Alkaloidit Rhazya stricta in vitro -viljelmissä

Rhazya stricta Decne. (Apocynaceae) is a traditional medicinal plant in the Middle East and South Asia. It produces a large number of terpenoid indole alkaloids(TIAs), some of which possess important pharmacological properties. This study focused on the establishment of biotechnological production tools of R. stricta, namely undifferentiated cell cultures, and an Agrobacterium rhizogenes-mediated transformation method to obtain hairy roots expressing heterologous genes from the early TIA pathway. As Rhazya alkaloids comprise a wide range of structures and polarities it was necessary first to develop different analytical methods to determine the alkaloid contents and changes in their profiles in transgenic cultures and after various treatments. Targeted and non-targeted analyses from cell andorgan cultures were carried out using gas chromatography-mass spectrometry(GC-MS), high performance liquid chromatography (HPLC), ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Callus cultures were successfully initiated from five different explants onmodified B5 medium containing phytohormones. The phenotypes of the calli varied, but as was expected the callus cultures accumulated lower levels of alkaloids than wild type hairy roots and adventitious roots. Surprisingly, calli derived from stems had elevated levels of strictosidine lactam compared to other cultures. Transformation experiments revealed that only leaves but not cotyledons, hypocotyls or stem segments were susceptible to Agrobacterium infection and subsequent root induction. The transformation efficiency varied from 22% to 83% depending on the gene. Wild type and gus hairy root clones contained twofold higher amounts of alkaloids than adventitious roots. A total of 17 TIAs, including glycosylated alkaloids, were identified from hairy root extracts by UPLC-MS. GC-MS analysis allowed the separation of the most volatile and non-polar alkaloids in a single run. The composition of typical non-polar alkaloids indicated the occurrence of 20 TIAs belonging to nine different groups. The quantities of these alkaloids varied between clones in the order eburenine, vincanine, vallesiachotamine and yohimbine isomer II. The occurrence of pleiocarpamine, fluorocarpamine, vincamine, ajmalicine, and yohimbine isomers, analysed by GC-MS, and serpentine and its isomer, tetrahydrosecodinol as well as tabersonine, analysed by UPLC-MS, is reported here for the first time from R. stricta. Methyl jasmonate, a well-known elicitor, caused a significant increase in the total alkaloid content of wild type hairy roots as determined by NMR analyses. Detailed targeted analyses by GC-MS showed that the contents of eight out of ten studied alkaloids increased compared to non-elicited cultures. Another studied elicitor, chitosan, did not have any effect on individual alkaloid contents. Transgenic hairy root clones did not exhibit phenotype differences. Multivariate analysis from NMR data showed a clear discrimination between transformed and wild type/gus cultures. This was most probably due to differences in primary metabolites, as the total alkaloid content did not vary between different hairy roots and controls. In general, the production of individual TIAs, analysed by HPLC, was repressed in hairy roots transformed with geraniol synthase(ges) and geraniol 8-oxidase (g8o) genes compared to the wild types. Overexpression of the strictosidine synthase (str) gene resulted in a higher accumulation of serpentine, whereas the production of strictosidine lactam was decreased. There were no significant differences in the contents of other alkaloids compared to the wild type hairy roots. In conclusion, a simple and efficient gene transfer method is reported for R. stricta for the first time. New analytical methods were established which enabled comprehensive investigation of the alkaloids. These data might serve as a basis for further utilization of biotechnological methods for R. stricta and its further metabolic engineering.Rhazya stricta Decne. (Apocynaceae) on Lähi-idästä ja Kaakkois-Aasiasta kotoisin oleva perinteinen lääkekasvi. Se tuottaa lukuisia