Synthesis of hydrolytic enzymes during production of tylosin byStreptomyces fradiae

Summary The exposure of a wild-type tylosin producing strain ofStreptomyces fradiae to mutagenic agents resulted in the isolation of several tylosin over-producing strains. Examination of three mutants, T4310, 612 and 3204 showed that improved tylosin production was associated with increased hydrolytic enzyme activity and cell growth. The wild-type strain showed lower levels of hydrolytic activity including, protease, amylase, lipase and esterase activities and attained a lower cell density than the mutants.     

Regulation of branched-chain amino acid biosynthesis in Streptomyces fradiae,a producer of tylosin

Synthesis of threonine dehydratase in Streptomyces fradiae was positively influenced by valine and negatively by isoleucine. However, these two amino acids had no effect on the activity of this enzyme. Synthesis of threonine dehydratase in -aminobutyrate resistant mutants of S. fradiae was pronouncedly less sensitive to the positive effect of valine and this change in regulation led to valine overproduction. Synthesis of acetohydroxy acid synthase is regulated in a similar manner to that of threonine dehydratase, however a lower level of expression was detected in -aminobutyrate resistant mutants. And again, no effect of branched-chain amino acids on acetohydroxy acid synthase activity was observed. It follows that in S. fradiae synthesis of threonine dehydratase is the main regulatory mechanism governing production and the mutual ratio of synthesized valine and isoleucine.Abbreviations -AB -aminobutyrate - AHAS acetohydroxy acid synthase - -KB -ketobutyrate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - TD threonine dehydratase - Trans. B. transaminase of branched-chain amino acids - VDH valine dehydrogenase     

Influence of dimethylsulfoxide on tylosin production in Streptomyces fradiae

The polyketide aglycone, tylactone (protylonolide), does not normally accumulate during tylosin production in Streptomyces fradiae, suggesting that the capacity of the organism to glycosylate tylactone exceeds the capacity for polyketide synthesis. Consistent with this model, tylosin yields were significantly increased (due to bioconversion of the added material) when exogenous tylactone was added to fermentations. However, tylosin yield improvements were also observed (albeit at lower levels) in solvent controls to which dimethylsulfoxide (DMSO) was added. At least in part, the latter effect resulted from stimulation of polyketide metabolism by DMSO. This was revealed when the solvent was added to fermentations containing the tylA mutant, S. fradiae GS14, which normally accumulates copious quantities of tylactone. Journal of Industrial Microbiology & Biotechnology (2001) 27, 46–51. Received 18 March 2001/ Accepted in revised form 29 May 2001     

The mycinose-biosynthetic genes of Streptomyces fradiae, producer of tylosin

The tylE-J region of the tylosin-biosynthetic gene cluster of Streptomyces fradiae contains six open reading frames. The products of tylJ and tylD are nucleoside diphospho (NDP)-deoxyhexose 3-epimerase and NDP-deoxyhexose 4-ketoreductase, respectively, involved in the synthesis of NDP-6-deoxyallose from NDP-4-keto, 6-deoxyglucose. After incorporation of deoxyallose at C23-OH of the polyketide lactone, tylosin biosynthesis is completed by the products of tylE and tylF, which convert the deoxyallosyl moiety to mycinose via bis-O-methylation at 2-OH and 3-OH, respectively. Hydroxylation of the polyketide lactone at C23 is catalysed by the cytochrome P450 enzyme, TylHl. The product of tylHll is a ferredoxin of unknown specificity that could conceivably act together with TylHl. Received 17 March 1999/ Accepted in revised form 20 June 1999     

Cloning of Transglutaminase Gene from Streptomyces fradiae and its Enhanced Expression in the Original Strain

The transglutaminase (TGase) gene of Streptomyces fradiae was cloned. It had an ORF of 1242 bp, encoding a presumed prepro-region of 82 amino acids and a mature TGase of 331 amino acids. Enhanced expression of the TGase was achieved by introducing another copy of TGase gene into the original host genome which was driven by the strong constitutive promoter, “ermE up”, and shown to be expressed at the mRNA and protein levels. TGase activity in the recombinant strain (3.2 U/ml) was improved 1.3-fold when compared to that normally expressed in the original strain (2.4 U/ml). The specific enzyme activity in the recombinant strain (3.8 U/mg) was double that of the original strain (1.9 U/mg).     

