Dipesh Dhakal

An Insight into the “-Omics” Based Engineering of Streptomycetes for Secondary Metabolite Overproduction

Microorganisms produce a range of chemical substances representing a vast diversity of fascinating molecular architectures not available in any other system. Among them, Streptomyces are frequently used to produce useful enzymes and a wide variety of secondary metabolites with potential biological activities. Streptomyces are preferred over other microorganisms for producing more than half of the clinically useful naturally originating pharmaceuticals. However, these compounds are usually produced in very low amounts (or not at all) under typical laboratory conditions. Despite the superiority of Streptomyces, they still lack well documented genetic information and a large number of in-depth molecular biological tools for strain improvement. Previous attempts to produce high yielding strains required selection of the genetic material through classical mutagenesis for commercial production of secondary metabolites, optimizing culture conditions, and random selection. However, a profound effect on the strategy for strain development has occurred with the recent advancement of whole-genome sequencing, systems biology, and genetic engineering. In this review, we demonstrate a few of the major issues related to the potential of “-omics” technology (genomics, transcriptomics, proteomics, and metabolomics) for improving streptomycetes as an intelligent chemical factory for enhancing the production of useful bioactive compounds.

Ag-BaMoO4: Er3+/Yb3+ photocatalyst for antibacterial application

Silver loaded and Er3+/Yb3+ doped BaMoO4 octahedron microcrystals were fabricated by microwave hydrothermal process. The synthesized samples were characterized by X-ray diffraction (XRD), Field emission scanning electron microscopy (FESEM), Energy dispersive X-ray spectroscopy (EDS), and Ultraviolet-visible diffuse reflection spectroscopy (UV-Vis DRS). The antibacterial application of samples were investigated by visible light irradiation and disk-diffusion method towards representative Gram-negative pathogen (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive pathogen (methicillin resistant Staphylococcus aureus). The complete inactivation of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were observed by Ag-BaMoO4: Er3+/Yb3+ photocatalyst within 1h, 4h, and 5h, respectively, under visible light irradiation. The high killing percentage and superior zone of inhibition revealed the excellent antibacterial performance. The FESEM images were used to visualize the morphology with the extent of damage in the phospholipid layer present in the cell membrane of bacteria. The synergistic effect of loaded silver particles and doped Er3+/Yb3+ ions in BaMoO4 contributed for efficient antibacterial performance in visible light as well as in the dark. The excellent antibacterial performance of Ag-BaMoO4: Er3+/Yb3+ photocatalyst makes the material suitable for smart weapon for multidrug-resistant microorganisms and disinfectants in biomedical application.

Enhanced Production of Nargenicin A 1 and Generation of Novel Glycosylated Derivatives

Nargenicin A1, an antibacterial polyketide macrolide produced by Nocardia sp. CS682, was enhanced by increasing the pool of precursors using different sources. Furthermore, by using engineered strain Nocardia sp. ACC18 and supplementation of glucose and glycerol, enhancement was ~7.1 fold in comparison to Nocardia sp. CS682 without supplementation of any precursors. The overproduced compound was validated by mass spectrometry and nuclear magnetic resonance analyses. The novel glycosylated derivatives of purified nargenicin A1 were generated by efficient one-pot reaction systems in which the syntheses of uridine diphosphate (UDP)-α-D-glucose and UDP-α-D-2-deoxyglucose were modified and combined with glycosyltransferase (GT) from Bacillus licheniformis. Nargenicin A1 11-O-β- D-glucopyranoside, nargenicin A1 18-O-β-D-glucopyranoside, nargenicin A111 18-O-β-D- diglucopyranoside, and nargenicin 11-O-β-D-2-deoxyglucopyranoside were generated. Nargenicin A1 11-O-β-D-glucopyranoside was structurally elucidated by ultra-high performance liquid chromatography-photodiode array (UPLC-PDA) conjugated with high-resolution quantitative time-of-flight-electrospray ionization mass spectroscopy (HR-QTOF ESI-MS/MS), supported by one- and two-dimensional nuclear magnetic resonance studies, whereas other nargenicin A1 glycosides were characterized by UPLC-PDA and HR-QTOF ESI-MS/MS analyses. The overall conversion studies indicated that the one-pot synthesis system is a highly efficient strategy for production of glycosylated derivatives of compounds like macrolides as well. Furthermore, assessment of solubility indicated that there was enhanced solubility in the case of glycoside, although a substantial increase in activity was not observed.