terpeeni-indolialkaloideja (TIA), joista useilla on tärkeitä farmakologisia ominaisuuksia. Tämä tutkimus keskittyi bioteknologisten menetelmien kehittämiseen R. stricta -kasville. Näihin menetelmiin lukeutuivat erilaistumattomien solukkoviljelmien perustaminen sekä Agrobacterium rhizogenes -bakteerin avulla aikaansaadut siirtogeeniset karva-juuret, jotka ilmensivät varhaisen TIA-synteesireitin geenejä. Rhazya-alkaloidit muodostavat laajan kirjon kemiallisia, erilaisen polaarisuuden omaavia rakenteita. Tästä syystä oli tärkeää kehittää uusia analyysimenetelmiä alkaloidi-pitoisuuksien määrittämiseen ja alkaloidiprofiilien muutosten seuraamiseen eri siirtogeenisten linjojen ja käsittelyiden välillä. Kohdennetut ja kohdentamattomat analyysit solu- ja solukkoviljelmistä suoritettiin kaasukromatografia-massaspektrometrialla (GM-MS), korkean erotuskyvyn neste-kromatografialla (HPLC), erittäin korkean suorituskyvyn nestekroma-tografialla (UPLC-MS) ja ydinmagneettisella resonanssispektroskopialla (NMR). Kallusviljelmät perustettiin viidestä eri kasvinosasta modifioidulle, kasvutekijöitä sisältävälle B5-kasvatusalustalle. Kallusten fenotyypit vaihtelivat, mutta kuten oletettua, ne tuottivat pienempiä määriä alkaloideja kuin villityypin juuret tai karvajuuret. Yllättäen varren soluista perustetut kallusviljelmät tuottivat suurempia määriä striktosidiinilaktaamia kuin muut soluviljelmät. Vain lehdet, toisin kuin sirkkalehdet, alkeisvarret tai varren osat, olivat alttiita agrobakteeri-infektiolle ja siten karvajuurten muodostumiselle. Transformaatio-tehokkuus vaihteli 22 ja 83 %:n välillä riippuen kohdegeenistä. Villityypin sekä gus-geeniä ilmentävät karvajuurilinjat tuottivat kaksinkertaisia määriä alkaloideja verrattuna tavallisiin juuriin. UPLC-MS -menetelmällä karvajuurista tunnistettiin kaikkiaan 17 terpeeni-indolialkaloidia, mukaan lukien glykosyloidut alkaloidit. GC-MS -analyysi mahdollisti myös kaikkein helpoimmin haihtuvien, poolittomien alkaloidien erottelun samalla kertaa. Tyypillisten poolittomien alkaloidien koostumus viittasi 20 TIA:n esiintymiseen, jotka voitiin edelleen jakaa yhdeksään ryhmään. Suurimmat vaihtelut alkaloidien pitoisuuksissa eri solulinjojen välillä havaittiin ebureniinin, vinkaniinin, vallesiakotamiinin ja johimbiinin isomeeri II:n kohdalla, tässä järjestyksessä. GC-MS:lla analysoitua pleiokarpamiinia, fluorokarpamiinia, vinkamiinia, ajmalisiinia ja johimbiini-isomeereja sekä UPLC-MS:lla analysoitua serpentiiniä ja sen isomeereja, tetrahydrosekodinolia ja tabersoniinia ei ole tätä ennen ole raportoitu R. stricta -kasvista. NMR-analyysi osoitti metyylijasmonaatin, joka on yleinen elisiittori, lisäävän merkittävästi alkaloidien kokonaismäärää villityypin karvajuurissa. Kohdennettu GC-MS -analyysi osoitti, että kahdeksan alkaloidin pitoisuus kymmenestä lisääntyi verrattuna käsittelemättömiin karvajuuriin. Toisaalta kitosaanilla, toisella elisiittorilla, ei havaittu olevan vaikutusta yhdenkään yksittäisen alkaloidin pitoisuuteen. Siirtogeeniset karvajuuret eivät poikenneet fenotyypiltään villityypin karvajuurista. NMR-datan monivarianssianalyysi osoitti kuitenkin selkeän eron siirtogeenisten ja villityypin tai gus-karvajuurien välillä. Tämä johtui todennäköisimmin eroista primäärimetaboliittien pitoisuuksissa, koska alkaloidien kokonaispitoisuudessa ei ollut eroa eri karvajuurilinjojen ja kontrollien välillä. Yksittäisten HPLC:lla analysoitujen terpeeni-indolialkaloidien tuotto oli yleisesti vähentynyt geraniolisyntaasi- (ges) sekä geranioli-8-oksidaasi-geenejä (g8o) ilmentävissä karvajuurilinjoissa verrattuna villityypin karvajuuriin. Striktosidiinisyntaasi-geenin (str) yli-ilmeneminen johti kohonneeseen serpentiini pitoisuuteen ja pienentyneeseen striktosidiinilaktaamin tuottoon. Muiden alkaloidien kohdalla ei havaittu merkittävää eroa villityypin karvajuuriin. Tässä työssä esitetään ensimmäistä kertaa yksinkertainen ja tehokas geenin-siirtomenetelmä R. stricta -kasville. Lisäksi kehitettiin joukko uusia analyysi-menetelmiä alkaloidien kokonaisvaltaiseen analysoimiseen. Nämä tulokset luovat pohjan bioteknologisten menetelmien laajemmalle käytölle R. stricta -kasvin kohdalla ja sen metabolian muokkaamiselle

