Enhancement of Ganoderic Acid Accumulation by Overexpression of an N-Terminally Truncated 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene in the Basidiomycete Ganoderma lucidum

ABSTRACTGanoderic acids produced byGanoderma lucidum, a well-known traditional Chinese medicinal mushroom, exhibit antitumor and antimetastasis activities. Genetic modification ofG. lucidumis difficult but critical for the enhancement of cellular accumulation of ganoderic acids. In this study, a homologous genetic transformation system forG. lucidumwas developed for the first time using mutatedsdhB, encoding the iron-sulfur protein subunit of succinate dehydrogenase, as a selection marker. The truncatedG. lucidumgene encoding the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) was overexpressed by using theAgrobacterium tumefaciens-mediated transformation system. The results showed that the mutatedsdhBsuccessfully conferred carboxin resistance upon transformation. Most of the integrated transfer DNA (T-DNA) appeared as a single copy in the genome. Moreover, deregulated constitutive overexpression of the HMGR gene led to a 2-fold increase in ganoderic acid content. It also increased the accumulation of intermediates (squalene and lanosterol) and the upregulation of downstream genes such as those of farnesyl pyrophosphate synthase, squalene synthase, and lanosterol synthase. This study demonstrates that transgenic basidiomyceteG. lucidumis a promising system to achieve metabolic engineering of the ganoderic acid pathway.

Download Full-text

Increased production of ganoderic acids by overexpression of homologous farnesyl diphosphate synthase and kinetic modeling of ganoderic acid production in Ganoderma lucidum

Microbial Cell Factories ◽

10.1186/s12934-019-1164-3 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 4

Author(s):

Yu Fei ◽

Na Li ◽

De-Huai Zhang ◽

Jun-Wei Xu

Keyword(s):

Kinetic Modeling ◽

Acid Production ◽

Ganoderma Lucidum ◽

Farnesyl Diphosphate ◽

Farnesyl Diphosphate Synthase ◽

Ganoderic Acid ◽

Ganoderic Acids ◽

Ganoderic Acid Production

Download Full-text

Enhanced Production of a Plant Monoterpene by Overexpression of the 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Catalytic Domain in Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.02987-09 ◽

2010 ◽

Vol 76 (19) ◽

pp. 6449-6454 ◽

Cited By ~ 61

Author(s):

Juan Rico ◽

Ester Pardo ◽

Margarita Orejas

Keyword(s):

Saccharomyces Cerevisiae ◽

Coenzyme A ◽

Catalytic Domain ◽

Laboratory Strain ◽

Strain Engineering ◽

Enhanced Production ◽

Wine Strain ◽

Coa Reductase ◽

Rate Limiting ◽

Coenzyme A Reductase

ABSTRACT Linalool production was evaluated in different Saccharomyces cerevisiae strains expressing the Clarkia breweri linalool synthase gene (LIS). The wine strain T73 was shown to produce higher levels of linalool than conventional laboratory strains (i.e., almost three times the amount). The performance of this strain was further enhanced by manipulating the endogenous mevalonate (MVA) pathway: deregulated overexpression of the rate-limiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) doubled linalool production. In a haploid laboratory strain, engineering of this key step also improved linalool yield.

Download Full-text

Effects of overproduction of the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase on squalene synthesis in Saccharomyces cerevisiae.

Applied and Environmental Microbiology ◽

10.1128/aem.63.9.3341-3344.1997 ◽

1997 ◽

Vol 63 (9) ◽

pp. 3341-3344 ◽

Cited By ~ 137

Author(s):

K A Donald ◽

R Y Hampton ◽

I B Fritz

Keyword(s):

Saccharomyces Cerevisiae ◽

Coenzyme A ◽

Catalytic Domain ◽

Coenzyme A Reductase

Download Full-text

Purification and identification of 7-O-ethyl ganoderic acid O from fermented mycelia of Ganoderma lucidum and its conversion to other ganoderic acids

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2009.08.218 ◽

2009 ◽

Vol 108 ◽

pp. S74

Author(s):

