Multiple transporters are involved in natamycin efflux in Streptomyces chattanoogensis L10

Antibiotic‐producing microorganisms have evolved several self‐resistance mechanisms to prevent auto‐toxicity. Overexpression of specific transporters to improve the efflux of toxic antibiotics has been found one of the most important and intrinsic resistance strategies used by many Streptomyces strains. In this work, two ATP‐binding cassette (ABC) transporter‐encoding genes located in the natamycin biosynthetic gene cluster, scnA and scnB, were identified as the primary exporter genes for natamycin efflux in Streptomyces chattanoogensis L10. Two other transporters located outside the cluster, a major facilitator superfamily transporter Mfs1 and an ABC transporter NepI/II were found to play a complementary role in natamycin efflux. ScnA/ScnB and Mfs1 also participate in exporting the immediate precursor of natamycin, 4,5‐de‐epoxynatamycin, which is more toxic to S. chattanoogensis L10 than natamycin. As the major complementary exporter for natamycin efflux, Mfs1 is up‐regulated in response to intracellular accumulation of natamycin and 4,5‐de‐epoxynatamycin, suggesting a key role in the stress response for self‐resistance. This article discusses a novel antibiotic‐related efflux and response system in Streptomyces, as well as a self‐resistance mechanism in antibiotic‐producing strains.     

Characterization of type II thioesterases involved in natamycin biosynthesis in Streptomyces chattanoogensis L10

The known functions of type II thioesterases (TEIIs) in type I polyketide synthases (PKSs) include selecting of starter acyl units, removal of aberrant extender acyl units, releasing of final products, and dehydration of polyketide intermediates. In this study, we characterized two TEIIs (ScnI and PKSIaTEII) from Streptomyces chattanoogensis L10. Deletion of scnI in S. chattanoogensis L10 decreased the natamycin production by about 43%. Both ScnI and PKSIaTEII could remove acyl units from the acyl carrier proteins (ACPs) involved in the natamycin biosynthesis. Our results show that the TEII could play important roles in both the initiation step and the elongation steps of a polyketide biosynthesis; the intracellular TEIIs involved in different biosynthetic pathways could complement each other.     

ABC转运蛋白SgnA/B促进纳他霉素胞外转运与高产

纳他霉素是一种天然、广谱、高效的多烯大环内酯类还原性抗真菌剂,广泛应用于食品真菌污染的防治和临床真菌感染的治疗。纳他霉素胞外转运效率可能是限制褐黄孢链霉菌(Streptomyces gilvosporeus)发酵高产纳他霉素的重要因素。通过生物信息学及分子对接技术分析纳他霉素胞外转运蛋白SgnA/B,发现SgnA和SgnB两个异源二聚体组成的ABC转运蛋白是内向开口构象的转运蛋白,且2个结合位点与纳他霉素结合能力有强弱差异,更有利于纳他霉素的胞外转运。本研究以纳他霉素生产菌株——褐黄孢链霉菌F607为出发菌株,构建了sgnA/B基因超表达菌株F-EX,以分析sgn A/B基因超表达对纳他霉素合成及胞外转运的影响。研究发现,纳他霉素对数合成期的F-EX菌株不仅提高了纳他霉素胞外/胞内比,其120 h发酵总产量也提高了12.5%,达到7.38 g/L。最后,通过转录组测序发现,sgnA/B基因超表达除提高纳他霉素胞外转运效率外,还影响了与多种氨基酸、丙酸盐、糖、五碳化合物代谢和TCA循环相关基因的表达。研究表明,强化纳他霉素胞外转运有利于纳他霉素的合成,是提高褐黄孢链霉菌纳他霉素产量的有效...     

