A Novel Approach to Enhancing Ganoderic Acid Production by Ganoderma lucidum Using Apoptosis Induction

Ganoderma lucidum is one of most widely used herbal medicine and functional food in Asia, and ganoderic acids (GAs) are its active ingredients. Regulation of GA biosynthesis and enhancing GA production are critical to using G. lucidum as a medicine. However, regulation of GA biosynthesis by various signaling remains poorly understood. This study investigated the role of apoptosis signaling on GA biosynthesis and presented a novel approach, namely apoptosis induction, to increasing GA production. Aspirin was able to induce cell apoptosis in G. lucidum, which was identified by terminal deoxynucleotidyl transferase mediated dUPT nick end labeling assay positive staining and a condensed nuclear morphology. The maximum induction of lanosta-7,9(11), 24-trien-3α-01-26-oic acid (ganoderic acid 24, GA24) production and total GA production by aspirin were 2.7-fold and 2.8-fold, respectively, after 1 day. Significantly lower levels of GA 24 and total GAs were obtained after regular fungal culture for 1.5 months. ROS accumulation and phosphorylation of Hog-1 kinase, a putative homolog of MAPK p38 in mammals, occurred after aspirin treatment indicating that both factors may be involved in GA biosynthetic regulation. However, aspirin also reduced expression of the squalene synthase and lanosterol synthase coding genes, suggesting that these genes are not critical for GA induction. To the best of our knowledge, this is the first report showing that GA biosynthesis is linked to fungal apoptosis and provides a new approach to enhancing secondary metabolite production in fungi.     

Biotechnological production and application of ganoderic acids

Ganoderic acids (GAs), a kind of highly oxygenated lanostane-type triterpenoids, are important bioactive constituents of the famous medicinal mushroom Ganoderma lucidum. They have received wide attention in recent years due to extraordinarily pharmacological functions. Submerged fermentation of G. lucidum is viewed as a promising technology for production of GAs, and substantial efforts have been devoted to process development for enhancing GA production in the last decade. This article reviews recent publication about fermentative production of GAs and their potential applications, especially the progresses toward manipulation of fermentation conditions and bioprocessing strategies are summarized. The biosynthetic pathway of GAs is also outlined.     

Effects of microbial volatile organic compounds on Ganoderma lucidum growth and ganoderic acids production in Co-v-cultures (volatile co-cultures)

Abstract

Co-v-culture (co-cultivations of physically separated microbes that only interact through the air) systems were designed to investigate the effects of microbial volatile organic compounds (mVOCs) from about 20 different microbes, on a medicinal fungus, Ganoderma lucidum. For more accuracy in co-cultivations, a novel synchronized cultivation approach was tested for culturing G. lucidum. The hyphal growth of G. lucidum and the content of its ganoderic acids (GAs) were measured. In almost all of the co-v-cultures, there was an inhibiting effect on hyphal growth and a promoting effect on GAs contents. In inducing GAs production, Bacillus cereus PTCC 1247 and Pseudomonas aeruginosa UTMC 1404 were the most effective ones, as, compared to control cultures, GAs content increased 2.8 fold. Comparing different co-v-cultivations demonstrated that the concentrations of mVOCs, oxygen, and carbon dioxide were the main players in co-v-cultures. No correlation was found between hyphal growth and GAs production. Strains of the same species imposed totally different effects on hyphal growth or GAs production. This study has investigated the effects of mVOCs on G. lucidum for the first time. Moreover, it suggests that co-v-cultivation may be a promising biotechnological approach to improve the production in G. lucidum.     

灵芝悬浮培养中添加微颗粒Talc对灵芝酸产生的影响

灵芝酸是灵芝中重要的药理活性物质,其低产量限制了它的广泛应用和深入研究,高效提高液体发酵中灵芝酸的含量十分必要。以CGMCC 5.65为材料,在悬浮培养条件下,研究添加微颗粒Talc对灵芝酸产生的影响。结果表明,微颗粒Talc添加显著减小了灵芝细胞粒径,对照组为(3.33±0.16)mm,15g/L微颗粒Talc添加组为(2.04±0.12)mm。灵芝细胞中单体灵芝酸和总灵芝酸的含量在微颗粒Talc添加条件下也显著提高。15g/L微颗粒Talc添加组的总灵芝酸含量达到(1.51±0.02)mg/100mg细胞干重,GA-Mk、GA-T和GA-Me的含量最高为(6.02±0.29)、(5.08±0.14)和(1.71±0.09)μg/100mg细胞干重,分别是对照的1.6、4、1.9和1.4倍。另外,15g/L微颗粒Talc添加条件下鲨烯和羊毛甾醇的最大积累量分别为(3.69±0.23)和(34.86±6.41)μg/100mg细胞干重,是对照的2.6和4.2倍;灵芝酸生物合成途径关键基因fps和cyp-5150l8的表达量最高为对照的2.35和1.53倍。     

Bioconversion of a ganoderic acid 3-hydroxy-lanosta-8,24-dien-26-oic acid by a crude enzyme from Ganoderma lucidum

Ganoderic acids (GAs) are oxygenated lanostane-type triterpenoids from the traditional medicinal mushroom Ganoderma lucidum and of significant biological activities. Although a ganoderic acid 3-hydroxy-lanosta-8,24-dien-26-oic acid (HLDOA) was found to be biosynthesized from lanosterol, further post-modification of HLDOA is yet unclear. In this work, by using HLDOA as the substrate and a crude enzyme from G. lucidum as the biocatalyst, we observed a new peak in liquid chromatography from the reaction system. The product was purified and identified to be 3-oxo-lanosta-8,24-dien-26-oic acid (OLDOA), which may be converted from HLDOA by a putative dehydrogenase of G. lucidum. The work is useful to future manufacture of GAs as well as their biosynthetic pathway elucidation.     