Thiosulfate production from cystine by the keratinolytic prokaryote Streptomyces fradiae

In cultures of Streptomyces fradiae on wool as the only source of nutrition inorganic thiosulfate (in amounts up to 0.5 mg of Na₂S₂O₃·5 H₂O/ml) was formed as the final product of metabolization of sulfur from cystine of keratin proteins. The presence of thiosulfate was proved by qualitative tests and thin-layer chromatography and estimated quantitatively by spectrophotometry, titrimetry, and capillary isotachophoresis. Metabolization of organic sulfur to thiosulfate excreted into the medium is a process not yet described in microorganisms.     

Production of tylosin in solid-state fermentation by Streptomyces fradiae NRRL-2702 and its gamma-irradiated mutant (γ-1)

Aims:  To develop solid-state fermentation system (SSF) for hyper production of tylosin from a mutant γ-1 of Streptomyces fradiae NRRL-2702 and its parent strain.
Methods and Results:  Various agro-industrial wastes were screened to study their effect on tylosin production in SSF. Wheat bran as solid substrate gave the highest production of 2500 μg of tylosin g⁻¹ substrate by mutant γ-1 against parent strain (300 μg tylosin g⁻¹ substrate). The tylosin yield was further improved to 4500 μg g⁻¹ substrate [70% moisture, 10% inoculum (v/w), pH 9·2, 30°C, supplemental lactose and sodium glutamate on day 9]. Wild-type strain displayed less production of tylosin (655 μg of tylosin g⁻¹ substrate) in SSF even after optimization of process parameters.
Conclusion:  The study has shown that solid-state fermentation system significantly enhanced the tylosin yield by mutant γ-1.
Significance and Impact of the Study:  This study proved to be very useful and resulted in 6·87 ± 0·30-fold increase in tylosin yield by this mutant when compared to that of wild-type strain.     

Studies on oil palm trunks as sources of infection in the field

Diseases of oil palm caused by Ganoderma boninense are of major economic importance in much of South-East Asia. This paper describes results from an ongoing field trial concerning the spread of the pathogen from artificially inoculated trunks used to simulate spread from windrowed trunks. Three planting distances for bait seedlings revealed that the closer the seedling was planted to the source of inoculum the sooner it succumbed to the disease. However, infection only occurred when the trunks were mounded (covered with soil), and seedlings planted around uncovered trunks (at any distance) have showed no symptoms of disease to date. Isolates are being collected from infected plants and molecular analysis is being undertaken to give more information on the spread of the pathogen.     

The lipopeptide antibiotic A54145 biosynthetic gene cluster from Streptomyces fradiae

Ca(2+)-dependent cyclic lipodepsipeptides are an emerging class of antibiotics for the treatment of infections caused by Gram-positive pathogens. These compounds are synthesized by nonribosomal peptide synthetase (NRPS) complexes encoded by large gene clusters. The gene cluster encoding biosynthetic pathway enzymes for the Streptomyces fradiae A54145 NRP was cloned from a cosmid library and characterized. Four NRPS-encoding genes, responsible for subunits of the synthetase, as well as genes for accessory functions such as acylation, methylation and hydroxylation, were identified by sequence analysis in a 127 kb region of DNA that appears to be located subterminally in the bacterial chromosome. Deduced epimerase domain-encoding sequences within the NRPS genes indicated a D: -stereochemistry for Glu, Lys and Asn residues, as observed for positionally analogous residues in two related compounds, daptomycin, and the calcium-dependent antibiotic (CDA) produced by Streptomyces roseosporus and Streptomyces coelicolor, respectively. A comparison of the structure and the biosynthetic gene cluster of A54145 with those of the related peptides showed many similarities. This information may contribute to the design of experiments to address both fundamental and applied questions in lipopeptide biosynthesis, engineering and drug development.     