Herboxidiene biosynthesis, production, and structural modifications: prospect for hybrids with related polyketide.

Herboxidiene is a polyketide with a diverse range of activities, including herbicidal, anti-cholesterol, and pre-mRNA splicing inhibitory effects. Thus, production of the compound on the industrial scale is in high demand, and various rational metabolic engineering approaches have been employed to enhance the yield. Directing the precursors and cofactors pool toward the production of polyketide compounds provides a rationale for developing a good host for polyketide production. Due to multiple promising biological activities, the production of a number of herboxidiene derivatives has been attempted in recent years in a search for the key to improve its potency and to introduce new activities. Structural diversification through combinatorial biosynthesis was attempted, utilizing the heterologous expression of substrate-flexible glucosyltransferase (GT) and cytochrome P450 in Streptomyces chromofuscus to generate structurally and functionally diverse derivatives of herboxidiene. The successful attempt confirmed that the strain was amenable to heterologous expression of foreign polyketide synthase (PKS) or post-PKS modification genes, providing the foundation for generating novel or hybrid polyketides.

Commentary: Toward a new focus in antibiotic and drug discovery from the Streptomyces arsenal

Infectious diseases caused by bacteria, particularly those gaining drug-resistance, are among the top causes of mortality in the world (Overbye and Barrett, 2005). The mechanisms of multidrug efflux systems, enzymatic modification and inactivation of drug molecules have enabled the resistant bacteria to reduce the potency of common antibiotics (van Hoek et al., 2011; Lin et al., 2015). Thus, the discovery of new antimicrobials and expansion of utility of existing antibiotics by overproduction or targeted modification is crucial to combat the ever-increasing antimicrobial resistance (Dhakal et al., 2015; Lin et al., 2015). Streptomyces is the major sources of natural products including effective antimicrobials (Chaudhary et al., 2013). Antoraz et al. (2015) have summarized advances on drug discovery from Streptomyces arsenal, illustrating different approaches for elicitation of antimicrobials by nutritional and hormonal signals or production of useful antimicrobials by co-culture and in situ culture technologies. The authors have flash-lighted on utility of different synthetic biological and system biological based metabolic engineering techniques for harnessing the biosynthetic capabilities of Streptomyces. It can give rise to superfluous possibilities for antibiotic discovery by using these precise genetic engineering techniques. Genome mining has revealed that Streptomyces can harbor numerous active or cryptic biosynthetic gene clusters encoding for diverse compounds including novel antimicrobials (Chaudhary et al., 2013). Fundamentally, identification of novel antimicrobials can be achieved by high throughput screening techniques, either (a) compound specific screening or (b) organism specific screening (Figure ​(Figure1A).1A). In the first approach, the structurally characterized molecule is tested against different pathogenic organisms. In next approach, the pathogenic organism is screened against different putative compounds and effects are assessed. Furthermore, bioinformatics tools assisting on studies rendering to structure-activity relationships (SAR) or quantitative structure activity relationship (QSAR) can assist in developing the effective drug molecules from microbial resources. Figure 1 Integrated approach for exploiting Streptomyces arsenal to combat the antibiotic resistance. (A) Extensive screening strategies for identifying lead antimicrobials: (a) Compound specific screening (b) Pathogenic organism specific screening. (B) Production ... Antoraz et al. (2015) indicated that the application of bioinformatics and “-omic” based engineering have important contribution in drug discovery. The key focus of these technologies is production and overproduction of important molecules or their structural/functional diversification for pharmaceutical value. The precise knowledge of biochemistry of secondary metabolites production supported with advanced system biological and integrated “-omic” techniques provide rational strategies for production and overproduction of targeted compounds in native/heterologous hosts (Gomez-Escribano and Bibb, 2011; Chaudhary et al., 2013; Hwang et al., 2014). The production profile can be modulated by interrogation with media optimization or medium component selection strategies (Jose et al., 2013). The application of synthetic biological tools such as construction of a complete genetic circuit or rewiring the transcriptional or post-transcriptional regulation, play significant role in production and overproduction strategies (Medema et al., 2011; Wohlleben et al., 2012; Weber et al., 2015) (Figure ​(Figure1B).1B). The optimization of bioprocessing and fermentation technology may help in harnessing the maximum yield of the desired compound. In addition, for compounds that are cryptic and not amenable to production from native host, a stable chassis can be utilized as heterologous host. The host can in turn be rationally engineered for maximum production using synthetic biological platform. Antoraz et al. (2015) illustrated that diverse synthetic biological approaches can be efficient method for precise modifications to attain a new molecules with better antimicrobial activities. These synthetic biological techniques in synergy with system biological impetus can be employed in two categories (a) living cell based (in vivo) or (b) chemical reagents based (in vitro) techniques (Figure ​(Figure1C).1C). The generation of natural product-derived libraries is assisted by synthetic chemistry, where novel structural diversity is generated by precursor directed biosynthesis or mutasynthesis or semi-synthesis approaches (Kirschning et al., 2007; Kennedy, 2008). In precursor-directed biosynthesis, the synthetic analogs of some scaffold moieties are fed and incorporated during metabolite production in wild type strains generating novel analogs. Mutasynthesis is a refined version of precursor directed biogenesis, where the producer strain is engineered curtailing the competitive pathways, yielding maximum production of desired compounds. In semi-synthesis, the designed chemical moiety is attached to the natural product by specific chemical reactions. In another approach termed “combinatorial biosynthesis,” the substrate promiscuity of natural or engineered enzymes and modulated pathways is utilized to produce “unnatural products” with potential pharmaceutical value (Sun et al., 2015). This approach can be employed by altering the precursors or enzyme level modifications through mutations. The domain alterations or complete pathway level recombination is another approach for combinatorial biosynthesis. In addition, the whole cell based in vivo biotransformation or reaction tube based in vitro catalysis contribute for generation of a repertoire of novel derivatives of existing antimicrobials (Figure ​(Figure1C1C). It seems we are running out our defensive options due to alarming increase in the drug resistant bug in contrast to decrease in the introduction of new antimicrobials. There is skepticism regarding potential post-antibiotic era when common infections may lead to mortality because of dwindling antibiotic arsenal. Antoraz et al. (2015) have illustrated the profound potential of S. arsenal as prolific source of antibiotics and drug discovery. Moreover, there are sufficiently availed details of biochemistry and physiology behind the pathogenicity of resistant bacteria as well as the biosynthetic ability of antimicrobial producers. Hence, it can be hoped that more efficacious antimicrobials can be developed from Streptomyces on the bases of all the available knowledge resources and technologies.

