Fast HPLC analysis of omeprazole, 5-hydroxyomeprazole and omeprazole sulfone in liquid culture medium using a monolithic column for application in biotransformation studies with fungi

A fast liquid chromatography method was developed and validated for the simultaneous determination of omeprazole (OMZ), 5-hydroxyomeprazole (5-HOMZ) and omeprazole sulphone (OMZ SUL) in liquid culture medium for application in biotransformation studies employing phytopathogenic and endophytic fungi. The separation was achieved using a monolithic Chromolith Fast gradient RP 18 endcapped column, using a mobile phase consisting of 0.15% (v/v) trifluoroacetid acid (TFA) in water (solvent A) and 0.15% (v/v) TFA in acetonitrile (solvent B), under linear gradient of 5 to 90% of B in 1 min, flow rate of 1.0 mL min-1, temperature at 30 ºC and detection at 220 nm. Sample preparation was performed by liquid-liquid extraction, with recoveries in the range of 62.3 to 76.6% for all analytes. The method was linear in the range of 0.2 to 10.0 µg mL-1 (r &#8805; 0.995). The values for intra- and inter-day precision (% coefficient of variation) and accuracy (% relative error) were < 15% for all analytes. The validated method was used to evaluate OMZ biotransformation to their mammalian metabolites by selected fungi. In general, the phytopathogenic fungi studied were more efficient to biotransform OMZ. The sulfonation reaction was more prevalent for all studied fungi.Um método rápido por cromatografia líquida foi desenvolvido para a determinação simultânea de omeprazol (OMZ), 5-hidroxiomeprazol (5-HOMZ) e omeprazol sulfona (OMZ SUL) em meio de cultura líquido, para aplicação em estudos de biotransformação empregando fungos fitopatogênicos e endofíticos. A separação foi realizada empregando uma coluna monolítica Chromolith Fast gradient RP 18 com a fase móvel constituída por ácido trifluoroacético (TFA) 0,15% (v/v) em água (solvente A) e TFA 0,15% (v/v) em acetonitrila (solvente B). Foi empregado um gradiente linear de 5 a 90% de B em 1 minuto, vazão de 1,0 mL min-1, temperatura de 30 ºC e detecção em 220 nm. A extração líquido-líquido foi empregada na preparação das amostras, com recuperações na faixa de 62,3-76,6% para todos os analitos. O método foi linear na faixa de 0,2-10,0 µg mL-1 (r &#8805; 0,995). Os valores de precisão e exatidão intra- e inter-dias (coeficiente de variação e erro relativo) foram inferiores a 15% para todos os analitos. O método validado foi utilizado para avaliar a biotransformação do OMZ em seus principais metabólitos humanos pelos fungos selecionados. Em geral, os fungos fitopatogênicos foram mais eficientes para biotransformar o OMZ. A reação de sulfonação foi mais prevalente em todos os fungos estudadosFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Biocatalytic preparation and absolute configuration of enantiomerically pure fungistatic anti-2-benzylindane derivatives. Study of the detoxification mechanism by Botrytis cinerea

Enantiomerically pure 2-benzylindane derivatives were prepared using biocatalytic methods and their absolute configuration determined. (1R,2S)-2-Benzylindan-1-ol ((1R,2S)-2) and (S)-2-benzylindan-1-one ((S)-3) were produced by fermenting baker’s yeast. Lipase-mediated esterifications and hydrolysis of the corresponding racemic substrates gave rise to the enantiopure compounds (1S,2R)-2-benzylindan-1-ol ((1S,2R)-2) and (1R,2S)-2-benzylindan-1-ol ((1R,2S)-2), respectively. The antifungal activity of these products against two strains of the plant pathogen Botrytis cinerea was tested. The metabolism of anti-(±)-2-benzylindan-1-ol (anti-(±)-2) by B. cinerea as part of the fungal detoxification mechanism is also described and revealed interesting differences in the genome of both strains

Genetic and molecular basis of botrydial biosynthesis: connecting cytochrome P450-encoding genes to biosynthetic intermediates

Over two hundred species of plants can be infected by the phytopathogenic fungus Botrytis cinerea under a range of different environmental conditions. In response to these, the fungus produces unique terpenoid and polyketide metabolites. Parts of the plants may be killed by the phytotoxin botrydial, enabling the fungus to feed on the dead cells. In this paper, we describe the genetic and molecular basis of botrydial biosynthesis and the function of the five genes of the genome of B. cinerea that together constitute the botrydial biosynthetic gene cluster. Genes BcBOT3 and BcBOT4, encoding two cytochrome P450 monooxygenases, were inactivated by homologous recombination and were shown to catalyze regio- and stereospecific hydroxylations at the carbons C-10 and C-4, respectively, of the presilphiperfolan-8β-ol skeleton. The null mutants, bcbot3Δ and bcbot4Δ, accumulated key intermediates in the botrydial biosynthesis enabling the complete genetic and molecular basis of the botrydial biosynthetic pathway to be established. Furthermore, the bcbot4Δ mutant overproduced a significant number of polyketides, which included, in addition to known botcinins, botrylactones and cinbotolide A, two new botrylactones and two new cinbotolides, cinbotolides B and C