Modified nitrocefin‐EDTA method to differentially quantify the induced L1 and L2 β‐lactamases in Stenotrophomonas maltophilia

Aim: To estimate the ethylene diamine tetraacetic acid (EDTA) concentration at which the L1 enzyme activity in the cell extracts of Stenotrophomonas maltophilia can be mostly inhibited. Methods and Results: The effective inhibition concentration of EDTA against the L1 enzyme in the cell extracts was firstly evaluated by using the L2 isogenic mutant of S. maltophilia KJ, KJΔL2, as the assayed strain. Approximately 92% L1 activity was inhibited by 10 mmol l^?1 EDTA, which is 100‐fold higher than that from previously reported protocols (0·1 mmol l^?1). Three phylogenetic clusters of L1 proteins were revealed from 11 clinical S. maltophilia isolates, with a L1 protein divergence of 0–11%. The EDTA concentration required to inhibit the L1 enzymes of different phylogenetic clusters was estimated to be 10 mmol l^?1. Conclusion: The previous nitrocefin‐EDTA protocol for differentially quantifying the L1 and L2 activity in the cell extracts has been modified by raising the added EDTA concentration to 10 mmol l^?1. Significance and Impact of the Study: A rapid and accurate method for determination of L1 and L2 activity will provide a convenient tool for enzyme characterization and induction mechanism study of S. maltophilia.     

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.     

Enhancement of natamycin production on Streptomyces gilvosporeus by chromosomal integration of the Vitreoscilla hemoglobin gene (vgb)

Oxygen deficiency is a critical factor during the fermentation production of natamycin. In order to alleviate oxygen limitation and enhance the yield of natamycin, the vgb gene, encoding Vitreoscilla hemoglobin (VHb) was inserted into pSET152 with its native promoter and integrated into the chromosome of Streptomyces gilvosporeus (S. gilvosporeus). The expression of VHb was determined by Western blotting. The activity of expressed VHb was confirmed by the observation of VHb-specific CO-difference spectrum with a maximal absorption at 419 nm for the recombinant. Integration of the empty plasmid pSET152 did not affect natamycin production of S. gilvosporeus. While the vgb-harboring strain exhibited high natamycin productivity, reaching 3.31 g/L in shake flasks and 8.24 g/L in 1-L fermenters. Compared to the wild strain, expression of VHb, increased the natamycin yield of the strain bearing vgb by 131.3 % (jar fermenter scale) and 175 % (shake flask scale), respectively, under certain oxygen-limiting condition. Addition of an extra copy of the vgb gene in S. gilvosporeus-vgb2 did not enhance the natamycin production obviously. These results provided a superior natamycin-producing strain which can be directly used in industry and a useful strategy for increasing yields of other metabolites in industrial strains.     

Heterologous coexpression of Vitreoscilla hemoglobin and Bacillus megaterium glucanase in Streptomyces lydicus A02 enhanced its production of antifungal metabolites

Streptomyces lydicus A02 is a novel producer of commercially important polyene macrocyclic antibiotic natamycin and a potential biocontrol agent to several plant fungal diseases, including wilt caused by Fusarium oxysporum f. spp. To improve the natamycin production and the antifungal activity of S. lydicus A02, we coexpressed gene vgb encoding Vitreoscilla hemoglobin (VHb) and bglC encoding Bacillus megaterium L103 glucanase, both under the control of the strong constitutive ermE* promoter, in S. lydicus A02. Our results showed that coexpressing VHb and glucanase improved cell growth, and the engineered strain produced 26.90% more biomass than the wild-type strain after 72 h fermentation in YSG medium. In addition, coexpressing genes encoding VHb and glucanase led to increased natamycin production, higher endogenous chitinase activity and exogenous glucanase activity, as well as enhanced antifungal activity in the engineered S. lydicus AVG02 and AGV02, regardless of the position of the two genes on the plasmids. Compared with model strains, few reports have successfully coexpressed VHb and other foreign proteins in industrial strains. Our results illustrated an effective approach for improving antifungal activity in an industrial strain by the rational engineering of combined favorable factors.     

