Individual Ganoderic Acids Research Articles

The Biological Activity of Ganoderma lucidum on Neurodegenerative Diseases: The Interplay between Different Active Compounds and the Pathological Hallmarks.

Neurodegenerative diseases represent a cluster of conditions characterized by the progressive degeneration of the structure and function of the nervous system. Despite significant advancements in understanding these diseases, therapeutic options remain limited. The medicinal mushroom Ganoderma lucidum has been recognized for its comprehensive array of bioactive compounds with anti-inflammatory and antioxidative effects, which possess potential neuroprotective properties. This literature review collates and examines the existing research on the bioactivity of active compounds and extracts from Ganoderma lucidum in modulating the pathological hallmarks of neurodegenerative diseases. The structural information and preparation processes of specific components, such as individual ganoderic acids and unique fractions of polysaccharides, are presented in detail to facilitate structure-activity relationship research and scale up the investigation of in vivo pharmacology. The mechanisms of these components against neurodegenerative diseases are discussed on multiple levels and elaborately categorized in different patterns. It is clearly presented from the patterns that most polysaccharides of Ganoderma lucidum possess neurotrophic effects, while ganoderic acids preferentially target specific pathogenic proteins as well as regulating autophagy. Further clinical trials are necessary to assess the translational potential of these components in the development of novel multi-target drugs for neurodegenerative diseases.

Breeding a new Ganoderma lucidum strain with increased contents of individual ganoderic acids by mono-mono crossing of genetically modified monokaryons.

Ganoderic acids (GAs) are major functional components of Ganoderma lucidum. The study aimed to breed a new G. lucidum strain with increased contents of individual GAs. Two mating-compatible monokaryotic strains, G. 260125 and G. 260124, were successfully isolated from the dikaryotic G. lucidum CGMCC 5.0026 via protoplast formation and regeneration. The Vitreoscilla hemoglobin gene (vgb) and squalene synthase gene (sqs) were overexpressed in the monokaryotic G. 260124 and G. 260125 strain, respectively. Mating between the G. 260124 strain overexpressing vgb and the G. 260125 strain overexpressing sqs resulted in the formation of the new hybrid dikaryotic G. lucidum strain sqs-vgb. The maximum contents of ganoderic acid (GA)-T, GA-Me, and GA-P in the fruiting body of the mated sqs-vgb strain were 23.1, 15.3, and 39.8 μg/g dry weight (DW), respectively, 2.23-, 1.75-, and 2.69-fold greater than those in G. lucidum 5.0026. The squalene and lanosterol contents increased 2.35- and 1.75-fold, respectively, in the fruiting body of the mated sqs-vgb strain compared with those in the G. lucidum 5.0026. In addition, the maximum expression levels of the sqs and lanosterol synthase gene (ls) were increased 3.23- and 2.13-fold, respectively, in the mated sqs-vgb strain. In summary, we developed a new G. lucidum strain with higher contents of individual GAs in the fruiting body by integrating genetic engineering and mono-mono crossing.

CRISPR-Cas9 assisted in-situ complementation of functional genes in the basidiomycete Ganoderma lucidum

Basidiomycetes can produce various valuable secondary metabolites with bioactivities. However, the low titer of metabolites has greatly limited their application. Currently, shortage of mutant complementation techniques, especially in-situ complementation, impeded the elucidation of their metabolites biosynthesis pathways. In this work, Ganoderma lucidum, a traditional Chinese medicinal basidiomycete, which can produce various bioactive ganoderic acids (triterpenoids), was used; and a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) mediated in-situ complementation system for two functional genes was established. For a disruptant of gl26097, which encodes a Cys2-His2 (C2H2)-type zinc finger transcription factor involved in calmodulin-calcineurin signaling, one in-situ complemented strain of 8 transformants was obtained,concluded by sequencing confirmation. For the function of gl26097, both the cell growth and content of four identified individual ganoderic acids (GAs) were restored in the complemented strain compared to the gl26097 disruptant. For a disruptant of cyp5150l8, which is responsible for transforming lanosterol to a GA, one in-situ complemented strain from 8 transformants was obtained as confirmed by sequencing. The titer of four identified GAs increased significantly in the complemented strain compared to the cyp5150l8 disruptant. To the best of our knowledge, this is the first research report on applying CRISPR-Cas9 to complement mutants in-situ in basidiomycetes. The CRISPR-Cas9 assisted gene complementation technology will be helpful to study gene functions and GA biosynthesis pathway in G. lucidum, which may be also applied to other basidiomycetes.

Enhanced production of individual ganoderic acids by integrating Vitreoscilla haemoglobin expression and calcium ion induction in liquid static cultures of Ganoderma lingzhi.