Effects of Rapeseed Oil on Activity of Methylmalonyl-CoA Carboxyltransferase in Culture of Streptomyces fradiae

To investigate why more tylosin was produced when Streptomyces fradiae T1558 was cultured in a rapeseed oil medium than in a glucose or starch medium, we measured the activity of methylmalonyl-CoA carboxyltransferase (EC 2.1.3.1) and intracellular propionic acid. The activity of the enzyme, which catalyzes the formation of the precursor of tylosin, protylonolide, was 0.19 U/mg protein in 5 days of culture in rapeseed oil medium, which was 2.5- and 1.3-fold that with the glucose or starch medium, respectively. The intracellular propionic acid concentration was 1.2 g/g of dry weight, which was 4.3- and 2.1-fold that with the glucose or starch medium, respectively. The addition of propionic acid increased tylosin production in batch culture: when 0.2 g/l (final concentration) propionic acid was added to the glucose medium, 3.8 g/l tylosin was produced in 10 days of culture, 4.7-fold the amount without propionic acid. These findings suggest that in glucose medium, intracellular propionic acid is a limiting factor because of the low activity of methylmalonyl-CoA carboxyltransferase of the tylosin biosynthesis pathway.     

Solvent-free glycerolysis of palm and palm kernel oils catalyzed by commercial 1,3-specific lipase from Humicola lanuginosa and composition of glycerolysis products

Glycerolysis of palm and palm kernel oils were conducted using a commercial 1,3-specific lipase from Humicola lanuginosa (trade name: SP 398) as catalyst (500 units lipase g^–1 oil) at 40°C and oil:glycerol (1:2 mol mol^–1) in a solvent-free system. After 24 h, the glycerolysis products of palm and palm kernel oils consisted of 23% triacylglycerols, 18% monoacylglycerols, 38% diacylglycerols and 18% triacylglycerols, 31% monoacylglycerols, 42% diacylglycerols, respectively. The monoacylglycerol fraction of the glycerolysis product of palm oil was enriched in oleic acid. Palmitic acid content of the monoacylglycerol fraction of the same product was less than that of the original oil. Under the same conditions, monacylglycerol fraction of the palm kernel oil glycerolysis product was enriched in palmitic, stearic and oleic acids.     

Change in colony morphology and kinetics of tylosin production after UV and gamma irradiation mutagenesis of Streptomyces fradiae NRRL-2702

Tylosin is a macrolide antibiotic used as veterinary drug and growth promoter. Attempts were made for hyper production of tylosin by a strain of Streptomyces fradiae NRRL-2702 through irradiation mutagenesis. Ultraviolet (UV) irradiation of wild-type strain caused development of six morphologically altered colony types on agar plates. After screening using Bacillus subtilis bioassay only morphological mutants indicated the production of tylosin. An increase of 2.7±0.22-fold in tylosin production (1500 mg/l) in case of mutant UV-2 in complex medium was achieved as compared to wild-type strain (550 mg/l). Gamma irradiation of mutant UV-2 using ⁶⁰Co gave one morphologically altered colony type γ-1, which gave 2500 mg/l tylosin yield in complex medium. Chemically defined media promoted tylosin production upto 3800 mg/l. Maximum value of q_p (3.34 mg/gh) was observed by mutant γ-1 as compared to wild strain (0.81 mg/gh). Moreover, UV irradiation associated changes were unstable with loss of tylosin activity whereas mutant γ-1 displayed high stability on subsequent culturing.     

Cyclic AMP regulation of tylosin biosynthesis and secondary metabolism in Streptomyces fradiae

Adenosine 3':5' cyclic monophosphate seems to regulate antibiotic biosynthesis and secondary metabolism in tylosin-producing cultures of Streptomyces fradiae C373.1. A dose-dependent response is observed by exogenous additions of dibutyryl cyclic AMP (cAMP), and is related to the nutritional status of the culture. Addition of cAMP to cultures growing in nutritionally lean media caused higher cumulative antibiotic tigers and some cellular differentiation compared with the control. In nutritionally rich media, a qualitatively different behavior resulted: an almost instantaneous shift toward secondary metabolism occurred. The response is characterized by extensive cellular differentiation with little growth and only a trace of antibiotic production. The possible role of cyclic AMP n the regulation of tylosin biosynthesis and secondary metabolism and its relation to specific nutrient limitations in synthetic, defined media in Streptomyces fradiae is discussed. (c) 1994 John Wiley & Sons, Inc.     