Marine Rare Actinobacteria: Isolation, Characterization, and Strategies for Harnessing Bioactive Compounds

Actinobacteria are prolific producers of thousands of biologically active natural compounds with diverse activities. More than half of these bioactive compounds have been isolated from members belonging to actinobacteria. Recently, rare actinobacteria existing at different environmental settings such as high altitudes, volcanic areas, and marine environment have attracted attention. It has been speculated that physiological or biochemical pressures under such harsh environmental conditions can lead to the production of diversified natural compounds. Hence, marine environment has been focused for the discovery of novel natural products with biological potency. Many novel and promising bioactive compounds with versatile medicinal, industrial, or agricultural uses have been isolated and characterized. The natural compounds cannot be directly used as drug or other purposes, so they are structurally modified and diversified to ameliorate their biological or chemical properties. Versatile synthetic biological tools, metabolic engineering techniques, and chemical synthesis platform can be used to assist such structural modification. This review summarizes the latest studies on marine rare actinobacteria and their natural products with focus on recent approaches for structural and functional diversification of such microbial chemicals for attaining better applications.

Structural modification of herboxidiene by substrate-flexible cytochrome P450 and glycosyltransferase

Herboxidiene is a natural product produced by Streptomyces chromofuscus exhibiting herbicidal activity as well as antitumor properties. Using different substrate-flexible cytochrome P450s and glycosyltransferase, different novel derivatives of herboxidiene were generated with structural modifications by hydroxylation or epoxidation or conjugation with a glucose moiety. Moreover, two isomers of herboxidiene containing extra tetrahydrofuran or tetrahydropyran moiety in addition to the existing tetrahydropyran moiety were characterized. The hydroxylated products for both of these compounds were also isolated and characterized from S. chromofuscus PikC harboring pikC from the pikromycin gene cluster of Streptomyces venezuelae and S. chromofuscus EryF harboring eryF from the erythromycin gene cluster of Saccharopolyspora erythraea. The compounds generated were characterized by high-resolution quadrupole-time-of-flight electrospray ionization mass spectrometry (HR-QTOF-ESI/MS) and 1H- and 13C-nuclear magnetic resonance (NMR) analyses. The evaluation of antibacterial activity against three Gram-positive bacteria, Micrococcus luteus, Bacillus subtilis, and Staphylococcus aureus, indicated that modification resulted in a transition from anticancer to antibacterial potency.

