZWA6 is homologous to a typical carbamoyltransferase from Streptomyces avermitilis, while ZWA5A and ZWA5B are homologs of cysteine synthetase and ornithine cyclodeaminase which jointly synthesize 2,3-diaminopropionate in the viomycin biosynthesis pathway, respectively. The identification of these three genes further supports the hypothesized ZwA biosynthesis pathway.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Functional conservation of the human <Emphasis Type="Italic">EXT1</Emphasis> tumor suppressor gene and its <Emphasis Type="Italic">Drosophila</Emphasis> homolog <Emphasis Type="Italic">tout</Emphasis><Emphasis Type="Italic">velu</Emphasis>

Heparan sulfate proteoglycans play a vital role in signaling of various growth factors in both Drosophila and vertebrates. In Drosophila, mutations in the tout velu (ttv) gene, a homolog of the mammalian EXT1 tumor suppressor gene, leads to abrogation of glycosaminoglycan (GAG) biosynthesis. This impairs distribution and signaling activities of various morphogens such as Hedgehog (Hh), Wingless (Wg), and Decapentaplegic (Dpp). Mutations in members of the exostosin (EXT) gene family lead to hereditary multiple exostosis in humans leading to bone outgrowths and tumors. In this study, we provide genetic and biochemical evidence that the human EXT1 (hEXT1) gene is conserved through species and can functionally complement the ttv mutation in Drosophila. The hEXT1 gene was able to rescue a ttv null mutant to adulthood and restore GAG biosynthesis.     

Improving heterologous polyketide production in <Emphasis Type="Italic">Escherichia coli</Emphasis> by overexpression of an <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase gene

An S-adenosylmethionine synthetase gene (metK) from Streptomyces spectabilis was cloned into an expression plasmid under the control of an inducible T7 promoter and introduced into a strain of Escherichia coli (BAP1(pBP130/pBP144)) capable of producing the polyketide product 6-deoxyerythronolide B (6-dEB). The metK coexpression in BAP1(pBP130/pBP144) improved the specific production of 6-dEB from 10.86 to 20.08 mg l⁻¹ . In an effort to probe the reason for this improvement, a series of gene deletion and expression experiments were conducted based on a metK metabolic pathway that branches between propionyl-CoA (a 6-dEB precursor) and autoinducer compounds. The deletion and expression studies suggested that the autoinducer pathway had a larger impact on improved 6-dEB biosynthesis. Supporting these results were experiments demonstrating the positive effect conditioned media (the suspected location of the autoinducer compounds) had on 6-dEB production. Taken together, the results of this study show an increase in heterologous 6-dEB production concomitant with heterologous metK gene expression and suggest that the mechanism for this improvement is linked to native autoinducer compounds.     

Double deletion of <Emphasis Type="Italic">dtsR1</Emphasis> and <Emphasis Type="Italic">pyc</Emphasis> induce efficient <Emphasis Type="SmallCaps">l</Emphasis>-glutamate overproduction in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis. The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC 13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis.     

<Emphasis Type="SmallCaps">L</Emphasis>-Tyrosine production by deregulated strains of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The excretion of the aromatic amino acid l-tyrosine was achieved by manipulating three gene targets in the wild-type Escherichia coli K12: The feedback-inhibition-resistant (fbr) derivatives of aroG and tyrA were expressed on a low-copy-number vector, and the TyrR-mediated regulation of the aromatic amino acid biosynthesis was eliminated by deleting the tyrR gene. The generation of this l-tyrosine producer, strain T1, was based only on the deregulation of the aromatic amino acid biosynthesis pathway, but no structural genes in the genome were affected. A second tyrosine over-producing strain, E. coli T2, was generated considering the possible limitation of precursor substrates. To enhance the availability of the two precursor substrates phosphoenolpyruvate and erythrose-4-phosphate, the ppsA and the tktA genes were over-expressed in the strain T1 background, increasing l-tyrosine production by 80% in 50-ml batch cultures. Fed-batch fermentations revealed that l-tyrosine production was tightly correlated with cell growth, exhibiting the maximum productivity at the end of the exponential growth phase. The final l-tyrosine concentrations were 3.8 g/l for E. coli T1 and 9.7 g/l for E. coli T2 with a yield of l-tyrosine per glucose of 0.037 g/g (T1) and 0.102 g/g (T2), respectively.     

