Use of Direct-Infusion Electrospray Mass Spectrometry To Guide Empirical Development of Improved Conditions for Expression of Secondary Metabolites from Actinomycetes

A major barrier in the discovery of new secondary metabolites from microorganisms is the difficulty of distinguishing the minor fraction of productive cultures from the majority of unproductive cultures and growth conditions. In this study, a rapid, direct-infusion electrospray mass spectrometry (ES-MS) technique was used to identify chemical differences that occurred in the expression of secondary metabolites by 44 actinomycetes cultivated under six different fermentation conditions. Samples from actinomycete fermentations were prepared by solid-phase extraction, analyzed by ES-MS, and ranked according to a chemical productivity index based on the total number and relative intensity of ions present in each sample. The actinomycete cultures were tested for chemical productivity following treatments that included nutritional manipulations, autoregulator additions, and different agitation speeds and incubation temperatures. Evaluation of the ES-MS data from submerged and solid-state fermentations by paired t test analyses showed that solid-state growth significantly altered the chemical profiles of extracts from 75% of the actinomycetes evaluated. Parallel analysis of the same extracts by high-performance liquid chromatography–ES-MS–evaporative light scattering showed that the chemical differences detected by the ES-MS method were associated with growth condition-dependent changes in the yield of secondary metabolites. Our results indicate that the high-throughput ES-MS method is useful for identification of fermentation conditions that enhance expression of secondary metabolites from actinomycetes.     

Rapid Method To Estimate the Presence of Secondary Metabolites in Microbial Extracts

Screening microbial secondary metabolites is an established method to identify novel biologically active molecules. Preparation of biological screening samples from microbial fermentation extracts requires growth conditions that promote synthesis of secondary metabolites and extraction procedures that capture the secondary metabolites produced. High-performance liquid chromatography (HPLC) analysis of fermentation extracts can be used to estimate the number of secondary metabolites produced by microorganisms under various growth conditions but is slow. In this study we report on a rapid (approximately 1 min per assay) surrogate measure of secondary metabolite production based on a metabolite productivity index computed from the electrospray mass spectra of samples injected directly into a spectrometer. This surrogate measure of productivity was shown to correlate with an HPLC measure of productivity with a coefficient of 0.78 for a test set of extracts from 43 actinomycetes. This rapid measure of secondary metabolite productivity may be used to identify improved cultivation and extraction conditions by analyzing and ranking large sets of extracts. The same methods may also be used to survey large collections of extracts to identify subsets of highly productive organisms for biological screening or additional study.     

Improved secondary metabolite production in the genus Streptosporangium by optimization of the fermentation conditions

The cultivation of strains of the genus Streptosporangium in batch fermentations demonstrated that the optimal conditions for secondary metabolite production are completely different to those of the closely related genus Streptomyces. The dissolved oxygen tension (pO(2)) was identified as an important parameter for optimal production of secondary metabolites in submerged cultures. Extreme variations of this parameter by changes in aeration (gas flow), agitation system and stirrer speed showed a tremendous impact in production yields of all investigated strains. Finally, a 20-fold increase in productivity was observed by conditions of controlled oxygen excess compared to optimal fermentation conditions for Streptomyces strains.     

Production of D-xylanases by thermophilic fungi using different methods of culture

Summary Seven thermophilic strains of fungi were examined for their ability to produce D-xylanase in Liquid and solid-state fermentations. It was confirmed that the best producers of xylanase, among microorganisms used, wereH. Lanuginosa andS. thermophile in liquid fermentation, andT. aurantiacus andH. lanuginosa in solid-state fermentations. The higher productivity of xylanase, namely 18,72 IU/ml, was obtained in liquid culture ofH. lanuginosa. The pH and temperature optima of enzymes from liquid and solid-state cultures of fungi used were also presented.     

