The Stimulation On Artemisinin Biosynthesis,the Bioconversion Of Artemisinic Acid And The Biodegradation Of Triclosan By An Endophytic Penicillium Oxalicum B4 From Artemisia Annua L.

Endophytes live in the inner of healthy plantswith symbiosis relationships with their hosts.Endophytes could promote plant growth,enhance the production of the secondary metabolites and coordinate the plants and environment.The special inner habitat enabled the endophytes to biosynthesize novel biologically active metabolites.Recently,the endophytes and their metabolites have been extensivly applied in fields of medicine hygienism,agriculture,forestry and environment research.Artemisia annua L.is a famous traditional medical herb in our country to yield antimalarial drug artemisinin.The rich endophytes in A.annua have been reported yet.In present study,we isolated an endophytic Penicillium oxalicum B4 from A.annua.The role of its enhancing the accumulation of artemisinin,biotranconcweaion of artemisinic acid and biodegradation of triclosan was investigated.The regulation mechanism of P.oxalicum B4 on the biosynthesis of artemisinin was revealed by the physiological changes,transcritomic and metabonomic analysis.New artemisinic acid derivatives with potentials of anticancer and anti-inflammatory were produced in the bioconversion system.The new application of this fungus in biodegradation of triclosan(TCS)was developed.The main results are showed below.Firstly,an endophytic fungus B4 was isolated from wild A.annua plant.Based on the morphological observation,ITS sequences of ribosomal DNA,and ethe phylogenetic tree,the endophytic fungus B4 was identified as a strain of Penicillium oxalicum,which exhibited 99%homology to genus Penicillium such as P.oxalicum EU434727.1.Its sequences had been deposited in the GenBank(the accession number FJ196840).The strain was utilized to inoculate the plantlet and seedling of A.annua in different culture conditions.The results indicated that the strain intruded A.annua through roots and colonized in intercellular space of root tissue.The growth of plant height and lateral root was inhibited by the inoculation.But there was no significant effect on the biomass of host.B4 treatment induced the change of the content of abscisic acid,which decreased and then increased in the later of growth stage.But there is no effect on the biosynthesis of indole-3-acetic acid.The stain B4 could stimulated artemisinin and artemisinic acid content.The content of artemisinin was enhanced 42.86%than that of control group after 23 days.The GC-TOF-MS was used to analyze the different metabolites between the B4 treatment and control.The principal component analysis(PCA)and orthogonal projections to latent structures-discriminant analysis(OPLS-DA)analysis showed there was a significant difference between B4 treatment and the control with the total of 85 different compounds.The content of ferulic acid and D-erythro-sphingosine of A.annua treated by B4 were 266822 fold and 12324 fold than that of control.The differentially expressed metabolites were related to the relevant 24 metabolic pathway such as valine,leucine and isoleucine biosynthesis,purine metabolism and phenylpropanol biosynthesis.The most relevant pathways were valine,leucine and isoleucine biosynthesis,glycine,serine and threonine metabolism,and starch,sucrose metabolism.At the same time,the transcriptional change of A.annua treated by B4 was analyzed by GeneChip.The total of 956 differentially expressed genes were identified with 411 transcripts increased and 545 decreased.GO analysis showed that the gene expression on protein phosphorylation,membrane and oxidation-reduction process had significant change.The differentially expressed genes were focused on plant-pathogen interaction,fatty acid metabolism,and biosynthesis of unsaturated fatty acids and sesquiterpenoid biosynthesis.Our results suggested that plant metabolic pathways had been rechanneled for producing more defensing compound including artemisinin,which was beneficial to for the both symbionts.We further investigated on the bioconversion of artemisinic acid by B4.After 10 days of incubation of B4 in biotransformation medium,artemisinic acid was conversed to 21 compounds.Among them,3a,14-dihydroxy artemisinic acid(th2-1),3-carbonyl,14-hydroxy artemisinic acid(th3),3-carbonyl,15-hydroxy artemisinic acid(th6),15-carboxyl artemisinic acid(th8)and 15-carboxyl-1,2,3,6-dehydroartemisinic acid(th7-2)were identified as new compounds.And 3 known compounds were obtained including 3?,15-dihydroxy artemisinic acid(th5-2),3a-hydroxy artemisinic acid(th9),9-oxo artemisinic acid(th10).The cytotoxicity activity of these compounds was evaluated against SMMC-7721,LS174T,A549 and HL60 cell lines.The novel artemisinic acid derivatives exhibited strong cytotoxicity activity,especially in HL60 cell line.The activity of these compounds in HL60 cells was showed as followed:th7-2(IC50=8.32± 0.19?g/mL)>th3(IC50=8.69±0.15?g/mL)>th2-1(IC50=9.63±0.55 ?g/mL)>th6(IC50=11.24±1.01?g/mL)>th8(IC50=14.00±0.46 pg/mL)>artemisinic acid(IC50=15.04±1.48 ?g/mL).And some of the derivatives were also exhibited anti-inflammatory activities in LPS-stimulated RAW264.Compared to the control group,the content of nitric oxide(NO)in the group treated with 5 ?M th9 was reduced by 42.83%.and the content of NO in the group treated with 10 ?M th5 and th8 were reduced by 25.16 and 43.91%.Artemisinic acid and its derivatives have no cytotoxicity to RAW264.7 cell lines.In our study,we also investigated the biodegradation by B4 on triclosan(TCS),a refractory organic pollutant.B4 showed the high capacity of TCS uptake,and the maximum adsorption capacity was 127.60 ± 8.57 mg/g dry weight(DW).TCS in medium was reduced rapidly by B4 in degradation medium in first 10 min after TCS addition.The content of TCS in medium was decreased from 5.00 mg/L to 0.41 mg/L and they all adsorbed on mycelium of B4.Then the TCS in mycelium was degraded from 0.45 to 0.05 mg/g DW after 1 h treatment.The GC-MS was used to analyze the degradation metabolites,and three compounds including 2-chlorohydroquinone,2,4-dichloropheno,and hydroquinone were detected in mycelia.At the same time,the medium showed no toxicity to Escherichia coli.The degradation capacity of B4 in synthetic wastewater was also investigated.TCS(5 mg/L)in synthetic wastewater was almost completely degraded within 2 h.To summarize,our research provides basis for the developing endophytic resource in A.annua.P.oxalicum B4 isolated from A.annua could enhance the accumulation of artemisinin,which could be applied in agricultural production of artemisinin in A.annua.It is a first time on the reports of artemisinic acid derivatives with anti-inflammatory activities,which gives a clue for medicinal utilization of endophytes.The rapid TCS biodegradation by B4 with higher adsorption and lower residual toxicity will provide a new bioremediation technology in wastewater and rivers.