Role of the <Emphasis Type="Italic">snorA</Emphasis> gene in nogalamycin biosynthesis by strain <Emphasis Type="Italic">Streptomyces nogalater</Emphasis> Lv65

Streptomyces nogalater Lv65 (= IMET 43360) is a producer of the anthracycline antitumor antibiotic nogalamycin. In this work, some aspects of the regulation of nogalamycin production by this strain were studied. Insertional inactivation of the snorA gene in the chromosome of the nogalamycin producer was carried out; as a result, strain S. nogalater A1 was obtained. This is the first successful gene knockout in S. nogalater. It was demonstrated that strain A1 is characterized by the absence of synthesis of nogalamycin and its precursors, as well as by the inability to form spores. As a result of the knockout complementation with an entire copy of the snorA gene, resumption of the nogalamycin synthesis by strain S. nogalater A1 was observed; in the case of the wild-type strain S. nogalater Lv65, insertion resulted in an increase in the antibiotic synthesis. Obtained results indicate that the snorA gene is involved in positive regulation of nogalamycin biosynthesis.     

Features of biosynthesis of chitinolytic enzymes by <Emphasis Type="Italic">Streptomyces griseus</Emphasis> var. <Emphasis Type="Italic">streptomycini</Emphasis>

The previously selected strain Streptomyces griseus var. streptomycini is able to hydrolyze colloid as well as crystal forms of chitin. During the submerged cultivation in the medium with crystal chitin, the chitinase activity achieved the maximal value after 46–50 h of culturing. Use of colloid chitin as an inductor allowed increasing the chitinolytic activity by 33%. Adding of mannose to the medium increased the chitinase activity of the producer by two times. It has been shown that the chitinase biosynthesis bears an inducible nature.     

Identification of the <Emphasis Type="Italic">bkdAB</Emphasis> gene cluster,a plausible source of the starter-unit for virginiamycin M production in <Emphasis Type="Italic">Streptomyces virginiae</Emphasis>

The bkdAB gene cluster, which encodes plausible E1 and E2 components of the branched-chain α-keto acid dehydrogenase (BCDH) complex, was isolated from Streptomyces virginiae in the vicinity of a regulatory island for virginiamycin production. Gene disruption of bkdA completely abolished the production of virginiamycin M (a polyketide-peptide antibiotic), while the production of virginiamycin S (a cyclodepsipeptide antibiotic) was unaffected. Complementation of the bkdA disruptant by genome-integration of intact bkdA completely restored the virginiamycin M production, indicating that the bkdAB cluster is essential for virginiamycin M biosynthesis, plausibly via the provision of isobutyryl-CoA as a primer unit. In contrast to a feature usually seen in the Streptomyces E1 component, namely, the separate encoding of the α and β subunits, S. virginiae bkdA seemed to encode the fused form of the α and β subunits, which was verified by the actual catalytic activity of the fused protein in vitro using recombinant BkdA overexpressed in Escherichia coli. Supply of an additional bkdA gene under the strong and constitutive promoter ermE* in the wild-type strain of S. virginiae resulted in enhanced production of virginiamycin M, suggesting that the supply of isobutyryl-CoA is one of the rate-limiting factors in the biosynthesis of virginiamycin M.     

<Emphasis Type="Italic">pspA</Emphasis> overexpression in <Emphasis Type="Italic">Streptomyces lividans</Emphasis> improves both Sec- and Tat-dependent protein secretion

Streptomyces is an interesting host for the secretory production of recombinant proteins because of its innate capacity to secrete proteins at high level in the culture medium. In this report, we evaluated the importance of the phage-shock protein A (PspA) homologue on the protein secretion yield in Streptomyces lividans. The PspA protein is supposed to play a role in the maintenance of the proton motive force (PMF). As the PMF is an energy source for both Sec- and Tat-dependent secretion, we evaluated the influence of the PspA protein on both pathways by modulating the pspA expression. Results indicated that pspA overexpression can improve the Tat-dependent protein secretion as illustrated for the Tat-dependent xylanase C and enhanced green fluorescent protein (EGFP). The effect on Sec-dependent secretion was less pronounced and appeared to be protein dependent as evidenced by the increase in subtilisin inhibitor (Sti-1) secretion but the lack of increase in human tumour necrosis factor (hTNFα) secretion in a pspA-overexpressing strain.     