SummaryGanoderic acids produced by Ganoderma exhibit anticancer and antimetastatic activities. A novel approach by combining Vitreoscilla haemoglobin (VHb) expression and calcium ion induction was developed to enhance ganoderic acid (GA) production in liquid static cultures of G. lingzhi. The maximum contents of GA‐O, GA‐S and GA‐Me were 1451.33 ± 67.50, 1431.23 ± 79.74 and 1283.81 ± 85.13 μg per 100 mg cell weight, respectively under the integrated approach, which are the highest contents as ever reported in Ganoderma. The contents of squalene and lanosterol were increased by 2.0‐ and 3.0‐fold in this case compared with those in the control. The transcription levels of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase, farnesyl‐diphosphate synthase, squalene synthase and cytochrome P450 CYP5150L8 were upregulated by 2.56‐, 3.31‐, 2.59‐ and 6.12‐fold respectively. Additionally, the expression of VHb improved the ratio of type I to type II GA in liquid static cultivation of G. lingzhi. The transcription levels of cyp512a2, cyp512v2 and cyp512a13, candidate cytochrome P450 genes involved in oxidative modification of the lanostane skeleton in GA biosynthesis, were also increased by 2.28‐, 2.65‐ and 3.54‐fold in the VHb‐expressing strain respectively. Our results illustrated that the approach described here efficiently improved GA production in G. lingzhi fermentation.

Enhancement of ganoderic acid production by constitutively expressing Vitreoscilla hemoglobin gene in Ganoderma lucidum

The Vitreoscilla hemoglobin (VHb) gene was expressed in Ganoderma lucidum to enhance antitumor ganoderic acid (GA) production. The effects of VHb expression on the accumulation of GAs and lanosterol (intermediate) and the transcription of GA biosynthesis genes were also investigated. In VHb-expressing G. lucidum, the maximum concentrations of four individual GAs (GA-S, GA-T, GA-Mk and GA-Me) were 19.1±1.8, 34.6±2.1, 191.5±13.1 and 45.2±2.8μg/100mg dry weight, respectively, which were 1.4-, 2.2, 1.9- and 2.0-fold higher than those obtained in the wild-type strain. Moreover, the maximum lanosterol concentration in the strain expressing VHb was 1.28-fold lower than that in the wild-type strain. The transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, and lanosterol synthase genes were up-regulated by 1.6-, 1.5-, and 1.6-fold, respectively, in the strain expressing VHb. This work is beneficial in developing an efficient fermentation process for the hyperproduction of GAs.

Enhanced accumulation of individual ganoderic acids in a submerged culture of Ganoderma lucidum by the overexpression of squalene synthase gene

Ganoderic acids (GAs) produced by Ganoderma lucidum exhibit antitumor and antimetastasis activities. This study aimed to improve the production of individual GAs by engineering the biosynthetic pathway of GAs in G. lucidum through the overexpression of squalene synthase (SQS) gene. SQS catalyzes the first enzymatic step from the mevalonate pathway toward triterpene biosynthesis. The effects of SQS gene overexpression on the accumulation of individual GAs and their intermediates (squalene and lanosterol) by a submerged culture of G. lucidum and on the transcription levels of GA biosynthesis genes in this mushroom were investigated. The maximum contents of GA-Mk, GA-T, GA-Me, and GA-S in G. lucidum overexpressing the SQS gene were 16, 40, 43, and 53μg/100mg dry cell weight, respectively, which were 2.86-, 2.67-, 1.95-, and 1.25-fold of those obtained in wild-type strain (WT). The maximum contents of squalene and lanosterol in the SQS gene-overexpression strain were 1.55- and 1.68-fold higher than those of the WT strain. The transcription levels of the biosynthetic genes encoding SQS and lanosterol synthase were up-regulated by 15.6- and 1.93-fold, respectively, in G. lucidum overexpressing the SQS gene, suggesting that increased GA biosynthesis may result from a higher expression of those genes.

Oxygen limitation improves ganoderic acid biosynthesis in submerged cultivation of Ganoderma lucidum

The effects of oxygen limitation on the production of ganoderic acid (GA), a secondary metabolite with antitumor activity, and on transcription levels of triterpene biosynthesis genes were investigated in liquid cultures of Ganoderma lucidum. A low oxygen supply level was beneficial to total GA biosynthesis, but negative to the cell growth. The higher GA production was obtained under micro-aerobic conditions (i.e. initial overall kLa values at 0.02 and 0.13/h). The maximum GA production of 272.3 ± 11.5 mg/L was obtained at an initial overall kLa of 0.13/h, which was 1.7-fold that at a normal cultivation condition (an initial kLa of 5.51/h). For four major individual GAs, the production level of GA-Mk, -T, -S, and -Me in the hypoxia-induced cells was increased by 50, 87, 62, and 111%, compared with that of the control, respectively. Meanwhile, the transcriptions of four key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate-5-pyrophosphate decarboxylase, squalene synthase and squalene epoxidase in the triterpene biosynthetic pathway were up-regulated under the hypoxia condition (at an initial kLa of 0.13/h). Reactive oxygen species was generated in response to hypoxia, which seemed to be involved in the regulation of GA biosynthesis. The information obtained provides an insight into the role of oxygen limitation in the GA biosynthesis, and will be helpful for optimizing the fermentation process on a large scale.