链霉菌CB02959中四霉素及四烯菌素的鉴定及培养基优化

【背景】四霉素(Tetramycin)和四烯菌素(Tetrin)是具有广谱抗真菌活性的四烯大环内酯类抗生素。链霉菌CB02959是一株雷纳霉素(Leinamycin)类化合物的潜在产生菌株,利用antiSMASH分析其基因组发现该菌株含有一个纳他霉素(Natamycin)类四烯大环内酯化合物的生物合成基因簇。【目的】对Streptomyces sp. CB02959中次级代谢产物进行研究,确定其是否可以产生四烯大环内酯化合物,对其发酵产物进行分离和结构鉴定,并进行初步的发酵优化以提高产量。【方法】基于生物信息学预测和高分辨质谱数据,推测CB02959中多烯化合物的结构;在不同发酵培养基中培养CB02959,确定适合大规模发酵的培养基;敲除tetrA基因以确定目标基因簇和四烯大环内酯化合物产生的相关性;分离和鉴定CB02959产生的主要代谢物的结构;通过改变培养基中葡萄糖、麦芽提取物和胰蛋白胨的含量,提高四烯大环内酯化合物的产量。【结果】通过对CB02959中纳他霉素类化合物生物合成基因簇的分析及16S rRNA基因序列的进化树分析,推测CB02959可能是一株新的四霉素和四烯菌素产生菌;在YEME发酵培养基中对CB02959进行大规模发酵,分离得到4个化合物,鉴定为四霉素A (1)、四霉素B (2)、四烯菌素A (3)、四烯菌素B (4);最后通过培养基的初步优化,将化合物1–4的产量分别提高至208.1、100.0、1 315.6、109.9 mg/L。【结论】通过基因组挖掘策略发现了一株新的四霉素和四烯菌素产生菌链霉菌CB02959,并通过培养基优化提升了其四烯大环内酯化合物的产量,此发现为这类抗真菌天然产物的后续开发奠定了基础。     

The catalase gene differentiates between some strains of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> ssp. <Emphasis Type="Italic">anaerobius</Emphasis>

Staphylococcus aureus ssp anaerobius strain S10 was isolated from an outbreak of sheep abscess disease. Sequence of the catalase gene of this strain showed 99 % identity to the catalase gene (katB) sequence of the reference strain (S. aureus ssp. anaerobius strain MVF213) with mismatching of three base pairs. An important substitution located 1036 nucleotides upstream of the initiation codon from “C” in katB to “T” in the catalase gene of strain S10 originated a stop codon. The deduced protein (345 amino acids) is 105 amino acids shorter than that of katB. Partial sequence of the catalase gene of other 8 local isolates in addition to another reference strain (DSM 20714/ATCC 35844) revealed the same mutations in all local (African) strains, whereas the sequence of the reference (European) strain was typical to that of katB. Sequence of the catalase gene of S. aureus ssp. anaerobius strain S10 was deposited in GenBank under accession no. EU281993.     

Efficient transformation and expression of the glucanase gene from Bacillus megaterium in the biocontrol strain Streptomyces lydicus A02

Streptomyces have been used extensively as the biocontrol agents due to their ability to produce various antimicrobial compounds, such as antibiotics and hydrolytic enzymes. Streptomyces lydicus strain A02, which was isolated from the soil of suburban forest field in Beijing (China), is capable of producing natamycin and has proved to be a potential biocontrol agent to several plant fungal diseases, including wilts caused by Fusarium oxysporum f. spp. However, hydrolytic enzymes like glucanase have not been detected in S. lydicus A02 on CMC-Na plates by congo red staining. Glucanase, a pathogenesis-related (PR) protein, degrades fungal cell walls and has been widely used as antifungal agent in plant protection. Therefore, a recombinant S. lydicus expressing a glucanase gene, which was cloned from the biocontrol strain Bacillus megaterium L103 and driven by the Streptomyces erythraea ermE* promoter, was constructed in this study. The engineered S. lydicus AG02 shared a similar yield of natamycin with the wild-type A02 strain. Compared to the wild-type strain A02, the engineered S. lydicus AG02 had a remarkably higher glucanase activity, as well as antifungal activity to F. oxysporum f. sp. conglutinans, F. oxysporum f. sp. niveum and Rhizoctonia cerealis. This demonstrated the improved biocontrol effect of S. lydicus AG02 attributed to transforming the exogenous glucanase from B. megaterium, which acted synergistically with natamycin to increase the antifungal activity of the strain.     