Overexpression of yeast <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase <Emphasis Type="Italic">metK</Emphasis> in <Emphasis Type="Italic">Streptomyces actuosus</Emphasis> leads to increased production of nosiheptide

S-Adenosylmethionine (SAM) is synthesized via the metabolic reaction involving adenosine triphosphate and l-methionine that is catalyzed by the enzyme S-adenosyl-l-methionine synthetase (SAM-s) and encoded by the gene metK. In the present study, metK with the absence of introns from Saccharomyces cerevisiae was introduced into Streptomyces actuosus, a nosiheptide (Nsh) producer. Intracellular SAM levels were determined by high-pressure liquid chromatography. Through optimizing the nutrient content of the medium, it was shown that increased SAM production induced by the overexpression of SAM-s leads to an increase in the intracellular cysteine pool and overproduction of Nsh in S. actuosus. This investigation shows that increased SAM promotes the elevated production of the non-ribosomal thiopeptide Nsh in Streptomyces sp.     

L-asparaginase production by <Emphasis Type="Italic">Streptomyces albidoflavus</Emphasis>

Attempts were made to optimize the cultural conditions for the production of L-asparaginase by Streptomyces albidoflavus under submerged fermentations. Enhanced level of L-asparaginase was found in culture medium supplemented with maltose as carbon source. Yeast extract (2%) was served as good nitrogen source for the production of L-asparaginase. The optimum pH for enzyme production was 7.5 and temperature was 35°C. The release of L-asparaginase from the cells of S. albidoflavus was high when strain was treated with cell disrupting agents like EDTA and lysozyme. The enzyme produced by the strain was purifi ed by ammonium sulfate, Sephadex G-100 and CM-Sephadex C-50 gel fi ltration and the molecular weight was apparently determined as 112 kDa.     

Proton-dependent transporter gene <Emphasis Type="Italic">lndJ</Emphasis> confers resistance to landomycin E in <Emphasis Type="Italic">Streptomyces globisporus</Emphasis> 1912

Sequence analysis of 2 kb BamHI-SmaI fragment of landomycin E (LaE) gene cluster in Streptomyces globisporus 1912 revealed one complete ORF marked as lndJ. Analysis of putative lndJ aminoacid sequence allowed us to suppose that it is proton-dependent antiporter which could be involved in resistance to LaE in the producing strain. Although disruption of lndJ had no significant influence on LaE production and resistance, its overexpression in wild type and LaE overproducing strains led to qualitative changes in landomycins biosynthesis and increased resistance to LaE. These data support the hypothesis about involvement of lndJ gene in landomycins export. The text was submitted by the authors in English.     

<Emphasis Type="Italic">Streptomyces lividans</Emphasis> and <Emphasis Type="Italic">Brevibacterium lactofermentum</Emphasis> as heterologous hosts for the production of X<Subscript>22</Subscript> xylanase from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>

The Aspergillus nidulans gene xlnA coding for the fungal xylanase X₂₂ has been cloned and expressed in two heterologous bacterial hosts: Streptomyces lividans and Brevibacterium lactofermentum. Streptomyces strains yielded 10 units/ml of xylanase when the protein was produced with its own signal peptide, and 19 units/ml when its signal peptide was replaced by the one for xylanase Xys1 from Streptomyces halstedii. B. lactofermentum was also able to produce xylanase X₂₂, affording 6 units/ml upon using either the Aspergillus xlnA signal peptide or Streptomyces xysA. These production values are higher than those previously reported for the heterologous expression of the A. nidulans xlnA gene in Saccharomyces cerevisiae (1 unit/ml). Moreover, the X₂₂ enzyme produced by Streptomyces lividans showed oenological properties, indicating that this Streptomyces recombinant strain is a good candidate for the production of this enzyme at the industrial scale.     