Production of fungal antibiotics using polymeric solid supports in solid-state and liquid fermentation

The use of inert absorbent polymeric supports for cellular attachment in solid-state fungal fermentation influenced growth, morphology, and production of bioactive secondary metabolites. Two filamentous fungi exemplified the utility of this approach to facilitate the discovery of new antimicrobial compounds. Cylindrocarpon sp. LL-Cyan426 produced pyrrocidines A and B and Acremonium sp. LL-Cyan416 produced acremonidins A–E when grown on agar bearing moist polyester–cellulose paper and generated distinctly different metabolite profiles than the conventional shaken or stationary liquid fermentations. Differences were also apparent when tenfold concentrated methanol extracts from these fermentations were tested against antibiotic-susceptible and antibiotic-resistant Gram-positive bacteria, and zones of inhibition were compared. Shaken broth cultures of Acremonium sp. or Cylindrocarpon sp. showed complex HPLC patterns, lower levels of target compounds, and high levels of unwanted compounds and medium components, while agar/solid support cultures showed significantly increased yields of pyrrocidines A and B and acremonidins A–E, respectively. This method, mixed-phase fermentation (fermentation with an inert solid support bearing liquid medium), exploited the increase in surface area available for fungal growth on the supports and the tendency of some microorganisms to adhere to solid surfaces, possibly mimicking their natural growth habits. The production of dimeric anthraquinones by Penicillium sp. LL-WF159 was investigated in liquid fermentation using various inert polymeric immobilization supports composed of polypropylene, polypropylene cellulose, polyester–cellulose, or polyurethane. This culture produced rugulosin, skyrin, flavomannin, and a new bisanthracene, WF159-A, after fermentation in the presence and absence of polymeric supports for mycelial attachment. The physical nature of the different support systems influenced culture morphology and relative metabolite yields, as determined by HPLC analysis and measurement of antimicrobial activity. The application of such immobilized-cell fermentation methods under solid and liquid conditions facilitated the discovery of new antibiotic compounds, and offers new approaches to fungal fermentation for natural product discovery.     

Sponge-associated sp. RM66 metabolome induction with N-acetylglucosamine: Antibacterial,antifungal and anti-trypanosomal activities

The marine sponge Amphimedon sp., collected from Hurghada (Egypt) was investigated for its sponge-derived actinomycetes diversity. Nineteen actinomycetes were cultivated and phylogenetically identified using 16S rDNA gene sequencing were carried out. The strains belong to genera Kocuria, Dietzia, Micrococcus, Microbacterium and Streptomyces. Many silent biosynthetic genes clusters were investigated using genome sequencing of actinomycete strains and has revealed in particular the genus Streptomyces that has indicated their exceptional capacity for the secondary metabolites production that not observed under classical cultivation conditions. In this study, the effect of N-acetylglucosamine on the metabolome of Streptomyces sp. RM66 was investigated using three actinomycetes media (ISP2, M1 and MA). In total, twelve extracts were produced using solid and liquid fermentation approaches. Liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) data were analysed using metabolomics tools to compare natural product production across all crude extracts. Our study highlighted the elicitation effect of N-acetylglucosamine on the secondary metabolite profiles of Streptomyces sp. RM66. These results highlight the of N-acetylglucosamine application as an elicitor to induce the cryptic metabolites and for increasing the chemical diversity. All the twelve extracts were tested for their antibacterial activity was tested against Staphylococcus aureus NCTC 8325, antifungal activity against Candida albicans 5314 (ATCC 90028) and anti-trypanosomal activity against Trypanosoma brucei brucei. Extract St1 showed the most potent one with activities 2.3, 3.2 and 4.7 ug/ml as antibacterial, antifungal and anti-trypanosomal, respectively.     

Fermentative production of self-toxic fungal secondary metabolites

Fungi are well known for their vast diversity of secondary metabolites that include many life-saving drugs and highly toxic mycotoxins. In general, fungal cultures producing such metabolites are immune to their toxic effects. However, some are known to produce self-toxic compounds that can pose production optimization challenges if the metabolites are needed in large amounts for chemical modification. One such culture, LV-2841, was identified as the lead for one of our exploratory projects. This culture was found to be a slow grower that produced trace amounts of a known metabolite, cercosporamide, under the standard flask fermentation conditions, and extensive medium optimization studies failed to yield higher titers. Poor growth of the culture in liquid media was attributed to the self-toxicity of cercosporamide to the producing organism, and the minimum inhibitory concentration (MIC) of cercosporamide was estimated to be in the range of 8–16 μg/ml. Fermentations carried out in media containing Diaion^® HP20 resin afforded significantly higher titers of the desired compound. While several examples of resin-based fermentations of soil streptomyces have been published, this approach has rarely been used for fungal fermentations. Over a 100-fold increase in the production titer of cercosporamide, a self-toxic secondary metabolite, was achieved by supplementing the production medium with a commercially available neutral adsorbent resin.     

