Activation studies and isolation of microbes

Abstract
Bioactive compounds derived from the natural world are responsible for many of the therapeutic effects that our ancestors experienced, and at present, these compounds serve as the framework by which scientists develop novel drugs. Streptomyces is known as the largest genus of Actinobacteria, and this genus belongs to the family Streptomycetaceae. Streptomycetes, just like other Actinobacteria have genomes with large amount of GC content and they are Gram-positive. They are typified by a compound secondary metabolism, and they generate more than â…“ of the clinically valuable antibiotics of natural origin such as chloramphenicol and neomycin. The purpose of this study was to activate secondary metabolites inStreptomycens spp. In normal laboratory conditions, the microbes would not produce secondary metabolites. Therefore, chemical activators (Lipopolysaccharideandmycolic acid) and biological activator (Tuberculosis cells or Tb) were used in this study to activate silent secondary metabolite genes. As the Streptomycenstakes 7-10 days to grow in the incubator, microbes were isolated from 4 terrestrial samples in order to do more work in the lab during the incubation time. On the whole, 39 strains of Streptomyceswere chosen from ACM, 9 of them did not grow. This could be because the media used did not suit them given that only ISP-2 was used for the initial cultivation. Nonetheless, some of them exhibited remarkable results with the activation studies and interesting chemical components with optimisation.
The isolation of microbes obtained from terrestrial soil samples is of major importance since it helps in identifying new species of microbes. These experiments are important in identifying known and unknown microbes which can be utilized for further studying to isolate vital natural products. In this study, it has been established that Streptomyces spp. contains secondary metabolite genes that are not transcriptionally activated under cultivation conditions, but that can be activated by natural chemical and biological cues such as tuberculosis (TB) cells, mycolic acid and lipopolysaccharide (LPS). This study is significant since the activation can be used to produce antibiotics which are vital in the pharmaceutical industry.
1 Introduction:
Natural products have had a close association with medicine since the dawn of mankind, and the field of pharmacology traces its origins to the use of medicinal plants, animals and microbes (Peláez, 2006). Early civilizations used products available from nature to deal with injuries and diseases, and in so doing, often attributed a mystical quality to their world. Bioactive compounds derived from the natural world are responsible for many of the therapeutic effects that our ancestors experienced, and today, these compounds serve as the framework by which scientists develop novel drugs (Butler &Buss, 2006). It is the vastness of the variety of molecules in the natural world that presents a fundamental research base for modern drug discovery.
The number of new medicines that are available today which are based on microorganisms, for example, is significant (Singh & Barrett, 2006; Newman &Cragg, 2007). Advances in biotechnology such as high throughput screening have enabled large numbers of biological samples to be screened in a short period of time. Improvements in the fields of genomics and analytical chemistry have enhanced the search for bioactive compounds from natural products. These new technologies have renewed the interest of scientists in pursuing natural product leads, and the increased success rate of drug discovery using natural products has offered the promise of new medicines from a near forgotten source (Nussbaum et al., 2006).
Under certain conditions, organisms can generate secondary metabolites which have potential biological activities that are useful in medicine. Changes in acidity, temperature, oxygen, available nutrients and/or toxins can act to activate silent genes and alter metabolic pathways to generate altered or novel compounds which may have beneficial attributes. In the following thesis, microbial natural products such as antibiotics, antitumor agents and immunosuppressants are described. This is followed by a description of the methods in which secondary metabolites can become activated. An in-depth description of Streptomyces and various approaches by which to activate secondary metabolism in this microorganism is provided. The aims of the project are discussed, followed by the experimental details. The thesis will conclude with a discussion of how secondary metabolite genes that become transcriptionally activated by chemical and biological cues can yield interesting chemical components which have the potential to benefit both scientific research and medicine.
