Role of sgcR3 in positive regulation of enediyne antibiotic C-1027 production of Streptomyces globisporus C-1027

Background  

C-1027, produced by Streptomyces globisporus C-1027, is one of the most potent antitumoral agents. The biosynthetic gene cluster of C-1027, previously cloned and sequenced, contains at least three putative regulatory genes, i.e. sgcR1, sgcR2 and sgcR3. The predicted gene products of these genes share sequence similarities to StrR, regulators of AraC/XylS family and TylR. The purpose of this study was to investigate the role of sgcR3 in C-1027 biosynthesis.     

Cloning, expression and characterization of gene sgcD involved in the biosynthesis of novel antitumor lidamycin

Lidamycin with high antitumor activity is a novel enediyne antitumor antibiotic produced by Streptomyces globisporus C1027. The 75 kb biosynthesis gene cluster of lidamycin containing 33 open reading frames has been cloned from S. globisporus C1027. In this paper, the function of sgcD (ORF24) is investigated. Gene disruption experiment proved that sgcD is involved in lidamycin biosynthesis. With homologous comparing analysis, we deduce that sgcD codes aminomutase catalyzing a-tyrosine to β-tyrosine which is one motif for lidamycin. To identify the function of enzyme coded by sgcD, sgcD is cloned into vector pET30a for inducing expression and the activity of expression product is analyzed. The result showed that the expression product of sgcD has the activity of aminomutase. Aminomutase coded by sgcD is the first characterized enzyme involved in the biosynthesis of enediyne antitumor antibiotics. Our research will be helpful to clarifying the biosynthesis mechanism of such kind of antibiotic and to producing new antitumor compounds.     

Engineered production of cancer targeting peptide (CTP)-containing C-1027 in Streptomyces globisporus and biological evaluation

Conjugation of cancer targeting peptides (CTPs) with small molecular therapeutics has emerged as a promising strategy to deliver potent (but typically nonspecific) cytotoxic agents selectively to cancer cells. Here we report the engineered production of a CTP (NGR)-containing C-1027 and evaluation of its activity against selected cancer cell lines. C-1027 is an enediyne chromoprotein produced by Streptomyces globisporus, consisting of an apo-protein (CagA) and an enediyne chromophore (C-1027). NGR is a CTP that targets CD13 in tumor vasculature. S. globisporus SB1026, a recombinant strain engineered to encode CagA with the NGR sequence fused at its C-terminus, directly produces the NGR-containing C-1027 that is equally active as the native C-1027. Our results demonstrate the feasibility to produce CTP-containing enediyne chromoproteins by metabolic pathway engineering and microbial fermentation and will inspire efforts to engineer other CTP-containing drug binding proteins for targeted delivery.     

Cloning and Nucleotide Sequencing of the Antitumor Antibiotic C-1027 Apoprotein Gene

The apoprotein gene for a chromoprotein antitumor antibiotic, C-1027, was cloned from the producer strain, Streptomyces globisporus C-1027, and sequenced. The process verified that; (1) the sequence included the entire structural gene directing a precursor of the apoprotein (pre-apoprotein having Met¹—Ala³³ leader peptide ahead of the apoprotein) and flanking regions, (2) the amino acid sequence of the apoprotein deduced from the base sequence perfectly matched the one based on protein analysis, ¹⁾ (3) 3rd letters of the codons were 88% G or C, while the 1st plus the 2nd letters were 63% G or C, (4) the structural gene had 57% homology with that of macromomycin apoprotein (mernA) while the flanking regions had little homology with the corresponding ones of mernA, except some homology at the – 10th and – 35th promoter regions, and (5) the gene was transcribed as a monocistronic mRNA in an early growth phase, independent of chromophore production.     

