Metabolic pathway engineering of actinomycetes for novel antibiotics discovery

Microbes harbouring a profound wealth of chemical space have instigated tremendous attention for developing crucial therapeutic drugs. The genes encoding the enzymes responsible for synthesizing these specialized metabolites are often organized in so-called biosynthetic gene clusters (BGCs). While these clusters are potentially capable of producing novel drug candidate, most of them remain dormant in natural environmental conditions (or settings). Microorganisms seldom synthesize substantial quantities of the desired molecules in natural settings. Harnessing the dormant compound production requires careful optimization of the host cellular machinery, which can be accomplished by thorough engineering of silent biosynthetic pathways. My target, the genus Streptomyces is endowed with tremendous abilities to secrete a diverse array of metabolites. Besides, insilico analysis of their genomic sequences reveals enormous potential to generate novel metabolites not biosynthesized in natural environmental settings. Realizing such bountiful resources, I attempt to unveil Streptomyces’ true potential to generate novel metabolites by using various approaches. In the present dissertation, various novel approaches have allowed me to unveil novel specialized metabolites encoded by otherwise silent biosynthetic clusters. For this, (i) I developed single cell mutant selection (SCMS) platform, where mutants harboring a silent promoter are probed with a double reporter system using classical mutagenesis techniques. Mutants were sorted using FACS based on expression of reporter genes and mutants generated a novel metabolite with a distinct chemical scaffold, referred to as mutaxanthene. (ii) Next approach involved binary physical interaction studies between dead yeast and Streptomyces where the contact induced production of prodigiosin. My studies identified a master-regulator, namely mbkZ, for its regulatory roles in prodigiosin production in different hosts (S. coelicolor and Streptomyces sp. MBK6). (iii) Third approach exploited CRISPR/Cas9 system to unveil the functional role of sdmA within the showdomycin biosynthetic pathway. (iv) Final approach revealed the characterization of new bacterial lineage (Streptomonospora sp. PA3) isolated from the high-salt environment, which helped me to isolate and identify a novel polyketide persiamycin A. Using these different approaches allowed me to unveil the secret knowledge sealed within biosynthetic pathways of the studied organisms. In a nutshell, adopting these techniques has helped me discover and characterize novel metabolites. I believe these strategies may aid in the fight against antimicrobial resistance and speed up the drug discovery process. Furthermore, this dissertation has not only implications for future engineering of Streptomyces to increase metabolites production, but it also illustrates a SCMS state-of-art approach for generating novel therapeutic leads.Aktinomykeettien aineenvaihduntareittien muokkaus uusien antibioottien löytämiseksi Mikrobien tuottamat monimuotoiset luonnonyhdisteet ovat herättäneet suurta kiinnostusta lääkekehityksessä. Näiden erikoistuneiden metaboliittien tuotannosta vastaavia entsyymejä koodaavat geenit ovat yleensä järjestyneet niin sanottuiksi biosynteettisiksi geeniryppäiksi (BGC, engl. biosynthetic gene cluster). Nämä geeniryppäät saattavat tuottaa vielä tuntemattomia metaboliitteja, mutta yleensä ne ovat hiljaisia luonnollisessa ympäristössä. Yleensä mikro-organismit eivät tuota haluttua yhdistettä merkittäviä määriä luonnollisissa olosuhteissa. Näiden hiljaisten yhdisteiden hyödyntäminen lääkeaineiden kehityksessä vaatii solukoneiston huolellista optimointia, mikä voidaan saavuttaa muokkaamalla hiljaisia biosynteesireittejä. Kohdeorganismimme Streptomyces -bakteerit kykenevät tuottamaan lukuisia erilaisia sekundäärimetaboliitteja, jotka ovat kemialliselta rakenteeltaan erittäin vaihtelevia. Tämän lisäksi genomisekvenssien analyysi on paljastanut lukuisia lupaavia hiljaisia geeniklustereita, jotka saattaisivat aktivoituina tuottaa aikaisemmin tuntemattomia yhdisteitä. Tämän työn tarkoitus on käyttää useita erilaisia tekniikoita tämän hiljaisen biosynteettisen potentiaalin valjastamiseen. Väitöskirjatyössä käytin useita uusia menetelmiä hiljaisten geeniryppäiden koodaamien uusien yhdisteiden tuottamiseksi. Tätä tarkoitusta varten (i) kehitin yksisolujen mutanttivalinta (SCMS, engl. single cell mutant selection) alusta – menetelmän, missä hiljaisen geeniryppään aktiivisuutta seurataan tuplareportterisysteemillä. Seuloin menetelmällä mutanttikirjastoja reportterigeenien ilmenemisen perusteella FACS –laitteistoilla ja tuotin erityisen kemiallisen rakenteen omaavia mutaxanthene -yhdisteitä. (ii) Seuraavaksi tutkin hiivojen ja streptomykeettien fyysisen vuorovaikutuksen vaikutusta prodigiosiini –yhdisteen tuottoon. Tutkimukseni paljasti säätelygeeni mbkZ:n roolin prodigiosiinien tuotossa kahdessa eri isäntäkannassa (S. coelicolor ja Streptomyces sp. MBK6). iii) Kolmannessa menetelmässä käytin CRISPR/Cas9-menetelmää selvittääkseni sdmA geenin roolin showdomysiinin biosynteesireitillä. iv) Viimeisenä menetelmänä eristin uuden halofiilisen bakteerikannan (Streptomonospora sp. PA3) korkean suolapitoisuuden kasvuympäristöstä meren pohjasta. Kannasta eristettiin uusi persiamysiini A polyketidi. Näiden erilaisten lähestymistapojen avulla pystyin paljastamaan tutkittujen organismien biosynteettisten reittien sisälle suljetut salaisuudet. Lyhyesti, olemme pystyneet löytämään ja karakterisoimaan uusia metaboliitteja käyttämiemme tekniikoiden avulla. Uskomme, että käyttämämme strategiat auttavat kamppailuissa antibioottiresistenssiä vastaan ja nopeuttamaan uusien lääkkeiden löytämistä. Tämän lisäksi tutkielman tulokset sekä auttavat sekundaarimetabolliittien tuotannon tehostamista tulevaisuudessa streptomykeettejä muokkaamalla, että havainnollistaa SCMS –menetelmän käyttökelpoisuuden uusien terapeuttisten yhdisteiden tuotossa