1 Natural Products as Drugs
In essence, natural products are mostly secondary metabolites, which are produced by living organisms through metabolic reactions but are not required for vital processes such as development, growth and reproduction. In 1961, the term "secondary metabolites" was coined by Bu’Lock to describe certain microbial products. Bennett and Bentley (1989) later evaluated the appropriateness of this term. Even though the history of the use of secondary metabolites dates back to ancient times (Bassett et al., 1980), the systematic detection and characterisation of these metabolites began in the 20th century.
Microorganisms, especially fungi and bacteria inhabiting soil, generate various substances that have small molecular masses and differing structures. It is obvious that the microbes generating these substances live in a complicated environment where they interact and compete with different living systems such as algae, fungi, bacteria, metazoans and protozoans (Goh et al., 2001; Losada et al., 2009). As only a small number of microorganisms have been identified and cultured so far (Yin et al., 2007), it is quite understandable that a huge number of microbial metabolites still need to be identified. Findings from the latest genome-sequencing projects also reveal the presence of this opportunity. Sometimes novel metabolites are discovered as soon as a new microbe is cultivated successfully. Nevertheless, our knowledge regarding the contributions of these metabolites to the environment in which they are produced is still deficient. In other words, what these metabolites actually do in the environment of the organism producing them when they are not essential to the organism still needs to be addressed specifically.
A huge number of secondary metabolites have demonstrated anticancer, antibacterial, antifungal, antiparasitic, herbicidal, immunosuppressing and cholesterol-reducing capabilities and have also proven to be useful for different tasks related to research and diagnostics. Certain bioactive secondary metabolites have made a remarkable contribution to preventing and treating different human diseases of increased severity, some of which were previously incurable. These promising features of microbial metabolites have spurred interest in their production on a commercial scale; however, microbes isolated from their natural environments produce only small quantities of these extremely useful secondary metabolites. Strategies aiming at increased productivity of microbial metabolites include optimization of culture conditions and genetic engineering, mainly involving recombinant DNA technology and selection and the utilisation of high-yielding mutant strains. The efforts being made to determine potential applications for identified metabolites and to discover novel secondary metabolites by studying identified organisms and microorganisms would definitely enable us to find treatment therapies for incurable diseases, thereby decreasing the rate of deaths. Some important metabolites that are of clinical significance are discussed below.
1 Antibiotics
Most of the agents currently used as antitumor drugs were previously identified as antibiotics. This is because several antibiotics are capable of demonstrating a wide range of activities (Newman & Shapiro, 2008). Some examples of such antibiotics are anthracenones like pentostatin, streptozotocin and mithramycin; glycopeptolides like phleomycin and bleomycin; anthracyclines like amrubicin, valrubicin, idarubicinn, pirirubicin, doxorubicin and daunorubicin; dactinomycin, themitosanemitomycin, and Mylotarg® (endiynecalcheamycin), linked with monoclonal antibodies.
β-lactams
The β-lactams (cephalosporins and penicillins) are considered the most popular antibiotics. Certain compounds having β-lactams are used as prodrugs for cancer therapy (Xing et al., 2008). These prodrugs target cancerous cells specifically. On the other hand, N-methylthiolated β-lactams demonstrate their anticancer activity by inducing apoptosis in them (Kuhn et al., 2004). Unlike necrosis, which involves the death of cells caused by acute injury, apoptosis refers to a natural phenomenon involving programmed cell death (Lederman, 2004). This regulated cell death is very important for proper growth, development, and homeostasis in living organisms because it eradicates cells that are not required by the body. Apoptosis induction has proven helpful in controlling cancer.
Tetracyclines
Prion diseases, including spongiform encephalopathy in cattle, scrapie in sheep, and fatal insomnia, Gerstmann–Sträussler–Scheinker disease and Creutzfeldt–Jakob disease in humans, can be treated with tetracyclines (Forloni et al., 2002) because they make aggregates of prions vulnerable to proteolytic degradation (Borman, 2002). These neurodegenerative diseases of animals and humans are communicable and hence also classified as transmissible spongiform encephalopathies (TSEs). Moreover, there is not any effective therapy for these diseases. The disease progresses quickly and proves fatal in all cases. Unlike other antiprions, likeanthracyclines, polyene antibiotics, polyanions, quinacrine, tetrapyrroles, polyamines, Congo red, chlorpromazine, and certain peptidesandantibodies, tetracylines are non-toxic and can cross the blood-brainbarrier.