Systematic and biotechnological aspects of halophilic and halotolerant actinomycetes

More than 70 species of halotolerant and halophilic actinomycetes belonging to at least 24 genera have been validly described. Halophilic actinomycetes are a less explored source of actinomycetes for discovery of novel bioactive secondary metabolites. Degradation of aliphatic and aromatic organic compounds, detoxification of pollutants, production of new enzymes and other metabolites such as antibiotics, compatible solutes and polymers are other potential industrial applications of halophilic and halotolerant actinomycetes. Especially new bioactive secondary metabolites that are derived from only a small fraction of the investigated halophilic actinomycetes, mainly from marine habitats, have revealed the huge capacity of this physiological group in production of new bioactive chemical entities. Combined high metabolic capacities of actinomycetes and unique features related to extremophilic nature of the halophilic actinomycetes have conferred on them an influential role for future biotechnological applications.     

Regulation of secondary metabolism in streptomycetes

While the biological functions of most of the secondary metabolites made by streptomycetes are not known, it is inconceivable that they do not play an adaptive ecological role. The biosynthesis of secondary metabolites under laboratory conditions usually occurs in a growth phase or developmentally controlled manner, but is also influenced by a wide variety of environmental and physiological signals, presumably reflecting the range of conditions that trigger their production in nature. The expression of secondary metabolic gene clusters is controlled by many different families of regulatory proteins, some of which are found only in actinomycetes, and is elicited by both extracellular and intracellular signalling molecules. The application of a variety of genetic and molecular approaches is now beginning to reveal fascinating insights into the complex regulatory cascades that govern this process.     

Hairy root culture for mass-production of high-value secondary metabolites

Plant cell cultivations are being considered as an alternative to agricultural processes for producing valuable phytochemicals. Since many of these products (secondary metabolites) are obtained by direct extraction from plants grown in natural habitat, several factors can alter their yield. The use of plant cell cultures has overcome several inconveniences for the production of these secondary metabolites. Organized cultures, and especially root cultures, can make a significant contribution in the production of secondary metabolites. Most of the research efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone free media. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Hairy root cultures offer promise for high production and productivity of valuable secondary metabolites (used as pharmaceuticals, pigments and flavors) in many plants. The main constraint for commercial exploitation of hairy root cultivations is the development and scaling up of appropriate reactor vessels (bioreactors) that permit the growth of interconnected tissues normally unevenly distributed throughout the vessel. Emphasis has focused on designing appropriate bioreactors suitable to culture the delicate and sensitive plant hairy roots. Recent reactors used for mass production of hairy roots can roughly be divided as liquid-phase, gas-phase, or hybrid reactors. The present review highlights the nature, applications, perspectives and scale up of hairy root cultures for the production of valuable secondary metabolites.     

放线菌核糖体工程的发展与应用

核糖体工程(ribosome engineering)是一项利用靶点位于细菌RNA聚合酶及核糖体功能因子的抗生素诱导细菌产生抗性突变,进而提升菌株次级代谢生产潜能的技术.该方法无需依赖菌株完善的遗传操作体系,可应用于发掘几乎所有放线菌菌株中潜在的宝贵活性次级代谢产物,并广泛应用于放线菌基因组挖掘和次级代谢产物增产优化....     

放线菌与枯草芽孢杆菌的共培养及其对活性次生代谢产物的影响

为探讨共培养对放线菌产生活性次生代谢产物的影响,结合抗菌活性测定及HPLC-PDA分析,研究了22株放线菌的单培养及其与枯草芽孢杆菌的共培养发酵代谢产物的差异,并选取抗菌活性较强的链霉菌FXJ2.014进一步研究其代谢产物。发现FXJ2.014、FXJ1.296、AS4.1252三株菌与枯草芽孢杆菌共培养时产生其在相同条件下单培养时没有的物质,其中链霉菌FXJ2.014单培养时主要产生醌霉素A,共培养时产物中增加了醌霉素结构类似物FXJ2.014-HB。进一步的抗菌、抗肿瘤活性测定结果表明,两者的生物活性有较显著的差异,且FXJ2.014-HB对多种肿瘤细胞系的抑制活性普遍弱于高毒性的醌霉素A,为有潜力的细胞毒性较小的抗生素。共培养是一条很有希望的发掘放线菌活性次生代谢产物的新途径。     