A Lactococcus lactis expression vector set with multiple affinity tags to facilitate isolation and direct labeling of heterologous secreted proteins

The gram-positive bacterium Lactococcus lactis is a useful host for extracellular protein production. A main advantage of L. lactis over other bacterial expression systems is that lactococcal cells display low levels of autolysis and proteolysis. Previously, we developed a set of vectors for nisin-inducible extracellular production of N- or C-terminally hexa-histidine (His₆)-tagged proteins. The present study was aimed at expanding our portfolio of L. lactis expression vectors for protein purification and site-specific labeling. Specifically, we present two new groups of vectors allowing N- or C-terminal provision of proteins with a Strep-tag II or AVI-tag. Vectors for AVI-tagging encode an additional His₆-tag for protein purification. Another set of vectors allows removal of N-terminal Strep- or His₆-tags from expressed proteins with the tobacco etch virus protease. Two possible applications of the developed vectors are presented. First, we show that Strep-tagged LytM of Staphylococcus aureus in the growth medium of L. lactis can be directly bound to microtiter plates coated with an affinity reagent and used for enzyme-linked immunosorbent assays. Second, we show that the AVI-tagged Sle1 protein from S. aureus produced in L. lactis can be directly biotinylated and fluorescently labeled. The fluorescently labeled Sle1 was successfully applied for S. aureus re-binding studies, allowing subcellular localization by fluorescence microscopy. In conclusion, we have developed a set of expression vectors that enhances the versatility of L. lactis as a system for production of proteins with tags that can be used for affinity purification and site-specific protein labeling.

     

Protein expression vector and secretion signal peptide optimization to drive the production, secretion, and functional expression of the bacteriocin enterocin A in lactic acid bacteria

Replacement of the leader sequence (LS) of the bacteriocin enterocin A (LS_entA) by the signal peptides (SP) of the protein Usp45 (SP_usp45), and the bacteriocins enterocin P (SP_entP), and hiracin JM79 (SP_hirJM79) permits the production, secretion, and functional expression of EntA by different lactic acid bacteria (LAB). Chimeric genes encoding the SP_usp45, the SP_entP, and the SP_hirJM79 fused to mature EntA plus the EntA immunity genes (entA + entiA) were cloned into the expression vectors pNZ8048 and pMSP3545, under control of the inducible P_nisA promoter, and in pMG36c, under control of the constitutive P₃₂ promoter. The amount, antimicrobial activity, and specific antimicrobial activity of the EntA produced by the recombinant Lactococcus lactis, Enterococcus faecium, E. faecalis, Lactobacillus sakei and Pediococcus acidilactici hosts varied depending on the signal peptide, the expression vector, and the host strain. However, the antimicrobial activity and the specific antimicrobial activity of the EntA produced by most of the LAB transformants was lower than expected from their production. The supernatants of the recombinant L. lactis NZ9000 (pNZUAI) and L. lactis NZ9000 (pNZHAI), overproducers of EntA, showed a 1.2- to 5.1-fold higher antimicrobial activity than that of the natural producer E. faecium T136 against different Listeria spp.     

Evaluation of Streptomyces spp. for biocontrol of gummy stem blight (Didymella bryoniae) and growth promotion of Cucumis melo L.

Gummy stem blight of Cucumis melo L. (melon) caused by Didymella bryoniae is a serious disease in the major production area of northwest China. Two Streptomyces isolates (Streptomyces pactum A12 and S. globisporus subsp. globisporus C28) previously isolated from the Qinghai-Tibet Plateau were investigated regarding their biocontrol of gummy stem blight and growth promotion of melon under controlled conditions. Streptomyces A12 and C28 indicated obvious antagonistic activity against D. bryoniae in vitro. Both A12 and C28 significantly decreased disease severity and AUDPC (area under the disease progress curve) of melon gummy stem blight in vivo (P < 0.05). Ten-fold dilution of C28 culture filtrate was more effective in controlling the disease compared with other treatments, the disease reduction effects were 41.0–64.2%. The mean fresh weights were increased by 40.4% for plants, 44.2% for roots, and 40.3% for aerial parts, when A12 was applied in both nursery soil and transplanted soil. Streptomyces C28 also increased the mean fresh weights of melon plants by 18.4–49.0% compared with the control in pot trial. Streptomyces A12 and C28 showed substantial colonization abilities in the rhizosphere and on the rhizoplane of melon plants. Results demonstrated that Streptomyces A12 and C28 were of positive effect on the biocontrol of gummy stem blight and growth promotion of Cucumis melo L.     