Aminoglycosides and Macrolides
Antibacterial aminoglycosides are one of the most renowned antibiotics. Neomycin, an aminoglycoside, was used as a broad-spectrum antibiotic between 1960 and 1980. Later, Pharmacia/Upjohn donated it to the World Health Organization. Its efficacy against leishmaniasis has been demonstrated by the Gates Foundation in collaboration with the World Health Organization. Unexpectedly, neomycin, which is an antibacterial agent, has demonstrated anti-angiogenic activity in the endothelial cells of humans (Hu, 1998). The process of the formation of new blood vessels from extant ones is known as angiogenesis. This process is involved in the development and advancement of several disorders such as diabetic retinopathy and cancer. It is essentially required for the development of malignant tumours.
Pigmented Antibiotics
Several gram-negative and gram-positive bacteria, including species of Pseudomonas, Alteromonas, Actinomadura, Vibrio, Pseudoalteromonas, Saccharopolyspora, Streptoverticillium, Serratia, Streptomyces and Hahella, produce pigmented antibiotics like prodigines, which refer to red tripyrroles with a pyrrolylpyrromethane skeleton. Aside from their antibacterial activity, they can also demonstrate antiprotozoal, antifungal, immunosuppressive and anticancer activities (Williamson et al., 2006). Owing to their ability to induce apoptosis in cancerous cells, they are under evaluation as anticancer agents. Over thirty genes take part in the production of prodigines (Péreg-Tomás et al., 2003).
The S. marcescensproducesprodigiosin, which demonstrates antitumour activity through stopping cell cycles and inducing cell death (Zhang et al., 2005) in cells affected by pancreatic, gastric, colorectal, and hematopoietic cancers. Pancreatic cancer is the fourth major type of cancer in the United States; 30,000 individuals die from pancreatic cancer every year. Only 3–5% of pancreatic cancer patients survive for five years; however, the median survival rate is less than half a year. Immunosuppressive activity of this antibiotic has also been reported (Pandey et al., 2007).
1-1-2 Antitumour Agents
Besides antibiotics that demonstrate antitumour activity, several other natural products can be used in this clinical area.
Taxol
One of the most prominent antitumour agents currently in use, particularly for breast cancer, is Pacitaxel (Taxol), which is essentially an alkaloid extracted from taxusbrevifolia (Pacific Yew tree) (Cissé& Mucke, 2009). Taxol inhibits microtubular depolymerisation during cell division of cancerous cells. Taxomycesandreanae, an endophytic fungus, was discovered in this tree in 1993. This fungus is able to produce taxol. Later, taxol-producing fungal endophytes were discovered in different higher plants. Taxol inhibits microtubular depolymerisation in fungi just as it does in cancerous cells. For this reason, it can be stated that taxol behaves as an antifungal agent in natural environments.
Epothilones
These macrolides are synthesised by myxobacteriumSorangiumcellulosum and have a ring structure with 16 members. They are also capable of demonstrating antifungal and anticancer activities. Moreover, the mechanism involved in their activity against breast cancer is the same as in the case of taxol; the only difference is that it binds to a different site on β-tubulin to demonstrate its activity. It is worth mentioning that epothilones demonstrate activity against tumours that are resistant to taxol. They were initially developed as antifungal agents, but their antitumour potential was acknowledged later. The FDA has approved an epothilone called ixabepilone. Epothilones bind with microtubules, which play an important role in cell division. They mediate a tumour’s cell cycle arrest at the GM2/M phase by inhibiting the disassembly of microtubules and initiating apoptosis (Goodin, 2008).
