Isolation, Identification And Synthesis Regulation Mechanism Of The Antifungal Substance Toxanthin From The Medicinal Plant Endophytic Bacteria HDXY-02

Fungal diseases are one of the important factors influencing agricultural production.More importantly,over 70%of the plant diseases are caused by fungi.Plant pathogenic fungi which lead to plant rot,cataplexis and plant mortality,can greatly reduce crop yields,for another,some pathogenic fungi can secrete toxins,causing food safety issues related to human health.Thus,great impacts on crop yields and human life caused by fungal diseases will be harmful to human beings.Furthermore,there are some fungi that are pathogenic to human beings.Conditional human fungal infections are mainly caused by Aspergillus,Candida and Cryptococcus.With the rising incidence of fungal infections and the emergence of drug-resistant strains in clinical practice,challenges to clinically limited antifungal drugs have gradually emerged.Moreover,the mortality of invasive fungal infections（IFIs）remains high.Therefore,it is of great significance to find new antifungal drugs or lead compounds.While considering endophytes of medical plants as a potential source with the ability to produce natural products with biological activities including antimicrobial activity,there is a lack of information about the antifungal abilities of endophytic bacteria in Lycoris plants.Therefore,endophytic bacteria isolated from Lycoris radiata,Lycoris aurea and Lycoris chinensis were screened for their antifungal abilities.Finally,30 endophytic bacteria that could antagonize pathogenic fungi were obtained.Among these strains,endophytic bacterium HDXY-02 isolated from medicinal plant Lycoris aurea showed a broad-spectrum antifungal activity against several plant and human fungal pathogens.Based on 16S r DNA and 16-23S r DNA spacer gene sequence analysis,combined with species-specific primer amplification,physiological and biochemical experiments,strain HDXY-02 was identified as Burkholderia gladioli.Furthermore,the purified component contributing to its antifungal activity was identified to be toxoflavin,a yellow compound possessing a pyrimido[5,4-e][1,2,4]triazine ring.In vitro bioactivity studies demonstrated that purified toxoflavin from B.gladioli HDXY-02 cultures had a significant antifungal activity against several pathogenic fungi,including Aspergillus fumigatus,Candida albicans,Cryptococcus neoformans and several phytopathogenic fungi.More importantly,toxoflavin was effective not only for azole-sensitive A.fumigatus,but also for azole-resistant mutants（cyp51A and non-cyp51A）of A.fumigatus,and the minimum inhibitory concentrations（MICs）of toxoflavin against azole-resistant strains were 64μg/m L.It was suggested that toxoflavin could be used as a candidate drug for drug-resistant A.fumigatus.In addition to its antifungal activity,toxoflavin also have good inhibitory activity on Lovo cells.Due to its various biological activities,toxoflavin is expected to be an alternative fungicide and antitumor drug after chemical modification.In order to improve the toxoflavin production of B.gladioli HDXY-02,the conditions and medium for toxoflavin fermentation of B.gladioli HDXY-02 was optimized.Moreover,under the determined optimal culture conditions,the toxoflavin yield by B.gladioli HDXY-02 performed with optimized medium composition was1533 mg/L in the shake flask verification experiment,which is 4.29 times compared with the starting KMB medium production of 357 mg/L.However,we found that the standard strain of B.gladioli,CICC10574 purchased from China Center of Industrial Culture Collection（CICC）produced almost no toxoflavin while B.gladioli HDXY-02 could produce toxoflavin under the same condition.Due to the difference between B.gladioli CICC10574 and B.gladioli HDXY-02,further study need to be carried out.Through gene amplification and sequence comparison,we found that genes related to toxoflavin synthesis in B.gladioli CICC10574 were intact.Therefore,it is believed that the difference in toxoflavin production between B.gladioli HDXY-02 and B.gladioli CICC10574 is not caused by the loss of genes and pathway related to toxoflavin synthesis as reported.Results of transcriptome sequencing and real-time quantitative PCR showed that the difference in the toxoflavin synthesis between B.gladioli HDXY-02 and B.gladioli CICC10574 was due to the different expression levels of genes related to toxoflavin synthesis.Thus,it suggested that the main reason for the high production of toxoflavin in B.gladioli HDXY-02 was due to the highly expressed genes related to toxoflavin synthesis.In addition,many of the differentially expressed genes were found to be related to signal transduction in B.gladioli HDXY-02.It was shown that Na Cl and KCl could activate the production of toxoflavin and the expression of genes related to toxoflavin production in B.gladioli CICC10574,while Na Cl KCl and Ca Cl₂ could raise the toxoflavin synthesis in B.gladioli HDXY-02 to some extent.Nitric Oxide（NO）can significantly promote toxoflavin synthesis and the expression of genes related to toxoflavin production in B.gladioli HDXY-02.At the same time,the intracellular cyclic diguanylate（c-di-GMP）level also increased,suggesting that the NO/c-di-GMP signaling pathway may be involved in activating the transcription of genes related to toxoflavin production.According to our research,B.gladioli HDXY-02 could not be co-cultured with other Gram-negative bacteria,including Escherichia coli Top10 and B.gladioli CICC10574.The motility of B.gladioli HDXY-02 was reduced,and it was more inclined to form biofilm.We speculated that the feature of B.gladioli HDXY-02 may be related to its living environment.As a result,B.gladioli HDXY-02 synthesized a large amount of toxoflavin to inhibit the growth of other organisms to survive.Presumably,the synthesis and fine regulation of toxoflavin in B.gladioli HDXY-02have been adjusted by certain universal and specific mechanism for survival.It could also be inferred from the above results that the high production of toxoflavin in B.gladioli HDXY-02 is likely to be related to its own defects.Although excessive synthesis of toxoflavin can inhibit the growth of other microorganisms,it may sacrifice something at the expense of other attributes.In conclusion,our findings provide a promising biosynthetic resource for producing a new antifungal reagent toxoflavin,from B.gladioli HDXY-02.On the other hand,the research on the regulation mechanisms of toxoflavin in B.gladioli may provide guidance for further engineering improvements of toxoflavin yield.It will also provide more information about the regulation of other bacterial secondary metabolite biosynthesis.