Silicon and nitrate differentially modulate the symbiotic performances of healthy and virus-infected Bradyrhizobium-nodulated cowpea (Vigna unguiculata), Yardlong Bean (V. unguiculata subsp. sesquipedalis) and Mung Bean (V. radiata)

The effects of 2 mM silicon (Si) and 10 mM KNO3 (N)—prime signals for plant resistanceto pathogens—were analyzed in healthy and Cowpea chlorotic mottle virus (CCMV) or Cowpea mildmottle virus (CMMV)-infected Bradyrhizobium-nodulated cowpea, yardlong bean and mung beanplants. In healthy plants of the three Vigna taxa, nodulation and growth were promoted in the orderof Si + N > N > Si > controls. In the case of healthy cowpea and yardlong bean, the addition of Siand N decreased ureide and -amino acids (AA) contents in the nodules and leaves in the order ofSi + N> N > Si > controls. On the other hand, the addition of N arrested the deleterious effects ofCCMV or CMMV infections on growth and nodulation in the three Vigna taxa. However, the additionof Si or Si + N hindered growth and nodulation in the CCMV- or CMMV-infected cowpea andyardlong bean, causing a massive accumulation of ureides in the leaves and nodules. Nevertheless,the AA content in leaves and nodules of CCMV- or CMMV-infected cowpea and yardlong bean waspromoted by Si but reduced to minimum by Si + N. These results contrasted to the counteractingeffects of Si or Si + N in the CCMV- and CMMV-infected mung bean via enhanced growth, nodulationand levels of ureide and AA in the leaves and nodules. Together, these observations suggest thefertilization with Si + N exclusively in virus-free cowpea and yardlong bean crops. However, Si + Nfertilization must be encouraged in virus-endangered mung bean crops to enhance growth, nodulationand N-metabolism. It is noteworthy to see the enhanced nodulation of the three Vigna taxa in thepresence of 10 mM KNO3.</p