Bryostatins
Bryostatinsare synthesised by Endobugulasertula, a gamma proteobacterialsymbiont. They are typically found in Bugulaneritina, which is a bryozoan. Bryostatins have proven to be cytotoxic and are being studied for application in anticancer therapy. Phase I and II clinical trials are in progress. Reports indicate that bryostatin-1 prevents cell division, causes differentiation, and facilitates programmed cell death in several haematological and solid tumour cell lines. As per the findings of preclinical studies, the effect of chemotherapy is augmented by bryostatin-1 (Kortmansky & Schwartz, 2003). Owing to their memory-enhancing activity, bryostatins can be used for treating Alzheimer’s disease. As per the results of studies on snail Hermissenda, they are effective for addressing both the cause and the symptoms of neurodegeneration. The Blanchette Rockefeller Neurosciences Institute (BRNI) has received approval from the FDA to carry out phase II trials of bryostatins for treating patients with Alzheimer’s disease (BRNI Press release; April 22, 2009).
1-1-3 Immunosuppressants
Organ transplantation depends largely on efficient immunosuppressants, which are microbial in origin. Several immunosuppressants, like cyclosporin A, tacrolimus (FK506) and rapamycin (sirolimus), were initially identified as antifungal agents (High, 1994). According to Cruz et al. (2000), immunosuppressive and antifungal activities can be demonstrated contrarily, which means high antifungal activity is seen with low immunosuppressing activity. Fungal growth is inhibited by a non-immunosuppressive analogue of tacrolimus. Similarly, the growth of Cryptococcus neoformans is inhibited by two analogues of cyclosporine, which are non-immunosuppressive. These analogues bind with cyclophilinA and prevent calcineurin activity in fungi.
Several microbial metabolites are also capable of lowering serum cholesterol levels in humans, especially the concentration of the LDL, thereby decreasing the risk of cardiovascular diseases. In this regard, fungal fermentation products like compactin (mevastatin), produced by Penicilliumspecies, and lovastatin (mevacor, monocolin K, mevinolin) from Monascusruber and Aspergillusterreus have proven to be highly active. These agents inhibit the synthesis of all substances of the mammalian polyisoprenoid pathway and strongly prevent activity of3-hydroxy-3-methylglutaryl-CoA reductase (Pandey et al., 2007).
During the last six decades, secondary metabolites have been largely used as antibiotics; however, other unpredicted activities of identified metabolites have cometo light more recently. For this reason, secondary metabolites are considered useful for a multitude of diseases, including cancer, AIDS, parasitic diseases, and cardiovascular diseases. Considering the low levels of toxicity to humans demonstrated by these secondary metabolites, it is highly anticipated that several currently incurable diseases could be treated by exploiting the remarkable activities of microbial secondary metabolites. One can hope for another miracle to benefit the medical field just like the miracle of the discovery of penicillin, which revolutionised the approach to treating several healthcare problems in the 1940s (Demain, 2013). It is highly likely that novel derivatives can be synthesised from extant substances and that the technology of molecular genetics could allow the efficient manipulation of microbial strains for producing new compounds for different clinical applications.
2 Activation of Secondary Metabolites
Streptomyces, mycobacteria, and fungi genome sequencinghave revealed that, even though there are genes in each strain that encode enzymes to produce an excess of possible secondary metabolites, during fermentation only a fraction of that amount is expressed. Therefore, significance has been developed in the establishment of these cryptic pathways. The rate of the detection of valuable compounds declined after 1970, even though there was a constant need for innovative and stronger drugs to battle the emerging infectious diseases and cancers. The major reason for this approach to gaining success was to determine methods to stimulate or increase the expression of pathways which are hidden or poorly expressed and to supply matter for structural clarification and biological testing.