Optimal culture conditions for keratinase production by a novel thermophilic Myceliophthora thermophila strain GZUIFR‐H49‐1

Aims: To investigate the effect of medium compositions and culture conditions on keratinase production by a novel thermophilic fungus Myceliophthora thermophila (Apinis) Oorschot strain GZUIFR‐H49‐1. Methods and Results: The thermophilic strain GZUIFR‐H49‐1 with keratinolytic ability was characterized and identified as a strain of M. thermophila on the basis of its morphological characters and molecular analysis of ITS1‐5.8S‐ITS2 rDNA sequence. Among the medium compositions tested, the soluble starch (SS), urea, sodium thiosulfate and CaCl₂ were the most effective C‐source, N‐source, S‐source and mineral ion, respectively, by employing the single‐factor experiment. The urea and pH value were the significant factors (P < 0·05) for the keratinase production in this experiment condition using Plackett–Burman factorial design. The conditions of keratinase production were further optimized by Box–Behnken design. Consequently, there was a 6·4‐fold increase (5100 U l^?1) in the keratinase activity than the initial value (800 U l^?1) by this optimal process. Conclusions: This study indicated that the optimization design proved a useful and powerful tool for the development of optimal medium compositions and culture conditions. Myceliophthora thermophila strain GZUIFR‐H49‐1 was a promising fungus strain for keratinase production. Significance and Impact of the Study: This study characterized a novel thermophilic M. thermophila strain GZUIFR‐H49‐1 with potential applications for keratinase production. These conditions of keratinase production obtained by means of optimization design will be accumulated as potential information for exploration and utilization to the new fungus isolate.     

The microalga Parachlorella kessleri––A novel highly efficient lipid producer

The alga Parachlorella kessleri, strain CCALA 255, grown under optimal conditions, is characterized by storage of energy in the form of starch rather than lipids. If grown in the complete medium, the cultures grew rapidly, producing large amounts of biomass in a relatively short time. The cells, however, contained negligible lipid reserves (1–10% of DW). Treatments inducing hyperproduction of storage lipids in P. kessleri biomass were described. The cultures were grown in the absence or fivefold decreased concentration of either nitrogen or phosphorus or sulfur. Limitation by all elements using fivefold or 10‐fold diluted mineral medium was also tested. Limitation with any macroelement (nitrogen, sulfur, or phosphorus) led to an increase in the amount of lipids; nitrogen limitation was the most effective. Diluted nutrient media (5‐ or 10‐fold) were identified as the best method to stimulate lipid overproduction (60% of DW). The strategy for lipid overproduction consists of the fast growth of P. kessleri culture grown in the complete medium to produce sufficient biomass (DW more than 10 g/L) followed by the dilution of nutrient medium to stop growth and cell division by limitation of all elements, leading to induction of lipid production and accumulation up to 60% DW. Cultivation conditions necessary for maximizing lipid content in P. kessleri biomass generated in a scale‐up solar open thin‐layer photobioreactor were described. Biotechnol. Bioeng. 2013; 110: 97–107. © 2012 Wiley Periodicals, Inc.     