Identification of a methyltransferase encoded by gene <Emphasis Type="Italic">stel16</Emphasis> and its function in Ebosin biosynthesis of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. 139

Streptomyces sp. 139 generates a novel exopolysaccharide (EPS) designated as Ebosin, which exerts an antagonistic effect on IL-1R in vitro and anti-rheumatic arthritis activity in vivo. A ste gene cluster for Ebosin biosynthesis consisting of 27 ORFs was previously identified in our laboratory. In this paper, ste16 was expressed in Escherichia coli BL21 and the recombinant protein was purified, which has the ability to catalyze the transfer of the methyl group from S-adenosylmethionine (AdoMet) to dTDP-4-keto-6-deoxy-D-glucos, which was thus identified as a methyltransferase. In order to determine the function of ste16 in Ebosin biosynthesis, the gene was disrupted with a double crossover via homologous recombination. The monosaccharide composition of EPS-m generated by the mutant strain Streptomyces sp. 139 (ste16) was found to differ from that of Ebosin. The IL-1R antagonist activity of EPS-m was markedly lower than that of Ebosin. These experimental results have shown that the ste16 gene codes for a methyltransferase which is involved in Ebosin biosynthesis. These authors contributed equally to this work.     

Mutational analysis of the <Emphasis Type="Italic">gum</Emphasis> gene cluster required for xanthan biosynthesis in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv <Emphasis Type="Italic">oryzae</Emphasis>

Genome sequence analysis of Xanthomonas oryzae pv. oryzae has revealed a cluster of 12 ORFs that are closely related to the gum gene cluster of Xanthomonas campestris pv. campestris. The gum gene cluster of X. oryzae encodes proteins involved in xanthan production; however, there is little experimental evidence supporting this. In this study, biochemical analyses of xanthan produced by a defined set of X. oryzae gum mutant strains allowed us to preliminarily assign functions to most of the gum gene products: biosynthesis of the pentasaccharide repeating unit for GumD, GumM, GumH, GumK, and GumI, xanthan polymerization and transport for GumB, GumC, GumE, and GumJ, and modification of the pentasaccharide repeating unit for GumF, GumG, and GumL. In addition, we found that the exopolysaccharides are essential but not specific for the virulence of X. oryzae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sang-Yoon Kim and Jeong-Gu Kim contributed equally to this work.     

Knockout of the gene (<Emphasis Type="Italic">ste15</Emphasis>) encoding a glycosyltransferase and its function in biosynthesis of exopolysaccharide in <Emphasis Type="Italic">Streptomyces</Emphasis> sp. 139

Streptomyces sp. 139 produces a novel exopolysaccharide (EPS) designated Ebosin which has antagonistic activity for IL-1R in vitro and remarkable anti-rheumatic arthritis activity in vivo. We previously identified a ste (Streptomyces eps) gene cluster consisting of 27 ORFs responsible for Ebosin biosynthesis. The gene product of ste15 shows high homology to known glycosyltransferases (GTFs). To elucidate its function in Ebosin biosynthesis, the ste15 gene was knocked out with a double crossover via homologous recombination. Our analysis of monosaccharide composition for EPS-m produced by the mutant strain Streptomyces sp. 139 (ste15 ⁻) showed that glucose was significantly diminished compared to its natural counterpart Ebosin. This derivative of Ebosin lost the antagonistic activity for IL-1R in vitro and its molecular mass was smaller than Ebosin. These results have demonstrated that the ste15 gene codes for a GTF for glucose, which is functionally involved in Ebosin biosynthesis.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>)

Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l⁻¹ phosphinothricin (PPT) and 150 mg l⁻¹ kanamycin, respectively, for shoot induction and 1 mg l⁻¹ PPT and 125 mg l⁻¹ kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T₀ and T₁ plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T₀ and T₁ transgenic plants.