Single cell mutant selection for metabolic engineering of actinomycetes

Actinomycetes are important producers of pharmaceuticals and industrial enzymes. However, wild type strains require laborious development prior to industrial usage. Here we present a generally applicable reporter-guided metabolic engineering tool based on random mutagenesis, selective pressure, and single-cell sorting. We developed fluorescence-activated cell sorting (FACS) methodology capable of reproducibly identifying highperforming individual cells from a mutant population directly from liquid cultures. Actinomycetes are an important source of catabolic enzymes, where product yields determine industrial viability. We demonstrate 5fold yield improvement with an industrial cholesterol oxidase ChoD producer Streptomyces lavendulae to 20.4 U g-1 in three rounds. Strain development is traditionally followed by production medium optimization, which is a time-consuming multi-parameter problem that may require hard to source ingredients. Ultra-high throughput screening allowed us to circumvent medium optimization and we identified high ChoD yield production strains directly from mutant libraries grown under preset culture conditions. Genome-mining based drug discovery is a promising source of bioactive compounds, which is complicated by the observation that target metabolic pathways may be silent under laboratory conditions. We demonstrate our technology for drug discovery by activating a silent mutaxanthene metabolic pathway in Amycolatopsis. We apply the method for industrial strain development and increase mutaxanthene yields 9-fold to 99 mg l- 1 in a second round of mutant selection. In summary, the ability to screen tens of millions of mutants in a single cell format offers broad applicability for metabolic engineering of actinomycetes for activation of silent metabolic pathways and to increase yields of proteins and natural products.Peer reviewe

Carbon footprint of Nepalese healthcare system: A study of Dhulikhel Hospital [version 1; peer review: 2 approved, 1 approved with reservations, 1 not approved]

Background: Though direct greenhouse gas emissions cannot be observed in health care sectors, there can exist indirect emissions contributing to global climate change. This study addresses the concept of the carbon footprint and its significance in understanding the environmental impact of human activities, with a specific emphasis on the healthcare sector through gate-to-gate (GtoG) life cycle assessment. Transportation, energy consumption, and solid waste generated by hospitals are the primary sources of carbon emissions. Methods: Different standards, guidelines and parameters were used to estimate emissions from both the primary and secondary data. All steps and sub-steps involved in GtoG were accessed and analyzed within the standard ISO 14040:44 guideline. An extensive review of existing literature was carried out for the evaluation and verification of secondary data. Results: The total carbon footprint of generators, electricity consumption, transportation activities, LPG cylinders, PV systems was found to be 58,780 kg-CO2-eq/yr, 519,794 kg-CO2-eq/yr, 272,375 kg-CO2-eq/yr, 44,494 kg-CO2-eq/yr, 35,283 kg-CO2-eq/yr respectively and the emissions from non-biodegradable solid waste was found to be 489,835 kg-CO2/yr. Local air pollutants such as PM10, CO, SO2, NOX, and VOCs generated by generators and transportation were also estimated. The CH4 emissions from liquid waste were 1177.344 kg CH4/BOD yr, and those from biodegradables were 3821.6954 kg CH4/yr. Conclusions: Healthcare professionals and policymakers can take action to reduce the sector's carbon footprint by implementing best practices and encouraging sustainable behavior. This study can be taken as foundation for further exploration of indirect emissions from healthcare sectors not only in Nepal but also in south Asian scenario

Genotyping-Guided Discovery of Persiamycin A From Sponge-Associated Halophilic Streptomonospora sp. PA3