Characterization of <Emphasis Type="Italic">Microbulbifer</Emphasis> Strain CMC-5, a New Biochemical Variant of <Emphasis Type="Italic">Microbulbifer elongatus</Emphasis> Type Strain DSM6810<Superscript>T</Superscript> Isolated from Decomposing Seaweeds

A Gram-negative, rod-shaped, non-spore forming, non-motile and moderate halophilic bacteria designated as strain CMC-5 was isolated from decomposing seaweeds by enrichment culture. The growth of strain CMC-5 was assessed in synthetic seawater-based medium containing polysaccharide. The bacterium degraded and utilized agar, alginate, carrageenan, xylan, carboxymethyl cellulose and chitin. The strain was characterized using a polyphasic approach for taxonomic identification. Cellular fatty acid analysis showed the presence of iso-C_15:0 as major fatty acid and significant amounts of iso-C_17:1ω9c and C_18:1ω7c . Phylogenetic analysis based on 16S rDNA sequence indicated that strain CMC-5 is phylogenetically related to Microbulbifer genus and 99% similar to type strain Microbulbifer elongatus DSM6810^T. However in contrast to Microbulbifer elongatus DSM6810^T, strain CMC-5 is non-motile, utilizes glucose, galactose, inositol and xylan, does not utilize fructose and succinate nor does it produce H₂S. Further growth of bacterial strain CMC-5 was observed when inoculated in seawater-based medium containing sterile pieces of Gracilaria corticata thalli. The bacterial growth was associated with release of reducing sugar in the broth suggesting its role in carbon recycling of polysaccharides from seaweeds in marine ecosystem.     

A potential bacterial biocontrol agent,strain S2V2 against pathogenic marine <Emphasis Type="Italic">Vibrio</Emphasis> in aquaculture

We isolated a marine bacterium strain S2V2 which inhibited the growth of pathogenic marine Vibrio spp. The aims of this research were to identify a new antibiotic-producing marine bacterium strain S2V2, and evaluate its spectrum activity and pathogenic property. Analysis of 16S rDNA sequence placed strain S2V2 in the genus Pseudoalteromonas, but the sequence similarity was low (95.46%) implying the strain might be a new species in this genus. Strain S2V2 inhibited the growth of 67.9% of 28 Vibrio strains tested. This strain inhibited V. alginolyticus, V. anguillarum, V. fluvialis, V. harveyi, V. metschnikovii, V. splendidus, V. ordalii, V. parahaemolyticus, and V. vulnificus, but inactive against V. campbellii, Aeromonas hydrophyla and Staphylococcus aureus. Strain S2V2 produced extracellular non proteinaceous antibacterial substances. The highest antibacterial activity was found when strain S2V2 was cultured for 96 h in ZoBell broth medium. An artificial infection to post larvae of Lithopenaeus vanname indicated that strain S2V2 was a non pathogenic bacterium. Non pathogenic property and specific antibacterial activity against a broad range of fish pathogenic marine Vibrio of strain S2V2 suggest that this strain is a prospective source of unique antibiotic and a potential biocontrol agent in marine aquaculture.     

Comparative analysis of the genomes of <Emphasis Type="Italic">Bombyx mandarina</Emphasis> and <Emphasis Type="Italic">Bombyx mori</Emphasis> nucleopolyhedroviruses

The Bombyx mandarina nucleopolyhedrovirus (BomaNPV) S1 strain can infect the silkworm, Bombyx mori, but is significantly less virulent than B. mori nucleopolyhedrovirus (BmNPV) T3 strain. The complete nucleotide sequence of the S1 strain of BomaNPV was determined and compared with the BmNPV T3 strain. The circular, double stranded DNA genome of the S1 strain was 126,770 nucleotides long (GenBank accession no. FJ882854), with a G+C content of 40.23%. The genome contained 133 potential ORFs. Most of the putative proteins were more than 96% identical to homologs in the BmNPV T3 strain, except for bro-a, lef-12, bro-c, and bro-d. Compared with the BmNPV T3 strain, however, this genome did not encode the bro-b and bro-e genes. In addition, hr1 lacked two repeat units, while hr2L, hr2R, hr3, hr4L, hr4R, and hr5 were similar to the corresponding hrs in the T3 strain. The sequence strongly suggested that BomaNPV and BmNPV are variants with each other, and supported the idea that baculovirus strain heterogeneity may often be caused by variation in the hrs and bro genes.     