Enhancing biological control of basal stem rot disease (<Emphasis Type="Italic">Ganoderma boninense</Emphasis>) in oil palm plantations

Basal Stem Rot (BSR) disease caused by Ganoderma boninense is the most destructive disease in oil palm, especially in Indonesia and Malaysia. The available control measures for BSR disease such as cultural practices and mechanical and chemical treatment have not proved satisfactory due to the fact that Ganoderma has various resting stages such as melanised mycelium, basidiospores and pseudosclerotia. Alternative control measures to overcome the Ganoderma problem are focused on the use of biological control agents and planting resistant material. Present studies conducted at Indonesian Oil Palm Research Institute (IOPRI) are focused on enhancing the use of biological control agents for Ganoderma. These activities include screening biological agents from the oil palm rhizosphere in order to evaluate their effectiveness as biological agents in glasshouse and field trials, testing their antagonistic activities in large scale experiments and eradicating potential disease inoculum with biological agents. Several promising biological agents have been isolated, mainly Trichoderma harzianum, T. viride, Gliocladium viride, Pseudomonas fluorescens, and Bacillus sp. A glasshouse and field trial for Ganoderma control indicated that treatment with T. harzianum and G. viride was superior to Bacillus sp. A large scale trial showed that the disease incidence was lower in a field treated with biological agents than in untreated fields. In a short term programme, research activities at IOPRI are currently focusing on selecting fungi that can completely degrade plant material in order to eradicate inoculum. Digging holes around the palm bole and adding empty fruit bunches have been investigated as ways to stimulate biological agents.     

Cryopreservation of oil palm (Elaeis guineensis Jacq.) somatic embryos involving a desiccation step

Summary The standard cryopreservation process previously developed for oil palm clones using shiny white, finger-like somatic embryos could be applied in some cases to standard cultures. Its efficiency was markedly improved by completing the 7-day pregrowth period on 0.75 M sucrose by an additional dehydration period carried out either by placing the embryos in the air current of the laminar flow cabinet or in an air tight box containing silica gel. This improved process was successfully applied to 7 different clones. It will facilitate the routine uof cryopreservation for oil palm cultures.     

Production of clavulanic acid and cephamycin C by Streptomyces clavuligerus in palm-oil medium

Palm and palm-kernel oils and their olein and stearin fractions were suitable as the main carbon sources for growth and production of clavulanic acid by Streptomyces clavuligerus. However, oleic and lauric acids were not utilized for growth. A spontaneous mutant, which was selected for higher cephamycin C production, also produced more clavulanic acid with these oils in the medium.     

油棕种壳厚度控制基因SHELL的SNP分子标记开发

油棕属棕榈科多年生木本油料作物,果实含油量高达50%,且单位面积产油量高,享有"世界油王"美誉。油棕果实由外果皮、中果皮、内果皮(种壳)、种子四个部分组成,产油部分主要是中果皮和种子,其中种壳厚度是影响果实含油量的重要因素。SHELL基因控制种壳厚度,是一类MADS-box同源基因,SHELL基因在厚壳种和无壳种中的变异主要是第一个外显子上的两个SNP位点。该研究根据两个SNP位点进行特异标记开发,根据已知的油棕SHELL基因的序列,设计了4对SNP引物。4对SNP引物以2个SNP位点设计,每个SNP位点设计2对SNP标记,并均在引物3'端第二位引入强错配碱基。以2份薄壳种油棕材料和2份厚壳种油棕材料DNA为模板,扩增筛选油棕SHELL基因SNP引物。通过PCR扩增发现,设计的SHELL基因特异SNP标记EgSh(N)-f/EgSh(SNP)-2r能够鉴别油棕厚壳种和薄壳种。再用24株油棕树进行特异性验证,发现该标记能较准确地判断油棕的厚薄壳。该研究结果表明SNP标记EgSh(N)-f/EgSh(SNP)-2r可用来进行油棕种质资源早期分子鉴定,为高产油棕品种选育提供了技术支撑。