Microbial natural products have been a cornerstone of the pharmaceutical industry, but the supply of novel bioactive secondary metabolites has diminished due to extensive exploration of the most easily accessible sources, namely terrestrialStreptomycesspecies. The Persian Gulf is a unique habitat for marine sponges, which contain diverse communities of microorganisms including marine Actinobacteria. These exotic ecosystems may cradle rare actinomycetes with high potential to produce novel secondary metabolites. In this study, we harvested 12 different species of sponges from two locations in the Persian Gulf and isolated 45 symbiotic actinomycetes to assess their biodiversity and sponge-microbe relationships. The isolates were classified intoNocardiopsis(24 isolates),Streptomyces(17 isolates) and rare genera (4 isolates) by 16S rRNA sequencing. Antibiotic activity tests revealed that culture extracts from half of the isolates displayed growth inhibitory effects against seven pathogenic bacteria. Next, we identified five strains with the genetic potential to produce aromatic polyketides by genotyping ketosynthase genes responsible for synthesis of carbon scaffolds. The combined data led us to focus onStreptomonosporasp. PA3, since the genus has rarely been examined for its capacity to produce secondary metabolites. Analysis of culture extracts led to the discovery of a new bioactive aromatic polyketide denoted persiamycin A and 1-hydroxy-4-methoxy-2-naphthoic acid. The genome harbored seven gene clusters involved in secondary metabolism, including a tetracenomycin-type polyketide synthase pathway likely involved in persiamycin formation. The work demonstrates the use of multivariate data and underexplored ecological niches to guide the drug discovery process for antibiotics and anticancer agents

Assessment of Pressure Ulcer Risk among Patients Admitted in Intensive Care Unit at a Tertiary Level Hospital

Background: Pressure ulcer continues to be a major health problem and prevention has been the main emphasis of patient care. Rigorous evaluation of patients in intensive care unit is necessary for early identification of those at risk of developing pressure ulcer. Multiple risk assessment scales are in practice for its prevention. This study aims to assess pressure ulcer risk of the patients admitted in intensive care unit using Braden Scale. Methods: A cross-sectional descriptive study was conducted among 272 patients admitted in intensive care unit of Nepal Medical College Teaching Hospital from August 2019 to January 2020. Consecutive sampling technique was used to collect data from the patients. Data was analyzed using chi-square test and multiple binary logistic regression in the statistical package for social sciences in version 16. Results: Among 272 patients, the mean Braden score of pressure ulcer risk was 18.23±3.51. Nearly half of the patients 127 (46.7%) had risk of developing pressure ulcer, while two of them eventually developed pressure ulcer. The mean age was 51.11±18.82 years. Majority of the patients 221 (81.2%) were admitted in intensive care unit with medical disorders. Risk of developing pressure ulcer was significantly associated with age, gender, fever, use of ventilator, pressure ulcer prevention device, total duration of the hospital stay and duration of Intensive Care Unit stay (p= <0.05). The predictors of pressure ulcer risk were mechanical ventilation (P=0.001, Adjusted Odds Ratio =6.99) and fever (p=0.011, Adjusted Odds Ratio =3.61). Conclusions: Routine use of Braden Scale helps in early identification of pressure ulcer risk. Nurses need to consider the patients with ventilatory support and fever as these are the strong predictors of pressure ulcer risk

The phytochemistry of Ganoderma species and their medicinal potentials

The Ganoderma genus is known for its diverse use as a functional food and therapeutic agent. This fungus has over 428 species, with Ganoderma lucidum being the most studied. The Ganoderma species produce several secondary metabolites and bioactive compounds like polysaccharides, phenols, and triterpenes, which are largely responsible for their therapeutic properties. Throughout this review, several extracts obtained from Ganoderma species have been studied to delve into their therapeutic characteristics and mechanisms. Such properties like immunomodulation, antiaging, antimicrobial, and anticancer activities have been demonstrated by several Ganoderma species and are supported by a large body of evidence. Although its phytochemicals play a vital role in its therapeutic properties, identifying the therapeutic potentials of fungal-secreted metabolites for human health-promoting benefits is a challenging task. Identification of novel compounds with distinct chemical scaffolds and their mechanism of action could help suppress the spread of rising pathogens. Thus, this review provides an updated and comprehensive overview of the bioactive components in different Ganoderma species and the underlying physiological mechanisms.peer-reviewe

Differential regulation of undecylprodigiosin biosynthesis in the yeast-scavenging Streptomyces strain MBK6