Enantiomeric oxidation of organic sulfides by the filamentous fungi Botrytis cinerea, Eutypa lata and Trichoderma viride

The biotransformations of a series of substituted sulfides were carried out with the filamentous fungi Botrytis cinerea, Eutypa lata and Trichoderma viride. Several products underwent microbial oxidation of sulfide to sulfoxide with medium to high enantiomeric purity. With regard to sulfoxide enantioselectivity, the (R)-enantiomer was favoured in biotransformations by T. viride and E. lata while the (S)-enantiomer was favoured in those by B. cinerea. A minor amount of sulfone product was also obtained

Relevance of trichothecenes in fungal physiology: Disruption of tri5 in Trichoderma arundinaceum

Trichothecenes are sesquiterpenoid mycotoxins produced mainly by Fusarium species. Harzianum A (HA), a non-phytotoxic trichothecene produced by Trichoderma arundinaceum, has recently been found to have antagonistic activity against fungal plant pathogens and to induce plant genes involved in defense responses. In the present work, we have shown that disruption of the T. arundinaceum tri5 gene, which encodes a terpene synthase, stops the production of HA, alters the expression of other tri genes involved in HA biosynthesis, and alters the expression of hmgR, dpp1, erg9, erg1, and erg7, all genes involved in terpene biosynthetic pathways. An increase in the level of ergosterol biosynthesis was also observed in the tri5 disrupted transformant in comparison with the wild type strain. The loss of HA also resulted in a drastic reduction of the biocontrol activity of the transformants against the phytopathogenic fungi Botrytis cinerea and Rhizoctonia solani. Finally, the effect of tri5 gene disruption on the regulation and balance of intermediates in terpene biosynthetic pathways, as well as the hypothetical physiological role of trichothecenes, both inter- and intracellularly, on regulation and biocontrol, are discussed

Identification of polyketide synthase genes required for aspinolide biosynthesis in Trichoderma arundinaceum

https://link.springer.com/article/10.1007/s00253-022-12182-9[EN] The fungus Trichoderma arundinaceum exhibits biological control activity against crop diseases caused by other fungi. Two mechanisms that likely contribute to this activity are upregulation of plant defenses and production of two types of antifungal secondary metabolites: the sesquiterpenoid harzianum A (HA) and the polyketide-derived aspinolides. The goal of the current study was to identify aspinolide biosynthetic genes as part of an effort to understand how these metabolites contribute to the biological control activity of T. arundinaceum. Comparative genomics identified two polyketide synthase genes (asp1 and asp2) that occur in T. arundinaceum and Aspergillus ochraceus, which also produces aspinolides. Gene deletion and biochemical analyses in T. arundinaceum indicated that both genes are required for aspinolide production: asp2 for formation of a 10-member lactone ring and asp1 for formation of a butenoyl subsituent at position 8 of the lactone ring. Gene expression and comparative genomics analyses indicated that asp1 and asp2 are located within a gene cluster that occurs in both T. arundinaceum and A. ochraceus. A survey of genome sequences representing 35 phylogenetically diverse Trichoderma species revealed that intact homologs of the cluster occurred in only two other species, which also produced aspinolides. An asp2 mutant inhibited fungal growth more than the wild type, but an asp1 mutant did not, and the greater inhibition by the asp2 mutant coincided with increased HA production. These findings indicate that asp1 and asp2 are aspinolide biosynthetic genes and that loss of either aspinolide or HA production in T. arundinaceum can be accompanied by increased production of the other metabolite(s).SIPublicación en abierto financiada por el Consorcio de Bibliotecas Universitarias de Castilla y León (BUCLE), con cargo al Programa Operativo 2014ES16RFOP009 FEDER 2014-2020 DE CASTILLA Y LEÓN, Actuación:20007-CL - Apoyo Consorcio BUCL

Hemisynthesis and Absolute Configuration of novel 6-pentyl-2H-pyran- 2-one derivatives from Trichoderma spp

A comparative study of the secondary metabolism of two Trichoderma spp. with that of the Thctf1 transcription factor gene null mutant of Trichoderma harzianum 34 was carried out in order to deepen our knowledge of the biosynthetic pathway and mode of action of 6-pentyl-2H-pyran-2-one (1) and its derivatives as biocontrol agents. New isolated metabolites have shed light on the detoxification mechanism of 6-pentyl-pyranone by Trichoderma spp. All new compounds were synthesized and their stereoisomer characterized. The absolute configuration of 6-[(10R,20S)-dihydroxypentyl]-2H-pyran-2-one and 6-((10S,20R)-20-propyloxiran-1-yl)-2H-pyran-2-one was determined by NMR analysis of the corresponding Mosher’s esters