Therefore, the enhanced expression of renowned and highly productive secondary metabolites through fermentation methods for the improvement of rapid strains will lead to new and enigmatic secondary metabolite pathway expressions (Baltz, 2011). With this method, certain early stage detection issues will be solved: (a) stimulating cryptic biosynthetic gene clusters at a certain level of expression [waking the sleeping genes] and (b) achieving adequate material to distinguish, chemically and biologically, the rapid increase in product yields [early stage yield enhancement]. Ribosome engineering, metabolic remodelling, rare earth elements, and cell-to-cell interaction are a few potential methods being used for the stimulation of enigmatic biosynthetic gene clusters in Streptomyces. The detection of new and/or inadequately understood antibiotics, including piperidamycin and stambomycin, is done using these approaches, and have useful features for an extensive variety of actinomycetes and possible saleability as a result of increased throughput.
Ribosome Engineering
The stimulation of silent genes can effectively be done with ribosome engineering. For example, 1,068 actinomycetes were isolated from a soil sample, from which a portion of both Streptomyces and non-Streptomyces were considered antibiotic non-producers, among which 43 and 6 % of these non-producing strains had the capability to produce antibacterials against Staphylococcus aureus once they underwent a procedure of selection generating unprompted mutations of rpsL or rpoB (Hosaka et al., 2009). When the strain Streptomyces mauvecolour 631689, which has antibacterial inactivity in all mediums, was assessed, it produced two mutations of rpoB (H437D or H437L); rpsL (K88R), two rpoB mutations (H437L), and a family of antibiotics, such as piperidamycins, which produced gentamicin-resistant (GenR) mutations (Fig. 1). The rpoB H437D or H437L mutations stimulated silent genes owing to improved attraction for silent gene promoters of mutant RNAP (Hosaka et al., 2009).
Antibiotic resistance can be achieved by merely choosing mutants on drug-containing plates with various antibiotics, such as streptomycin, aiming for the ribosome, which mutates despite a fraction of the mutants being affected in membrane permeability. Likewise, by growing bacteria on plates with rifampicin that aims for the RNAP, RNAP mutants can be created. Various triumphant examples of ribosome engineering have been produced through this possibility, such as the improved manufacturing of secondary metabolites and enzymes, along with better forbearance towards toxic compounds, including 4-hydroxybenzoate (Table 1). The improvement in the production of secondary metabolites, such as polyketides, macrolides, amino glycosides, and nucleosides, in a variety of structural classes was effective using ribosome engineering.
MetabolismRemodelling
Even though the stimulation of biosynthetic genes is significant at the transcriptional level for using beneficial metabolites, it is also by modulating that it causes a perturbation of biosynthesis. The provision of precursors may also be a hopeful approach to improve a cell’s ability to manufacture secondary metabolites, in a method called metabolic engineering (Olano et al., 2008). The modulation of the biosynthesis of fatty acid by employing minute molecules is another technique for improving a secondary metabolite's production. For example, when extensive (>30,000) minute molecules were screened, 19 compounds were detected as candidates in S. coelicolor that might ‘remodel’the production of actinorhodin (Craney et al., 2012). When four of these 19 molecules underwent further assessment, it revealed 2, 3, 4, and 5antibiotic remodelling compounds (ARC) where (1) ARC2 low concentrations improved the production of actinorhodin, (2) and in other actinomycetes the ARC2 effect can be seen, suggestive of preservation of the molecular target and method of the ARC2 in various actinomycetes (3). Moreover, mostly the similarity of the structure in active molecules to triclosan is found, which is not only an acknowledged antimicrobial agent but also an inhibitor of the synthesis of fatty acid. Hence, the effects of the ARC2 involve the inhibition of an enzyme that in fatty acid biosynthesis catalyses the concluding and rate-limiting step: FabI enoyl reductase activity. Since fatty acid and the synthesis of polyketide share both the precursors acetyl-CoA and malonyl-CoA, fatty acids incomplete inhibition could employ those acyl-CoAs mostly to the biosynthesis of polyketide.