Jia Le Wang ◽

Ying Bo Li ◽

Ru Ming Liu ◽

Cui Xia Liang ◽

Jian Jiang Zhong

Keyword(s):

Ganoderma Lucidum ◽

Ganoderic Acid ◽

Ganoderic Acids

Download Full-text

Heat Stress Modulates Mycelium Growth, Heat Shock Protein Expression, Ganoderic Acid Biosynthesis, and Hyphal Branching of Ganoderma lucidum via Cytosolic Ca2+

Applied and Environmental Microbiology ◽

10.1128/aem.01036-16 ◽

2016 ◽

Vol 82 (14) ◽

pp. 4112-4125 ◽

Cited By ~ 33

Author(s):

Xue Zhang ◽

Ang Ren ◽

Meng-Jiao Li ◽

Peng-Fei Cao ◽

Tian-Xi Chen ◽

...

Keyword(s):

Signal Transduction ◽

Heat Stress ◽

Heat Shock ◽

Ganoderma Lucidum ◽

Potential Model ◽

Acid Biosynthesis ◽

Ganoderic Acid ◽

Mycelium Growth ◽

Hyphal Branching ◽

Content Type

ABSTRACTHeat stress (HS) influences the growth and development of organisms. Thus, a comprehensive understanding of how organisms sense HS and respond to it is required.Ganoderma lucidum, a higher basidiomycete with bioactive secondary metabolites, has become a potential model system due to the complete sequencing of its genome, transgenic systems, and reliable reverse genetic tools. In this study, we found that HS inhibited mycelium growth, reduced hyphal branching, and induced the accumulation of ganoderic acid biosynthesis and heat shock proteins (HSPs) inG. lucidum. Our data showed that HS induced a significant increase in cytosolic Ca2+concentration. Further evidence showed that Ca2+might be a factor in the HS-mediated regulation of hyphal branching, ganoderic acid (GA) biosynthesis, and the accumulation of HSPs. Our results further showed that the calcium-permeable channel gene (cch)-silenced and phosphoinositide-specific phospholipase gene (plc)-silenced strains reduced the HS-induced increase in HSP expression compared with that observed for the wild type (WT). This study demonstrates that cytosolic Ca2+participates in heat shock signal transduction and regulates downstream events in filamentous fungi.IMPORTANCEGanoderma lucidum, a higher basidiomycete with bioactive secondary metabolites, has become a potential model system for evaluating how environmental factors regulate the development and secondary metabolism of basidiomycetes. Heat stress (HS) is an important environmental challenge. In this study, we found that HS inhibited mycelium growth, reduced hyphal branching, and induced HSP expression and ganoderic acid biosynthesis inG. lucidum. Further evidence showed that Ca2+might be a factor in the HS-mediated regulation of hyphal branching, GA biosynthesis, and the accumulation of HSPs. This study demonstrates that cytosolic Ca2+participates in heat shock signal transduction and regulates downstream events in filamentous fungi. Our research offers a new way to understand the mechanism underlying the physiological and metabolic responses to other environmental factors inG. lucidum. This research may also provide the basis for heat shock signal transduction studies of other fungi.

Download Full-text

Cross Talk between Nitric Oxide and Calcium-Calmodulin Regulates Ganoderic Acid Biosynthesis in Ganoderma lucidum under Heat Stress

Applied and Environmental Microbiology ◽

10.1128/aem.00043-18 ◽

2018 ◽

Vol 84 (10) ◽

pp. e00043-18 ◽

Cited By ~ 18

Author(s):

Rui Liu ◽

Liang Shi ◽

Ting Zhu ◽

Tao Yang ◽

Ang Ren ◽

...