Dual roles of CagA protein in Helicobacterpylori-induced chronic gastritis in mice

Cytotoxin-associated gene A (CagA) acts directly on gastric epithelial cells. However, the roles of CagA in host adaptive immunity against Helicobacter pylori (H. pylori) infection are not fully understood. In this study, to investigate the roles of CagA in the development of H. pylori-induced chronic gastritis, we used an adoptive-transfer model in which spleen cells from C57BL/6 mice with or without H. pylori infection were transferred into RAG2^−/− mice, with gastric colonization of either CagA⁺H. pylori or CagA⁻H. pylori. Colonization of CagA⁺H. pylori but not CagA⁻H. pylori in the host gastric mucosa induced severe chronic gastritis in RAG2^−/− mice transferred with spleen cells from H. pylori-uninfected mice. In addition, when CagA⁺H. pylori-primed spleen cells were transferred into RAG2^−/− mice, CD4⁺ T cell infiltration in the host gastric mucosa were observed only in RAG2^−/− mice infected with CagA⁺H. pylori but not CagA⁻H. pylori, suggesting that colonization of CagA⁺H. pylori in the host gastric mucosa is essential for the migration of H. pylori-primed CD4⁺ T cells. On the other hand, transfer of CagA⁻H. pylori-primed spleen cells into CagA⁺H. pylori-infected RAG2^−/− mice induced more severe chronic gastritis with less Foxp3⁺ regulatory T-cell infiltration as compared to transfer of CagA⁺H. pylori-primed spleen cells. In conclusion, CagA in the stomach plays an important role in the migration of H. pylori-primed CD4⁺ T cells in the gastric mucosa, whereas CagA-dependent T-cell priming induces regulatory T-cell differentiation, suggesting dual roles for CagA in the pathophysiology of H. pylori-induced chronic gastritis.     

Minimal polyketide pathway expression in an actinorhodin cluster-deleted and regulation-stimulated <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

Along with traditional random mutagenesis-driven strain improvement, cloning and heterologous expression of Streptomyces secondary metabolite gene clusters have become an attractive complementary approach to increase its production titer, of which regulation is typically under tight control via complex multiple regulatory networks present in a metabolite low-producing wild-type strain. In this study, we generated a polyketide non-producing strain by deleting the entire actinorhodin cluster from the chromosome of a previously generated S. coelicolor mutant strain, which was shown to stimulate actinorhodin biosynthesis through deletion of two antibiotic downregulators as well as a polyketide precursor flux downregulator (Kim et al. in Appl Environ Microbiol 77:1872–1877, 2011). Using this engineered S. coelicolor mutant strain as a surrogate host, a model minimal polyketide pathway for aloesaponarin II, an actinorhodin shunt product, was cloned in a high-copy conjugative plasmid, followed by functional pathway expression and quantitative metabolite analysis. Aloesaponarin II production was detected only in the presence of a pathway-specific regulatory gene, actII-ORF4, and its production level was the highest in the actinorhodin cluster-deleted and downregulator-deleted mutant strain, implying that this engineered polyketide pathway-free and regulation-optimized S. coelicolor mutant strain could be used as a general surrogate host for efficient expression of indigenous or foreign polyketide pathways derived from diverse actinomycetes in nature.     

Extracellular enzyme production and fungal mycelia degradation of antagonistic Streptomyces induced by fungal mycelia preparation of cucurbit plant pathogens

Streptomyces are beneficial soil microorganisms and potential candidates for biocontrol agents against soilborne pathogenic fungi of cucurbit plants. Extracellular enzymes such as cellulase, chitinase and glucanase produced by Streptomyces are important components of actinomycete-fungus antagonism. This study aimed to investigate the influence on extracellular enzymes production and fungal mycelia degradation by antagonistic Streptomyces of mycelia preparation of pathogenic fungi (MPPF) of cucurbit plants. The results showed that the antagonistic Streptomyces displayed high extracellular enzyme activities to varying degrees when MPPF was used as the sole carbon source. The MPPF from Fusarium proliferatum, Fusarium oxysporum f. sp. niveum and Alternaria tenuissima were the most effective carbon sources in enhancing the cellulase activity of Streptomyces globisporus C7, Streptomyces globisporus subsp. globisporus C28 and Streptomyces kanamyceticus C49, respectively. S. globisporus subsp. globisporus C28, Streptomyces pactum A12 and S. kanamyceticus C49 cultured in the medium containing MPPF from Fusarium equiseti showed the highest chitinase activity (12.35, 12.50 and 15.06 U, respectively) of all the MPPF treatments. Glucanase activity of Streptomyces carnosus A11 was enhanced greatly (9.26 U) when MPPF from A. tenuissima was used as the sole carbon source. A hyphal intertwining and degradation phenomenon was observed when the antagonistic Streptomyces came across the pathogenic fungal mycelia, which was due to a synergistic effect of the extracellular enzymes produced by the antagonistic Streptomyces.     