Not only was the production of known antibiotics improved, but the ARC2 series of molecules also stimulated the yield of undisclosed compounds in Streptomyces peuceticus which could not be identified without the ARC2 (Craney et al., 2012). It is possible that a low expression level is present in the genes encoding the enzymes to produce these compounds (i.e., nearly but not entirely silent) so that strengthening due to a proficient supply of substrate to the biosynthetic process made its recognition possible. On the other hand, another unidentified mechanism could also be involved. Theoretically, by combination with the transcription-associated approaches, this approach may be improved (Ochi & Okamoto, 2012). For other classes of antibiotics, in order to gain access to the complete spectrum of secondary metabolites, it may be most effective to formulate identical methods, such as amino glycosides and non-ribosomal peptides.
Cell-to-Cell Interactions
In the third method, the normal ecology where microbes live together in complex microbial communities is imitated. The features of these micro-habitats involve the struggle for inadequate resources and opposition, which is beneficial for various defence mechanisms that are mostly dependent on bioactive secondary metabolites [30]. Within these ecological frames, bacterial and fungal antibiotic manufacturing means that microbes that are chemically protected will be chosen despite ambiguity [31–35] [36–38]. In a laboratory scale, co-cultivation of two or more diverse microbes try to imitate this situation. Generally, three main strategies were followed: (a) diverse fungi co-cultivation; (b) bacteria and fungi co-cultivation and (c) diverse bacteria co-cultivation.
For example, the biosynthesis of Streptomyces spp with its hidden natural goods could be affected by mycolic acid-containing bacteria. By co-culture and mycolic acid containing the bacteria Tsukamurella pulmonis, the manufacture of red colour by S. lividans could be stimulated. Significantly, in 88% of the Streptomyces strains, a co-culture with T. Pulmonis enhanced or inhibited the natural product biosynthesis when isolated from soil. In 37% of strains, the manufacture of novel secondary metabolites was identified, while in 55% of strains increased metabolite production was seen. In 87 and 90% of the Streptomyces strains, the remaining mycolic acid-containing bacteria, Rhodococcus erythropolis and Corynebacterium glutamicum, enhanced or inhibited biosynthesis.In 32 and 24% of strains the production of new secondary metabolites was identified. The latest antibiotic, alchivemycin, was recognised by the T. pulmonis and Streptomyces endus co-culture. No effect on antibiotic manufacturing was observed by adding mycolic acid into a pure S. lividans culture's medium, which was indicative of the fact that the secondary metabolism in Streptomyces mycolic acid was effected by the bacteria which remain restricted to the boundary of the inducer, producing undeviating cell-to-cell contact within the two strains of bacteria (Onaka et al.,2011). For stimulating the manufacture of cryptic antibiotics, this technique is also scalable, as in the pure culture of an actinomycete; a mycolic acid-containing bacterium is added.
Rare Earth Elements
Even though rare earth elements (REEs) are quite useful for the chemical industry, there is a lack of information regarding their biological effects on living cells. However, lately these elements have been occupied with the overproduction of antibiotics and with the stimulation of bacteria's silent or poorly expressed genes. There are 17 elements in REEs, including scandium, yttrium, and the lanthanides (i.e., the 15 elements from lanthanum (La) to lutetium on the periodic table). The antibiotic production was enhanced 2–25-fold when a decreased (10–100 μM) scandium (Sc) concentration was added to S. coelicolor (an actinorhodin producer), Streptomyces antibioticus (an actinomycin producer), and S. griseus (a streptomycin producer) (Kawai et al., 2007). At the level of transcription of pathway-specific positive regulatory genes, the effect of Sc was applied, as verified by S. coelicolor and the marked-up regulation of act II-ORF4. Particularly, in Streptomyces, the REEs were efficient in stimulating silent and poorly expressed secondary metabolite biosynthetic genes. The expression of nine genes improved 2.5- to 12-fold owing to the addition of Sc’s or La's medium to decreased concentrations of S. coelicolor's nine secondary metabolite– biosynthetic gene clusters (Tanaka et al., 2010). It was proved by an ethyl acetate-extractable metabolite's HPLC analysis that...