Effects of long-term preservation on growth and productivity of Panax ginseng and Catharanthus roseus cell cultures

Cell cultures of Panax ginseng and Catharanthus roseus producing secondary metabolites were preserved in liquid nitrogen or under mineral oil for six months. The growth behaviour and the ability of the cultures to produce ginsenosides or indole alkaloids were measured after a recovery period and compared with cultures maintained by frequent subcultivation during the same period. Neither growth kinetics nor the degree of vacuolization during growth were affected by the long term preservation. Some changes in secondary metabolism were however found, indicating that preservation under mineral oil does not preserve the productivity of cell cultures whereas the cryogenic method does.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethylsulphoxide - HPLC high performance liquid chromatography     

Increase in eremomycin production by regeneration and UV-irradiation of Amycolatopsis orientalis subsp. eremomycini protoplasts]

Protoplast regeneration of Amycolatopsis orientalis subsp. eremomycini producing eremomycin leads to the change of cultural and morphological properties as well as synthesis of secondary metabolites. Formation of plus-variants with enchanced antibiotic production was promoted by UV-irradiation of protoplasts. These plus-variants can be successfully used for repeating protoplasting--UV-irradiation of protoplasts with further increasing of the strain productivity. Finally activity of the initial A. orientalis culture was increased 7-8 times. Proposed method is recommended for the improvement of actinomycetes strains producing antibiotics especially in the case of cultures with poor sporulation.     

Hairy Root Culture for Mass-Production of High-Value Secondary Metabolites

ABSTRACT

Plant cell cultivations are being considered as an alternative to agricultural processes for producing valuable phytochemicals. Since many of these products (secondary metabolites) are obtained by direct extraction from plants grown in natural habitat, several factors can alter their yield. The use of plant cell cultures has overcome several inconveniences for the production of these secondary metabolites. Organized cultures, and especially root cultures, can make a significant contribution in the production of secondary metabolites. Most of the research efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone free media. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Hairy root cultures offer promise for high production and productivity of valuable secondary metabolites (used as pharmaceuticals, pigments and flavors) in many plants. The main constraint for commercial exploitation of hairy root cultivations is the development and scaling up of appropriate reactor vessels (bioreactors) that permit the growth of interconnected tissues normally unevenly distributed throughout the vessel. Emphasis has focused on designing appropriate bioreactors suitable to culture the delicate and sensitive plant hairy roots. Recent reactors used for mass production of hairy roots can roughly be divided as liquid-phase, gas-phase, or hybrid reactors. The present review highlights the nature, applications, perspectives and scale up of hairy root cultures for the production of valuable secondary metabolites.     

东乡野生稻内生放线菌分离及菌株S123次级代谢产物分析

【目的】从东乡野生稻(Oryza rufipogon)中分离和鉴定内生放线菌,对其进行抗菌活性筛选,并分析高抗菌活性菌株S123的次级代谢产物。【方法】采用S培养基对东乡野生稻内生放线菌进行分离、纯化,并构建16S rRNA基因序列系统发育进化树进行菌株鉴定。以琼脂扩散法和菌丝生长速率法进行抗菌活性筛选,同时设计简并引物检测菌株I型聚酮合酶(PKS-I)基因。对具广谱抗菌活性的菌株S123进行分批大量发酵,运用多种色谱方法对发酵产物进行分离、纯化,利用MS和NMR分析鉴定化合物的结构。【结果】从东乡野生稻中共分离到11株内生放线菌,分别属于链霉菌属(8株)和假诺卡氏属(3株)。其中有8株具有抗菌活性,8株呈现I型PKS阳性。从高抑菌活性菌株S123中分离到化合物Nigericin和17-O-demethylgeldanamycin,其中Nigericin对金黄色葡萄球菌、枯草芽孢杆菌及水稻纹枯病菌均有抑制活性。【结论】对东乡野生稻内生放线菌进行了分离、鉴定和抗菌活性筛选,并从中分到两种与I型PKS基因相关活性的化合物Nigericin和17-O-demethylgeldanamycin,为研究东乡野生稻内生放线菌的多样性和次级代谢产物的分离提供依据。     