Effect of solid-medium coupled with reactive oxygen species on ganoderic acid biosynthesis and MAP kinase phosphorylation in Ganoderma lucidum

Ganoderma lucidum is used as a functional food and medicinal mushroom in Asia. Biosynthesis of its active ingredients, the ganoderic acids (GAs), together with signal transduction related to the fungal biology of G. lucidum, are critical for this application. However, regulation of G. lucidum GA biosynthesis and signal transduction remains enigmatic. We investigated the role of reactive oxygen species (ROS) on biosynthesis of individual GAs and on MAPK (mitogen-activated protein kinase) signaling. Antioxidant treatment decreased lanosta-7,9(11), 24-trien-3α-01-26-oic acid (ganoderic acid 24) and total GA production. Cultures incubated with the pro-oxidant CDNB (1-chloro-2,4-dinitrobenzene) or H2O2, increased production of ganoderic acid 24 and total GAs were found. Interestingly, CDNB reduced RNA expression of the squalene synthase and lanosterol synthase, indicating that both genes may not be bottlenecks in GA induction. H2O2 induced phosphorylation of the proteins Hog-1 and Fus3, which are homologs of the mammalian MAPKs p38 and ERK (extracellular signal-related kinases), respectively. CDNB also enhanced phosphorylation of Hog1 but had no effect on Fus3. To the best of our knowledge, this is the first report indicating that ROS regulate the biosynthesis of individual GAs and affect MAPK signaling in G. lucidum. This study also identifies the potential application of ROS to the G. lucidum functional food industry.

Enhanced biosynthetic gene expressions and production of ganoderic acids in static liquid culture of Ganoderma lucidum under phenobarbital induction

Static liquid culture of Ganoderma lucidum, a traditional Chinese medicinal mushroom, is a proven technology for producing ganoderic acids, which are secondary metabolites that possess antitumor properties. In this work, the addition of phenobarbital, a P450 inducer, was used to enhance the production of total and individual ganoderic acids in a two-stage cultivation involving a period of initial shake flask culture followed by static liquid culture of G. lucidum. The dosage and time of phenobarbital induction were critical for the enhanced production of ganoderic acids. The addition of 100 muM (final concentration) phenobarbital on day 5 after the shake flask culture was converted to the static liquid culture was found to be optimal, resulting in a maximal amount of total ganoderic acids of 41.4 +/- 0.6 mg/g cell dry weight and increases in the levels of ganoderic acid-Mk, -T, -S, and -Me in the treated cells by 47%, 28%, 36%, and 64%, respectively. Meanwhile, the accumulation of lanosterol, a key intermediate, was found to decrease and transcriptions of three key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, and lanosterol synthase in the triterpene biosynthetic pathway were up-regulated under phenobarbital induction. This work demonstrated a useful strategy for the enhanced production of ganoderic acids by G. lucidum.

Comparison of individual ganoderic acids production and identification of differentially expressed genes between liquid static culture and shaking culture of Ganoderma lucidum

Comparison of individual ganoderic acids production and identification of differentially expressed genes between liquid static culture and shaking culture of Ganoderma lucidum

Effect of oxygen concentration in gas phase on sporulation and individual ganoderic acids accumulation in liquid static culture of Ganoderma lucidum

Effects of oxygen concentration within 21-100% in gaseous phase on the morphology and ganoderic acids (GAs) production by Ganoderma lucidum in liquid static culture were studied. A higher oxygen concentration increased individual GAs production, and more spores and higher total GA content were obtained at an oxygen level of 80%.

Production of individual ganoderic acids and expression of biosynthetic genes in liquid static and shaking cultures of Ganoderma lucidum

Two-stage culture was efficient in enhancing total ganoderic acid (GA) production by Ganoderma lucidum (Fang and Zhong, Biotechnol Prog 18:51-54, 2002). As different GAs have different bioactivities, it is critical to understand the kinetics of individual GA production during fermentation, but no related information is yet available. To understand the regulation of GA biosynthesis, investigation of the accumulation of intermediate (lanosterol) and by-product (ergosterol) and of the expression of three important biosynthetic genes was also conducted in liquid shaking and static cultures of G. lucidum. The results showed that the content of individual GAs increased rapidly in the liquid static culture, and their maximum value was 6- to 25-fold that of shaking culture while lanosterol content in the former was lower than the latter. The transcript of squalene synthase (SQS), lanosterol synthase and 3-hydroxy-3-methylglutaryl coenzyme A reductase in liquid static culture was 4.3-, 2.1-, and 1.9-fold that of the shaking culture, respectively. Higher GA content in liquid static culture was related to increased transcription of those genes especially SQS. The work is helpful to the production of individual GAs and provided an insight into why the liquid static culture was superior to the shaking culture in view of biosynthetic gene expression.