Heterologous expression of the novel α-helical hybrid peptide PR-FO in Bacillus subtilis

PR-FO is a novel α-helical hybrid antimicrobial peptide (AMP) with strong antimicrobial activities and high stability, and the potential to develop into a new generation of antimicrobial agents. In this study, the encoded gene sequence of SMT3-PR-FO was designed and transformed into B. subtilis WB800N. Fusion proteins with concentrations of 16 mg L⁻¹ (SP_amyQ) and 23 mg L⁻¹ (SP_sacB) were obtained after purification by a Ni–NTA resin column. A total of 3 mg (SP_amyQ) and 4 mg (SP_sacB) of PR-FO with a purity of 90% was obtained from 1 L fermentation cultures. Recombinant PR-FO exhibited high inhibition activities against both gram-negative bacteria and gram-positive bacteria, and low haemolytic activity against human red blood cells. These results indicated that the rSMT3-PR-FO could be expressed under the guidance of SP_amyQ and SP_sacB, and the maltose-induced expression strategy might be a safe and efficient method for the soluble peptides production in B. subtilis.

     

A ubiquitin carboxyl extension protein secreted from a plant‐parasitic nematode Globodera rostochiensis is cleaved in planta to promote plant parasitism

Nematode effector proteins originating from esophageal gland cells play central roles in suppressing plant defenses and in formation of the plant feeding cells that are required for growth and development of cyst nematodes. A gene (GrUBCEP12) encoding a unique ubiquitin carboxyl extension protein (UBCEP) that consists of a signal peptide for secretion, a mono‐ubiquitin domain, and a 12 amino acid carboxyl extension protein (CEP12) domain was cloned from the potato cyst nematode Globodera rostochiensis. This GrUBCEP12 gene was expressed exclusively within the nematode's dorsal esophageal gland cell, and was up‐regulated in the parasitic second‐stage juvenile, correlating with the time when feeding cell formation is initiated. We showed that specific GrUBCEP12 knockdown via RNA interference reduced nematode parasitic success, and that over‐expression of the secreted Gr^ΔSPUBCEP12 protein in potato resulted in increased nematode susceptibility, providing direct evidence that this secreted effector is involved in plant parasitism. Using transient expression assays in Nicotiana benthamiana, we found that Gr^ΔSPUBCEP12 is processed into free ubiquitin and a CEP12 peptide (GrCEP12) in planta, and that GrCEP12 suppresses resistance gene‐mediated cell death. A target search showed that expression of RPN2a, a gene encoding a subunit of the 26S proteasome, was dramatically suppressed in Gr^ΔSPUBCEP12 but not GrCEP12 over‐expression plants when compared with control plants. Together, these results suggest that, when delivered into host plant cells, Gr^ΔSPUBCEP12 becomes two functional units, one acting to suppress plant immunity and the other potentially affecting the host 26S proteasome, to promote feeding cell formation.     

Development of dual-inducible duet-expression vectors for tunable gene expression control and CRISPR interference-based gene repression in Pseudomonas putida KT2440

The development of P. putida as an industrial host requires a sophisticated molecular toolbox for strain improvement, including vectors for gene expression and repression. To augment existing expression plasmids for metabolic engineering, we developed a series of dual-inducible duet-expression vectors for P. putida KT2440. A number of inducible promoters (P_lac, P_tac, P_tetR/tetA and P_bad) were used in different combinations to differentially regulate the expression of individual genes. Protein expression was evaluated by measuring the fluorescence of reporter proteins (GFP and RFP). Our experiments demonstrated the use of compatible plasmids, a useful approach to coexpress multiple genes in P. putida KT2440. These duet vectors were modified to generate a fully inducible CRISPR interference system using two catalytically inactive Cas9 variants from S. pasteurianus (dCas9) and S. pyogenes (spdCas9). The utility of developed CRISPRi system(s) was demonstrated by repressing the expression of nine conditionally essential genes, resulting in growth impairment and prolonged lag phase for P. putida KT2440 growth on glucose. Furthermore, the system was shown to be tightly regulated, tunable and to provide a simple way to identify essential genes with an observable phenotype.     