Streptomyces are efficient chemists with a capacity to generate diverse and potent chemical scaffolds. The secondary metabolism of these soil-dwelling prokaryotes is stimulated upon interaction with other microbes in their complex ecosystem. We observed such an interaction when a Streptomyces isolate was cultivated in a media supplemented with dead yeast cells. Whole-genome analysis revealed that Streptomyces sp. MBK6 harbors the red cluster that is cryptic under normal environmental conditions. An interactive culture of MBK6 with dead yeast triggered the production of the red pigments metacycloprodigiosin and undecylprodigiosin. Streptomyces sp. MBK6 scavenges dead-yeast cells and preferentially grows in aggregates of sequestered yeasts within its mycelial network. We identified that the activation depends on the cluster-situated regulator, mbkZ, which may act as a cross-regulator. Cloning of this master regulator mbkZ in S. coelicolor with a constitutive promoter and promoter-deprived conditions generated different production levels of the red pigments. These surprising results were further validated by DNA-protein binding assays. The presence of the red cluster in Streptomyces sp. MBK6 provides a vivid example of horizontal gene transfer of an entire metabolic pathway followed by differential adaptation to a new environment through mutations in the receiver domain of the key regulatory protein MbkZ

Maculosin, a non-toxic antioxidant compound isolated from Streptomyces sp. KTM18

Context Streptomyces species are prolific sources of bioactive secondary metabolites known especially for their antimicrobial and anticancer activities. Objective This study sought to isolate and characterize antioxidant molecules biosynthesized by Streptomyces sp. KTM18. The antioxidant potential of an isolated compound and its toxicity were accessed. Materials and methods The compound was purified using bioassay-guided chromatography techniques. Nuclear magnetic resonance (NMR) experiments were carried out for structure elucidation. The antioxidant potential of the isolated compound was determined using DPPH free radical scavenging assay. The toxicity of the isolated compound was measured using a brine shrimp lethality (BSL) assay. Results Ethyl acetate extract of Streptomyces sp. KTM18 showed more than 90% inhibition of DPPH free radical at 50 mu g/mL of the test concentration. These data were the strongest among 13 Streptomyces isolates (KTM12-KTM24). The active molecule was isolated and characterized as maculosin (molecular formula, C14H16N2O3 as determined by the [M + H](+) peak at 261.1259). The DPPH free radical scavenging activity of pure maculosin was higher (IC50, 2.16 +/- 0.05 mu g/mL) than that of commercial butylated hydroxyanisole (BHA) (IC50, 4.8 +/- 0.05 mu g/mL). No toxicity was observed for maculosin (LD50, <128 mu g/mL) in brine shrimp lethality assay (BSLA) up to the compound's antioxidant activity (IC50) concentration range. The commercial standard, berberine chloride, showed toxicity in BSLA with an LD50 value of 8.63 +/- 0.15 mu g/mL. Conclusions Maculosin may be a leading drug candidate in various cosmetic and therapeutic applications owing to its strong antioxidant and non-toxic properties

Single cell mutant selection for metabolic engineering of actinomycetes

Actinomycetes are important producers of pharmaceuticals and industrial enzymes. However, wild type strains require laborious development prior to industrial usage. Here we present a generally applicable reporter-guided metabolic engineering tool based on random mutagenesis, selective pressure, and single-cell sorting. We developed fluorescence-activated cell sorting (FACS) methodology capable of reproducibly identifying high-performing individual cells from a mutant population directly from liquid cultures. Actinomycetes are an important source of catabolic enzymes, where product yields determine industrial viability. We demonstrate 5-fold yield improvement with an industrial cholesterol oxidase ChoD producer Streptomyces lavendulae to 20.4 U g−1 in three rounds. Strain development is traditionally followed by production medium optimization, which is a time-consuming multi-parameter problem that may require hard to source ingredients. Ultra-high throughput screening allowed us to circumvent medium optimization and we identified high ChoD yield production strains directly from mutant libraries grown under preset culture conditions. Genome-mining based drug discovery is a promising source of bioactive compounds, which is complicated by the observation that target metabolic pathways may be silent under laboratory conditions. We demonstrate our technology for drug discovery by activating a silent mutaxanthene metabolic pathway in Amycolatopsis. We apply the method for industrial strain development and increase mutaxanthene yields 9-fold to 99 mg l−1 in a second round of mutant selection. In summary, the ability to screen tens of millions of mutants in a single cell format offers broad applicability for metabolic engineering of actinomycetes for activation of silent metabolic pathways and to increase yields of proteins and natural products.</p