Synthesis of Trichodermin Derivatives and Their Antimicrobial and Cytotoxic Activities

Trichothecene mycotoxins are recognized as highly bioactive compounds that can be used in the design of new useful bioactive molecules. In Trichoderma brevicompactum, the first specific step in trichothecene biosynthesis is carried out by a terpene cyclase, trichodiene synthase, that catalyzes the conversion of farnesyl diphosphate to trichodiene and is encoded by the tri5 gene. Overexpression of tri5 resulted in increased levels of trichodermin, a trichothecene-type toxin, which is a valuable tool in preparing new molecules with a trichothecene skeleton. In this work, we developed the hemisynthesis of trichodermin and trichodermol derivatives in order to evaluate their antimicrobial and cytotoxic activities and to study the chemo-modulation of their bioactivity. Some derivatives with a short chain at the C-4 position displayed selective antimicrobial activity against Candida albicans and they showed MIC values similar to those displayed by trichodermin. It is important to highlight the cytotoxic selectivity observed for compounds 9, 13, and 15, which presented average IC50 values of 2 g/mL and were cytotoxic against tumorigenic cell line MCF-7 (breast carcinoma) and not against Fa2N4 (non-tumoral immortalized human hepatocytes)

Synthesis of Trichodermin Derivatives and Their Antimicrobial and Cytotoxic Activities

[EN] Trichothecene mycotoxins are recognized as highly bioactive compounds that can be used in the design of new useful bioactive molecules. In Trichoderma brevicompactum, the first specific step in trichothecene biosynthesis is carried out by a terpene cyclase, trichodiene synthase, that catalyzes the conversion of farnesyl diphosphate to trichodiene and is encoded by the tri5 gene. Overexpression of tri5 resulted in increased levels of trichodermin, a trichothecene-type toxin, which is a valuable tool in preparing new molecules with a trichothecene skeleton. In this work, we developed the hemisynthesis of trichodermin and trichodermol derivatives in order to evaluate their antimicrobial and cytotoxic activities and to study the chemo-modulation of their bioactivity. Some derivatives with a short chain at the C-4 position displayed selective antimicrobial activity against Candida albicans and they showed MIC values similar to those displayed by trichodermin. It is important to highlight the cytotoxic selectivity observed for compounds 9, 13, and 15, which presented average IC50 values of 2 µg/mL and were cytotoxic against tumorigenic cell line MCF-7 (breast carcinoma) and not against Fa2N4 (non-tumoral immortalized human hepatocytes)S

Botrydial and botcinins produced by Botrytis cinerea regulate the expression of Trichoderma arundinaceum genes involved in trichothecene biosynthesis

Trichoderma arundinaceum IBT 40837 (Ta37) and Botrytis cinerea produce the sesquiterpenes harzianum A (HA) and botrydial (BOT), respectively, and also the polyketides aspinolides and botcinins (Botcs), respectively. We analysed the role of BOT and Botcs in the Ta37-B. cinerea interaction, including the transcriptomic changes in the genes involved in HA (tri) and ergosterol biosynthesis, as well as changes in the level of HA and squalene-ergosterol. We found that, when confronted with B. cinerea, the tri biosynthetic genes were up-regulated in all dual cultures analysed, but at higher levels when Ta37 was confronted with the BOT non-producer mutant bcbot2Δ. The production of HA was also higher in the interaction area with this mutant. In Ta37-bcbot2Δ confrontation experiments, the expression of the hmgR gene, encoding the 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is the first enzyme of the terpene biosynthetic pathway, was also up-regulated, resulting in an increase in squalene production compared with the confrontation with B. cinerea B05.10. Botcs had an up-regulatory effect on the tri biosynthetic genes, with BotcA having a stronger effect than BotcB. The results indicate that the interaction between Ta37 and B. cinerea exerts a stimulatory effect on the expression of the tri biosynthetic genes, which, in the interaction zone, can be attenuated by BOT produced by B. cinerea B05.10. The present work provides evidence for a metabolic dialogue between T. arundinaceum and B. cinerea that is mediated by sesquiterpenes and polyketides, and that affects the outcome of the interaction of these fungi with each other and their environmentSIFunding was obtained from the Junta de Castilla y Leon (SA260A11-2,LE125A12-2 and LE228U14) and Spanish Government grants MICINN-AGL2009-13431-C02-02, MINECO-AGL2012-40041-C02-01, AGL2012-40041-C02-02 and AGL2012-39798-C02-01. MGM and II-B were granted fellowships from the Spanish Ministry of Science and Innovation(AP2007-02835, BES-2013-063411