Methyljasmonate Elicitation Increases Terpenoid Indole Alkaloid Accumulation in Rhazya stricta Hairy Root Cultures

Methyl jasmonate is capable of initiating or improving the biosynthesis of secondary metabolites in plants and therefore has opened up a concept for the biosynthesis of valuable constituents. In this study, the effect of different doses of methyl jasmonate (MeJA) elicitation on the accumulation of terpenoid indole alkaloids (TIAs) in the hairy root cultures of the medicinal plant, Rhazya stricta throughout a time course (one-seven days) was investigated. Gas chromatography-mass spectrometry (GC-MS) analyses were carried out for targeted ten major non-polar alkaloids. Furthermore, overall alterations in metabolite contents in elicited and control cultures were investigated applying proton nuclear magnetic resonance (H-1 NMR) spectroscopy. Methyl jasmonate caused dosage- and time course-dependent significant rise in the accumulation of TIAs as determined by GC-MS. The contents of seven alkaloids including eburenine, quebrachamine, fluorocarpamine, pleiocarpamine, tubotaiwine, tetrahydroalstonine, and ajmalicine increased compared to non-elicited cultures. However, MeJA-elicitation did not induce the accumulation of vincanine, yohimbine (isomer II), and vallesiachotamine. Furthermore, principal component analysis (PCA) of H-1 NMR metabolic profiles revealed a discrimination between elicited hairy roots and control cultures with significant increase in total vindoline-type alkaloid content and elevated levels of organic and amino acids. In addition, elicited and control samples had different sugar and fatty acid profiles, suggesting that MeJA also influences the primary metabolism of R. stricta hairy roots. It is evident that methyl jasmonate is applicable for elevating alkaloid accumulation in "hairy root" organ cultures of R. strica.Peer reviewe

Methyljasmonate Elicitation Increases Terpenoid Indole Alkaloid Accumulation in Rhazya stricta Hairy Root Cultures

Methyl jasmonate is capable of initiating or improving the biosynthesis of secondary metabolites in plants and therefore has opened up a concept for the biosynthesis of valuable constituents. In this study, the effect of different doses of methyl jasmonate (MeJA) elicitation on the accumulation of terpenoid indole alkaloids (TIAs) in the hairy root cultures of the medicinal plant, Rhazya stricta throughout a time course (one-seven days) was investigated. Gas chromatography-mass spectrometry (GC-MS) analyses were carried out for targeted ten major non-polar alkaloids. Furthermore, overall alterations in metabolite contents in elicited and control cultures were investigated applying proton nuclear magnetic resonance (1H NMR) spectroscopy. Methyl jasmonate caused dosage- and time course-dependent significant rise in the accumulation of TIAs as determined by GC-MS. The contents of seven alkaloids including eburenine, quebrachamine, fluorocarpamine, pleiocarpamine, tubotaiwine, tetrahydroalstonine, and ajmalicine increased compared to non-elicited cultures. However, MeJA-elicitation did not induce the accumulation of vincanine, yohimbine (isomer II), and vallesiachotamine. Furthermore, principal component analysis (PCA) of 1H NMR metabolic profiles revealed a discrimination between elicited hairy roots and control cultures with significant increase in total vindoline-type alkaloid content and elevated levels of organic and amino acids. In addition, elicited and control samples had different sugar and fatty acid profiles, suggesting that MeJA also influences the primary metabolism of R. stricta hairy roots. It is evident that methyl jasmonate is applicable for elevating alkaloid accumulation in “hairy root” organ cultures of R. strica

Methyljasmonate Elicitation Increases Terpenoid Indole Alkaloid Accumulation in Rhazya stricta Hairy Root Cultures

Methyl jasmonate is capable of initiating or improving the biosynthesis of secondary metabolites in plants and therefore has opened up a concept for the biosynthesis of valuable constituents. In this study, the effect of different doses of methyl jasmonate (MeJA) elicitation on the accumulation of terpenoid indole alkaloids (TIAs) in the hairy root cultures of the medicinal plant, Rhazya stricta throughout a time course (one-seven days) was investigated. Gas chromatography-mass spectrometry (GC-MS) analyses were carried out for targeted ten major non-polar alkaloids. Furthermore, overall alterations in metabolite contents in elicited and control cultures were investigated applying proton nuclear magnetic resonance (1H NMR) spectroscopy. Methyl jasmonate caused dosage- and time course-dependent significant rise in the accumulation of TIAs as determined by GC-MS. The contents of seven alkaloids including eburenine, quebrachamine, fluorocarpamine, pleiocarpamine, tubotaiwine, tetrahydroalstonine, and ajmalicine increased compared to non-elicited cultures. However, MeJA-elicitation did not induce the accumulation of vincanine, yohimbine (isomer II), and vallesiachotamine. Furthermore, principal component analysis (PCA) of 1H NMR metabolic profiles revealed a discrimination between elicited hairy roots and control cultures with significant increase in total vindoline-type alkaloid content and elevated levels of organic and amino acids. In addition, elicited and control samples had different sugar and fatty acid profiles, suggesting that MeJA also influences the primary metabolism of R. stricta hairy roots. It is evident that methyl jasmonate is applicable for elevating alkaloid accumulation in “hairy root” organ cultures of R. strica