Isolation of a novel high erythritol-producing <Emphasis Type="Italic">Pseudozyma tsukubaensis</Emphasis> and scale-up of erythritol fermentation to industrial level

This study isolated a novel erythritol-producing yeast strain, which is capable of growth at high osmolarity. Characteristics of the strain include asexual reproduction by multilateral budding, absence of extracellular starch-like compounds, and a negative Diazonium blue B color reaction. Phylogenetic analysis based on the 26S rDNA sequence and physiological analysis indicated that the strain belongs to the species Pseudozyma tsukubaensis and has been named P. tsukubaensis KN75. When P. tsukubaensis KN75 was cultured aerobically in a fed-batch culture with glucose as a carbon source, it produced 245 g/L of erythritol, corresponding to 2.86 g/L/h productivity and 61% yield, the highest erythritol yield ever reported by an erythritol-producing microorganism. Erythritol production was scaled up from a laboratory scale (7 L fermenter) to pilot (300 L) and plant (50,000 L) scales using the dissolved oxygen as a scale-up parameter. Erythritol production at the pilot and plant scales was similar to that at the laboratory scale, indicating that the production of erythritol by P. tsukubaensis KN75 holds commercial potential.     

Establishment of Arabidopsis thaliana ribosomal protein RPL23A-1 as a functional homologue of Saccharomyces cerevisiae ribosomal protein L25

Arabidopsis thaliana ribosomal protein (r-protein) RPL23A-1 shows 54% amino acid sequence identity to the Saccharomyces cerevisiae equivalent r-protein, L25. AtRPL23A-1 also shows high amino acid sequence identity to members of the L23/L25 r-protein family in other species. R-protein L25 in S. cerevisiae has been identified as a primary rRNA-binding protein that directly binds to a specific site on yeast 26S rRNA. It is translocated to the nucleolus where it binds to 26S rRNA during early large ribosome subunit assembly; this binding is thought to play an important role in ribosome assembly. The S. cerevisiae mutant strain YCR61 expresses L25 when grown on galactose, but not glucose, medium. Transformation of YCR61 with a shuttle vector containing the AtRPL23A-1 cDNA allowed transformed colonies to grow in and on glucose selection medium. R-protein AtRPL23A-1 can complement the L25 mutation, demonstrating the functional equivalence of the two r-proteins and introducing AtRPL23A-1 as the first plant member of the L23/L25 r-protein family.     

Thermoactive extracellular proteases of <Emphasis Type="Italic">Geobacillus caldoproteolyticus</Emphasis>, sp. nov., from sewage sludge

A proteolytic thermophilic bacterial strain, designated as strain SF03, was isolated from sewage sludge in Singapore. Strain SF03 is a strictly aerobic, Gram stain-positive, catalase-positive, oxidase-positive, and endospore-forming rod. It grows at temperatures ranging from 35 to 65°C, pH ranging from 6.0 to 9.0, and salinities ranging from 0 to 2.5%. Phylogenetic analyses revealed that strain SF03 was most similar to Saccharococcus thermophilus, Geobacillus caldoxylosilyticus, and G. thermoglucosidasius, with 16S rRNA gene sequence identities of 97.6, 97.5 and 97.2%, respectively. Based on taxonomic and 16S rRNA analyses, strain SF03 was named G. caldoproteolyticus sp. nov. Production of extracellular protease from strain SF03 was observed on a basal peptone medium supplemented with different carbon and nitrogen sources. Protease production was repressed by glucose, lactose, and casamino acids but was enhanced by sucrose and NH₄Cl. The cell growth and protease production were significantly improved when strain SF03 was cultivated on a 10% skim-milk culture medium, suggesting that the presence of protein induced the synthesis of protease. The protease produced by strain SF03 remained active over a pH range of 6.0–11.0 and a temperature range of 40–90°C, with an optimal pH of 8.0–9.0 and an optimal temperature of 70–80°C, respectively. The protease was stable over the temperature range of 40–70°C and retained 57 and 38% of its activity at 80 and 90°C, respectively, after 1 h.