Insight Into Malachite Green Degradation, Mechanism and Pathways by Morphology-Tuned α-NiMoO4 Photocatalyst

The microwave hydrothermal process was used for the synthesis of various morphologies of α‐NiMoO4 by simply adjusting the pH during experimental conditions. The effect of morphology/size on the photocatalytic performances for degradation of malachite green (MG) has been investigated under UV‐Vis/visible light irradiation. Nanorod morphology has strong tendency to degrade (88.18%) the MG as compared to spherical quantum‐sized (57.65%) and layered square microsheet (37.98%) under UV‐Vis irradiation in 180 min. The active species trapping experiment revealed that active species (OH•, O2•− and h+) play a crucial role for MG degradation. The high BET surface area, greater amount of oxygen defect and efficient separation of electron–hole pair are responsible for MG degradation. About seventeen types of organic fragments of MG were confirmed by high resolution‐quadruple time of flight electrospray ionization mass spectroscopy (HR‐QTOF ESI/MS) technique on the basis of retention time and molecular masses. Degradation mechanism and pathways were proposed that follow the demethylation, nitration, decarboxylation, hydrolysis, decarboxylation and oxidation reaction. The reduction of total organic carbon revealed the mineralization of MG during photocatalytic degradation process. Therefore, this article represents the investigation of MG degradation by various morphology of α‐NiMoO4 and detailed degradation mechanism and pathways.

Substrate Scope of O -Methyltransferase from Streptomyces peucetius for Biosynthesis of Diverse Natural Products Methoxides

Methylation is a common post-modification reaction that is observed during the biosynthesis of secondary metabolites produced by plants and microorganisms. Based on the sequence information from Streptomyces peucetius ATCC27952, a putative O-methyltransferase (OMT) gene SpOMT7740 was polymerase chain reaction amplified and cloned into E. coli BL21 (DE3) host to test the substrate promiscuity and conduct functional characterization. In vitro and in vivo reaction assays were carried out over various classes of substrates: flavonoids (flavonol, flavones, and isoflavonoid), chalcones, anthraquinones, anthracyclines, and sterol molecules, and the applications in synthesizing diverse classes of O-methoxy natural products were also illustrated. SpOMT7740 catalyzed the O-methylation reaction to form various natural and non-natural O-methoxides, includes 7-hydroxy-8-O-methoxy flavone, 3-O-methoxy flavone, three mono-, di-, and tri-O-methoxy genistein, mono-O-methoxy phloretin, mono-O-methoxy luteolin, 3-O-methoxy β-sitosterol, and O-methoxy anthraquinones (emodin and aloe emodin) and O-methoxy anthracycline (daunorubicin) exhibiting diverse substrate flexibility. Daunorubicin is a native secondary metabolite of S. peucetius. Among the compounds tested, 7,8-dihydroxyflavone was the best substrate for bioconversion to 7-hydroxy-8-O-methoxy flavone, and it was structurally elucidated. This enzyme showed a flexible catalysis over the given ranges of temperature, pH, and divalent cationic conditions for O-methylation.

Coalition of Biology and Chemistry for Ameliorating Antimicrobial Drug Discovery

Natural products (NPs) are superior starting point for the major antimicrobials used in clinical trials (Newman and Cragg, 2012; Butler et al., 2014). Such antimicrobial NPs can be obtained from different microorganisms (Polpass and Jebakumar, 2013) and plants (Atanasov et al., 2015). They can be broadly classified as (i) native NPs, (ii) derivatives of NPs, or (iii) synthetic products based on structures of NPs (Demain and Sanchez, 2009). While NPs exhibit a wide range of pharmacophores and a high degree of stereochemistry (Harvey et al., 2015), novel NPs with better activities still need to be developed. Versatile biological knowledge based synthetic-biology approaches, system-biology guided metabolic engineering techniques, enzymatic modifications, and synthetic chemistry methods can be utilized to maximize the benefit of NPs from the source organism (Dhakal et al., 2015, 2016). Thus, the optimum application of NPs can only be improved with considerable effort based on precise screening, higher production, and desirable structural diversification (Dhakal and Sohng, 2015).