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

THE EFFECT OF A VARNISH CONTAINING SELF-CURING RESIN ON THE SOFTNESS OF TWO TYPES OF TISSUE CONDITIONERS

Objectives: One of the limitations of tissue conditioners (TC) is the gradual hardening of the material in a short time after insertion in the mouth. This study aimed to determine the softness of two different tissue conditioners with and without the coating made up of 1,1,1trichloroethan and self-curing acrylic resin.Materials and Methods: In this experimental study, Acrosoft (Marlic, Tehran, Iran) and GC (GC corporation, Tokyo, Japan) tissue conditioners were examined. 28 discs of 20 x 3 mm dimensions were prepared for each tissue conditioner (n=14). Half of the samples in each group were coated with varnish coating made up of 1,1,1trichloroethan and self-curing acrylic resin. The hardness of all samples was measured at five intervals of 1,3,7,14, and28 days by a Shore-A Durometer with a conical indenter. The data were analyzed by descriptive statistics and Friedman analyses. P<0.05 was considered statistically significant.Results: The mean hardness of the GC and Acrosoft tissue conditioners on days 1, 3, 7, 14, and 28 in both varnish-coated and non-varnish-coated groups were statistically different and Acrosoft tissue conditioner was harder than the GC. In the paired mean hardness comparison on days 1, 3, 7, 14, and 28 in the GV and G0 groups: this trend was the same in AV and A0 groups. The comparison of hardness in the GV and G0 groups at each time interval indicated that only on day 3, the control group(G0) was harder than the surface coating group(GV). The comparison of the hardness in the AV and A0 groups showed that on days 3 and 7, the hardness in the control group (A0) was higher than the surface coating group(AV).Conclusions: The varnish containing self-curing resin can soften the Acrosoft and GC tissue conditioner in a short time. Moreover, this varnish can be clinically applied in the borders between the soft liner and acrylic denture, which is usually the starting point for debonding

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

Analysis of indole alkaloids from Rhazya stricta hairy roots by ultra-performance liquid chromatography-mass spectrometry

Rhazya stricta Decne. (Apocynaceae) contains a large number of terpenoid indole alkaloids (TIAs). This study focused on the composition of alkaloids obtained from transformed hairy root cultures of R. stricta employing ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). In the UPLC-MS analyses, a total of 20 TIAs were identified from crude extracts. Eburenine and vincanine were the main alkaloids followed by polar glucoalkaloids, strictosidine lactam and strictosidine. Secodine-type alkaloids, tetrahydrosecodinol, tetrahydro- and dihydrosecodine were detected too. The occurrence of tetrahydrosecodinol was confirmed for the first time for R. stricta. Furthermore, two isomers of yohimbine, serpentine and vallesiachotamine were identified. The study shows that a characteristic pattern of biosynthetically related TIAs can be monitored in Rhazya hairy root crude extract by this chromatographic method.Peer reviewe

Methyljasmonate Elicitation Increases Terpenoid Indole Alkaloid Accumulation in Rhazya stricta Hairy Root Cultures

Methyl jasmonate is capable of initiating or improving the biosynthesis of secondary metabolites in plants and therefore has opened up a concept for the biosynthesis of valuable constituents. In this study, the effect of different doses of methyl jasmonate (MeJA) elicitation on the accumulation of terpenoid indole alkaloids (TIAs) in the hairy root cultures of the medicinal plant, Rhazya stricta throughout a time course (one-seven days) was investigated. Gas chromatography-mass spectrometry (GC-MS) analyses were carried out for targeted ten major non-polar alkaloids. Furthermore, overall alterations in metabolite contents in elicited and control cultures were investigated applying proton nuclear magnetic resonance (H-1 NMR) spectroscopy. Methyl jasmonate caused dosage- and time course-dependent significant rise in the accumulation of TIAs as determined by GC-MS. The contents of seven alkaloids including eburenine, quebrachamine, fluorocarpamine, pleiocarpamine, tubotaiwine, tetrahydroalstonine, and ajmalicine increased compared to non-elicited cultures. However, MeJA-elicitation did not induce the accumulation of vincanine, yohimbine (isomer II), and vallesiachotamine. Furthermore, principal component analysis (PCA) of H-1 NMR metabolic profiles revealed a discrimination between elicited hairy roots and control cultures with significant increase in total vindoline-type alkaloid content and elevated levels of organic and amino acids. In addition, elicited and control samples had different sugar and fatty acid profiles, suggesting that MeJA also influences the primary metabolism of R. stricta hairy roots. It is evident that methyl jasmonate is applicable for elevating alkaloid accumulation in "hairy root" organ cultures of R. strica

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