Stimulatory Effects of Oleci Acid and Fungal Elicitor on Betulinic Acid Production by Submerged Cultivation of Medicinal Mushroom Inonotus obliquus

To evaluate the novel strategy of oleic acid and fungal elicitor (made from Aspergillus niger) to elicit betulinic acid biosynthesis in medicinal mushroom Inonotus obliquus, we conduct the stimulatory effects investigation for synthesizing betulinic acid from betulin. HPLC results indicated oleic acid and fungal elicitor were effective stimulators. The supplementation of 1.0 g/L oleic acid led to the highest increase of betulinic acid either in dry mycelia or fermentation broth by 2-fold of the control. Fungal elicitor at 45 mg/L markedly increases mycelia growth by 146.0% and enhance intracellular betulinic acid accumulation by 429.5% as compared to the controls. Quantification of transcription levels determined that oleic acid, fungal elicitor and their combinations could induce the expressions of key genes involved in betulinic acid biosynthesis, such as HMG-CoA reductase and squalene synthase. These findings indicated that oleic acid and fungal elicitor could enhance betulinic acid metabolism by up-regulating key genes expression.

Transcriptome analysis of the regulation of natamycin biosynthesis in Streptomyces natalensis HW-2 by fungal elicitor

Background Natamycin is a polyene macrolide polyketide antibiotics and used in 150 countries as a natural food preservative. Streptomyces natalensis is an important producer. Elicitation had been approved to be an effective method to improve the biosynthesis of secondary metabolites. Fungal elicitor from Penicillium chrysogenum AS 3.5163 showed inductive effect on the biosynthesis of natamycin in S. natalensis HW-2 fermentation. However, regarding the global gene expression of natamycin in response to fungal elicitor is not still reported. Results RNA-Seq analysis showed that there were 1265 differential expression genes (DEGs) at 40 h and 2196 DEGs at 80 h. The fungal elicitor had stronger effects on the transcription level of S. natalensis HW-2 at 80 h than that at 40 h. Gene Ontology (GO) enrichment analysis of DEGs showed significant enrichment in biological processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the fungal elicitor mainly affected the expression levels of some genes about cellular process, metabolism and genetic information, especially in pentose phosphate pathway (PPP), glycolytic pathway (EMP) and tricarboxylic acid cycle (TCA). KEGG pathway showed that fungal elicitor had a greater influence on the metabolism of branched-chain amino acids (BCAAs). Among them, 23 DEGs associated with BCAAs metabolism were up-regulated or down-regulated. The supplementation experiment with BCAAs confirmed that 0.2 g/L of L-Ile and 0.5 g/L of L-Val increased natamycin yield by 17.6% and 37.8%, respectively. Fungal elicitor also up-regulated the transcriptional levels of most of the enzymes associated with the biosynthesis of natamycin and two important transcription regulators ( pimR and pimM ). To confirm the accuracy of RNA-Seq, the results of qPCR showed that these gene expression levels were in agreement with the transcription changes by RNA-Seq. Conclusion In this study, the change of transcriptional levels in S. natalensis HW-2 under treated with the fungal elicitor was firstly reported. The major finding of our comparative transcriptome analysis is that the fungal elicitor improves the supply of precursor, and alters the expression of natamycin related genes and regulator of secondary metabolism. From our results, we conclude that regulatory alterations are important factors for the enhancement of natamycin.

Isolation and Characterization of Plant Growth-Promoting Endophytic Fungi from the Roots of Dendrobium moniliforme

The present study aims to identify the diverse endophytic fungi residing in the roots of Dendrobium moniliforme and their role in plant growth and development. Nine endophytic fungi were isolated from the root sections and characterized by molecular technique. Quantification of the indole acetic acid (IAA) compound by these endophytes was done. Further, Chemical profiling of R11 and R13 fungi was done by Gas Chromatography-Mass Spectroscopy (GC-MS). Asymbiotic seed derived protocorms of Rhynchostylis retusa was used for the plant growth assay to investigate the growth promoting activities of the fungal elicitor prepared from the isolated fungi from D. moniliforme. Among the isolated fungi, the relative dominant fungus was Fusarium sp. The R13 and R6 fungi were identified only at the genus level which concludes the fungi are of new species or strain. The indole acetic acid production was relatively higher in R10. Bioactive compound diversity was observed in the organic extract of R11 and R6. The presence of phenolic compound and essential oil suggest their contribution for the antimicrobial and antioxidant properties to their host plant, D. moniliforme. The plant growth assay result concluded, the fungal elicitor prepared from R10, Colletotrichum alatae was the best among all other for the plant growth activities.

Effect of Aspergillus flavus Fungal Elicitor on the Production of Terpenoid Indole Alkaloids in Catharanthus roseus Cambial Meristematic Cells

This study reported the inducing effect of Aspergillus flavus fungal elicitor on biosynthesis of terpenoid indole alkaloids (TIAs) in Catharanthus roseus cambial meristematic cells (CMCs) and its inducing mechanism. According to the results determined by HPLC and HPLC-MS/MS, the optimal condition of the A. flavus elicitor was as follows: after suspension culture of C. roseus CMCs for 6 day, 25 mg/L A. flavus mycelium elicitor were added, and the CMC suspensions were further cultured for another 48 h. In this condition, the contents of vindoline, catharanthine, and ajmaline were 1.45-, 3.29-, and 2.14-times as high as those of the control group, respectively. Transcriptome analysis showed that D4H, G10H, GES, IRS, LAMT, SGD, STR, TDC, and ORCA3 were involved in the regulation of this induction process. The results of qRT-PCR indicated that the increasing accumulations of vindoline, catharanthine, and ajmaline in C. roseus CMCs were correlated with the increasing expression of the above genes. Therefore, A. flavus fungal elicitor could enhance the TIA production of C. roseus CMCs, which might be used as an alternative biotechnological resource for obtaining bioactive alkaloids.