Influence of abiotic stress signals on secondary metabolites in plants

Plant secondary metabolites are unique sources for pharmaceuticals, food additives, flavors, and industrially important biochemicals. Accumulation of such metabolites often occurs in plants subjected to stresses including various elicitors or signal molecules. Secondary metabolites play a major role in the adaptation of plants to the environment and in overcoming stress conditions. Environmental factors viz. temperature, humidity, light intensity, the supply of water, minerals, and CO₂ influence the growth of a plant and secondary metabolite production. Drought, high salinity, and freezing temperatures are environmental conditions that cause adverse effects on the growth of plants and the productivity of crops. Plant cell culture technologies have been effective tools for both studying and producing plant secondary metabolites under in vitro conditions and for plant improvement. This brief review summarizes the influence of different abiotic factors include salt, drought, light, heavy metals, frost etc. on secondary metabolites in plants. The focus of the present review is the influence of abiotic factors on secondary metabolite production and some of important plant pharmaceuticals. Also, we describe the results of in vitro cultures and production of some important secondary metabolites obtained in our laboratory.     

THE BEGINNINGS OF ANTARCTIC MACROALGAL CHEMICAL ECOLOGY: DEFENSES AGAINST HERBIVORES IN A NITROGEN REPLETE,CARBON LIMITED OCEAN

Amsler, C. D.¹, Iken, K. B.¹, McClintock, J. B.¹, Furrow, F. B.², & Baker, B. J.^{2 1}Department of Biology, University of Alabama at Birmingham, Birmingham AL 35294-1170 USA; ²Department of Chemistry, Florida Institute of Technology, Melbourne, FL 32901 USA We examined palatability of Antarctic Peninsula macroalgae in feeding bioassays with three common, sympatric macroalgal-consuming omnivores (amphipod, fish, sea star). Antarctic macroalgae have low C:N ratios, high nitrogen contents, and are usually growth limited by carbon (light). The Carbon Nutrient Balance Hypothesis (CNBH) predicts that macroalgae would produce nitrogenous secondary metabolites for defense rather than high levels of non-nitrogenous defenses under these conditions. To date, feeding bioassays have been performed on fragments of thallus from 26 macroalgal species and 21 (81%) were rejected by at least one omnivore. Organic extracts from 13 macroalgal species rejected as thallus were used in feeding bioassays. At least one extract from 12 species (92%) was rejected by at least one omnivore, suggesting that chemical defenses against herbivores probably are present in at least some of the macroalgal species. We have identified a number of specific, non-nitrogenous secondary metabolites in these extracts and previous workers have also reported non-nitrogenous secondary metabolites from antarctic macroalgae. Additional extracts targeting nitrogenous metabolites from 25 species were subjected to thin layer chromatography and visualized by stains specific for nitrogenous compounds. No nitrogenous secondary metabolites were identified by this or other methods. Nitrogenous secondary metabolites are also extremely rare in macroalgae from other areas of the world. Consequently, although our bioassays suggest that chemical defenses probably do occur, our data cast doubt on the applicability of the CNBH for predicting the chemical composition of macroalgal defenses under carbon limited conditions. (NSF OPP9814538, OPP9901076)     

Secondary metabolism of hairy root cultures in bioreactors

Summary In vitro cultures are being considered as an alternative to agricultural processes for producing valuable secondary metabolites. Most efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Bioreactors used to culture hairy roots can be roughly divided into three types: liquid-phase, gas-phase, or hybrid reactors that are a combination of both. The growth and productivity of hairy root cultures are reviewed with an emphasis on successful bioreactors and important culture considerations. The latter include strain selection, production of product in relation to growth phase, media composition, the gas regime, use of elicitors, the role of light, and apparent product loss. Together with genetic engineering and process optimization, proper reactor design plays a key role in the development of successful large scale production of secondary metabolites from plant cultures.