Significance of protein elicitor isolated from Tuber melanosporum on the production of ganoderic acid and Ganoderma polysaccharides during the fermentation of Ganoderma lucidum

Under the elicitation of protein elicitor isolated from the culture mycelia of Tuber melanosporum, the biosynthesis of ganoderic acids (GA) was significantly stimulated during Ganoderma lucidum fermentation. Compared with our previous results that, GA content was inhibited by polysaccharide elicitor isolated from T. melanosporum, while improved by the elicitor of polysaccharide and protein, protein was identified to be the exact component inducing GA biosynthesis in this work. G. lucidum cell growth was significantly inhibited by elicitor of polysaccharide and protein, and polysaccharide elicitor did not inhibit the cell growth. In this work, the remarkable inhibition on the cell growth was considerably eliminated under the elicitation of protein elicitor isolated from T. melanosporum. These suggested maybe the interaction of polysaccharide and protein components existed in the inhibition on the cell growth of G. lucidum. Not only GA content but also total GA accumulation obtained the highest values after the elicitation of protein elicitor. The maximal GA production of 260.5 ± 5.6 mg/L was 31.2% higher than the control. Under the elicitation of protein elicitor, the production of extracellular polysaccharide (EPS) and the content of intracellular polysaccharide (IPS) were also enhanced; however, total IPS accumulation was lower. GA biosynthesis was also significantly affected by the addition time of protein elicitor, whose optimal value was the culture of day 4.     

Efficient biosynthesis of antitumor ganoderic acid HLDOA using a dual tunable system for optimizing the expression of CYP5150L8 and a Ganoderma P450 reductase

Ganoderic acid 3-hydroxy-lanosta-8,24-dien-26-oic acid (GA-HLDOA), an antitumor triterpenoid from the traditional Chinese medicinal higher fungus Ganoderma lucidum, is considered as a key precursor for biosynthesizing other ganoderic acids (GAs) with superior antitumor activities. Our previous study identified CYP5150L8 from G. lucidum as a lanosterol oxidase, and achieved heterologous biosynthesis of GA-HLDOA in Saccharomyces cerevisiae. However, low production of GA-HLDOA in either G. lucidum or heterologous host hindered its further investigation and application. In this study, we constructed a dual tunable system for balancing the expression of CYP5150L8 and a Ganoderma P450 reductase iGLCPR, and performed a comprehensive optimization of CYP5150L8 expression, iGLCPR expression, and glycerol usage. Then, we investigated the fermentation behavior of the best strain in optimized condition in flask and achieved 154.45 mg/L GA-HLDOA production, which was 10.7-fold higher compared with previous report. This study may facilitate the wide-spread application of GA-HLDOA and the discovery of unknown cytochrome P450s in downstream GAs biosynthesis.     

Isolation and characterization of a novel Ganoderma lucidum gene that differentially expressed between shaking culture and liquid static culture

Suppression subtractive hybridization (SSH) was preformed to investigate the differences of gene expression between the shaking culture mode and the liquid static culture mode which favors ganoderic acids production in Ganoderma lucidum. One novel gene preferentially expressed in liquid static culture was identified and analyzed. Its full length cDNA sequence and the 5′-flanking region were then obtained by rapid amplification of cDNA ends (RACE) and self-formed adaptor PCR (SEFA-PCR), respectively. Nucleotide sequence of the gene is not homologous to any of the known Ganoderma genes. The sequence analysis revealed that the open reading frame of this gene encodes a protein of 371 amino acids that has high homology with the mitogen-activated protein kinase (MAPK) of other five species-Postia (97%), Coprinopsis (91%), Neurospora (86%), Aspergillus (83%), and Saccharomyces (80%)-so that it can be defined as a G. lucidum MAPK gene (GenBank accession number: JF781125). Computer assisted analysis revealed that this new G. lucidum MAPK gene contains thirteen exons and twelve introns. The quantitative real-time RT-PCR (qRT-PCR) analysis showed that this new gene had a much higher expression level in liquid static culture than in traditional shaking culture. Results of this research established a good foundation for further study on the functions of the G. lucidum MAPK at the molecular level.     

Production of ganoderic acid by Ganoderma lucidum RCKB-2010 and its therapeutic potential

The newly isolated basidiomycetous fungus, identified as Ganoderma lucidum RCKB-2010 was tested for production of ganoderic acid (GA) under submerged fermentation conditions. Production of GA under liquid static cultivation condition was found to be 2,755.88 mg L^?1 on the 25th day of incubation, whereas under shaking cultivation conditions the maximum production of GA was observed to be 373.75 mg L^?1. ¹H NMR analysis revealed clearly that the fungal extracts possessed a lanostane skeleton, confirming the presence of GA. Interestingly, GA was found to have potential to inhibit the proliferation of HeLa cells and U87 human glioma cells in a dose dependent manner. In addition, GA was also found to possess antibacterial activity, exhibiting a minimal inhibitory concentration of 0.25 mg mL^?1 against standard strains of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis. GA produced in the present study holds potential as a potent anticancer agent.