STP2201, a Chromosomal Promoter Sequence of Streptococcus thermophilus

Analysis of the structural and functional properties of chromosomal DNA fragments of Streptococcus thermophilus ST128 delineated the promoter sequence ST_P2201 and identified its −35, −10 and Shine-Dalgarno regions. ST_P2201 was used in cloning vectors derived from small resident plasmids pER8 (2094 bp) and pER371 (2672 bp) of S. thermophilus strains to facilitate expression of a Streptomyces sp. marker gene (cholesterol oxidase) in lactic acid bacteria. Cell extracts of ST128 transformants converted up to 75% of cholesterol into 4-cholesten-3-one during 8 h of incubation. Received: 8 February 1997 / Accepted: 20 March 1997     

Improvement of human interferon alpha secretion by <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

To improve the secretion and expression of human interferon alpha 2b (IFN) in Lactococcus lactis, a synthetic pro-peptide, LEISSTCDA (LEISS), was fused to the N-terminus of IFN. This gave a higher secretion efficiency (12% vs. 5%) and yield (~2.8-fold) of IFN. The signal peptide, SP_SlpA (SlpA, an S-layer protein of Lactobacillus brevis), was also tested to secrete IFN instead of SP_Usp45 (Usp45, the main secreted protein in L. lactis). This gave increased IFN secretion (~3-fold) but lower total production. All the recombinant IFN had appropriate bioactivities in an antiviral assay.     

Biologically Active Endophytic Streptomycetes from <Emphasis Type="Italic">Nothofagus</Emphasis> spp. and Other Plants in Patagonia

Endophytic streptomycetes have been isolated and characterized from several species of Nothofagus and other plants growing in the southern reaches of Patagonia. No endophytic streptomycete was obtained from any plant species studied in Northern Patagonia. However, from Southern Patagonia, biologically active Streptomyces spp. from several plant species were isolated. Each isolate, as studied by scanning electron microscopy (SEM), has small hyphae, some produce typical barrel-shaped spores in culture and each has some unique hyphal surface structures. Interestingly, although none has any detectable antibacterial killing properties, each has demonstrable killing activity against one or more pathogenic fungi including representative plant pathogenic organisms such as Phytophthora erythroseptica, Pythium ultimum, Sclerotinia sclerotiorum, Mycosphaerella fijiensis, and Rhizoctonia solani. The 16S rDNA sequences of the isolates were distinct from all other genetic accessions of Streptomyces in GenBank. However, isolate C-2 from Chiliotrichum diffusum (Compositae) is identical, in all respects, to isolate C-4 obtained from Misodendrum punctulatum (Loranthaceae). These results confirm that endophytic streptomycetes represent a novel source of biologically active microorganisms.     

Improving heterologous protein expression in transfected Drosophila S2 cells as assessed by EGFP expression

Drosophila melanogaster S2 cells were co-transfected with plasmid vectors containing the enhanced green fluorescent protein gene (EGFP), under the control of metallothionein promoter (pMt), and the hygromycin selection gene, in view of establishing parameters for optimized gene expression. A protocol of transfection was worked out, leading after hygromycin selection, to ∼90% of S2MtEGFP fluorescent cells at day 5 after copper sulfate (CuSO₄) induction. As analyzed by confocal microscopy, S2MtEGFP cell cultures were shown to be quite heterogeneous regarding the intensity and cell localization of fluorescence among the EGFP expressing cells. Spectrofluorimetry kinetic studies of CuSO₄ induced S2MtEGFP cells showed the EGFP expression at 510 nm as soon as 5 h after induction, the fluorescence increasing progressively from this time to attain values of 4.6 × 10⁵ counts/s after 72 h of induction. Induction with 700 μM of CuSO₄ performed at the exponential phase of the S2MtEGFP culture (10⁶ cells/mL) led to a better performance in terms of cell growth, percent of fluorescent cells and culture intensity of fluorescence. Sodium butyrate (NaBu) treatment of CuSO₄ induced S2MtEGFP cell cultures, although leading to a loss of cell culture viability, increased the percent of EGFP expressing cells and sharply enhanced the cell culture fluorescence intensity. The present study established parameters for improving heterologous protein expression in stably transfected Drosophila S2 cells, as assessed by the EGFP expression.