Keyword(s):

Nitric Oxide ◽

Heat Stress ◽

Ganoderma Lucidum ◽

Ganoderic Acid ◽

No Donor ◽

Content Type ◽

Fungal Biology ◽

Close Relationship ◽

Ga Biosynthesis

ABSTRACT We previously reported that high temperature impacts ganoderic acid (GA) biosynthesis in Ganoderma lucidum via Ca2+. Therefore, to further understand the signal-regulating network of the organism's response to heat stress (HS), we examined the role of nitric oxide (NO) under HS. After HS treatment, the NO level was significantly increased by 120% compared to that under the control conditions. The application of a NO scavenger resulted in a 25% increase in GA compared with that found in the sample treated only with HS. Additionally, the application of a NO donor to increase NO resulted in a 30% lower GA content than that in the sample treated only with HS. These results show that the increase in NO levels alleviates HS-induced GA accumulation. Subsequently, we aimed to detect the effects of the interaction between NO and Ca2+ on GA biosynthesis under HS in G. lucidum. Our pharmacological approaches revealed that the NO and Ca2+ signals promoted each other in response to HS. We further constructed the silenced strain of nitrate reductase (NR) and calmodulin (CaM), and the results are in good agreement with the silenced strain and pharmacological experiment. The cross-promotion between NO and Ca2+ signals is involved in the regulation of HS-induced GA biosynthesis in G. lucidum, and this finding is supported by studies with NR-silenced (NRi) and CaM-silenced (CaMi) strains. However, Ca2+ may have a more direct and significant effect on the HS-induced GA increase than NO. These data indicate that NO functions in signaling and has a close relationship with Ca2+ in HS-induced GA biosynthesis. IMPORTANCE HS is an important environmental stress affecting the growth and development of organisms. We previously reported that HS modulates GA biosynthesis in G. lucidum via Ca2+. However, the signal-regulating network of the organism's response to HS has not yet been elucidated. In this study, we found that NO relieved HS-induced GA accumulation, and NO and Ca2+ could exert promoting effects on each other in response to HS. Further research on the effect of NO and Ca2+ on the production of GAs in response to HS indicated that Ca2+ has a notably more direct and significant effect on the HS-induced GA increase than NO. Our results improve our understanding of the mechanism of HS signal transduction in fungi. A greater understanding of the regulation of secondary metabolism in response to environmental stimuli will provide clues regarding the role of these products in fungal biology.

Download Full-text

Ornithine Decarboxylase-Mediated Production of Putrescine Influences Ganoderic Acid Biosynthesis by Regulating Reactive Oxygen Species in Ganoderma lucidum

Applied and Environmental Microbiology ◽

10.1128/aem.01289-17 ◽

2017 ◽

Vol 83 (20) ◽

Cited By ~ 5

Author(s):

Chen-Gao Wu ◽

Jia-Long Tian ◽

Rui Liu ◽

Peng-Fei Cao ◽

Tian-Jun Zhang ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Ornithine Decarboxylase ◽

Ganoderma Lucidum ◽

Oxygen Species ◽

Ganoderic Acid ◽

Gene Encoding ◽

Transcript Levels ◽

Content Type ◽

Future Studies

ABSTRACT Putrescine is an important polyamine that participates in a variety of stress responses. Ornithine decarboxylase (ODC) is a key enzyme that catalyzes the biosynthesis of putrescine. A homolog of the gene encoding ODC was cloned from Ganoderma lucidum. In the ODC-silenced strains, the transcript levels of the ODC gene and the putrescine content were significantly decreased. The ODC-silenced strains were more sensitive to oxidative stress. The content of ganoderic acid was increased by approximately 43 to 46% in the ODC-silenced strains. The content of ganoderic acid could be recovered after the addition of exogenous putrescine. Additionally, the content of reactive oxygen species (ROS) was significantly increased by approximately 1.3-fold in the ODC-silenced strains. The ROS content was significantly reduced after the addition of exogenous putrescine. The gene transcript levels and the activities of four major antioxidant enzymes were measured to further explore the effect of putrescine on the intracellular ROS levels. Further studies showed that the effect of the ODC-mediated production of putrescine on ROS might be a factor influencing the biosynthesis of ganoderic acid. Our study reports the role of putrescine in large basidiomycetes, providing a basis for future studies of the physiological functions of putrescine in microbes. IMPORTANCE It is well known that ODC and the ODC-mediated production of putrescine play an important role in resisting various environmental stresses, but there are few reports regarding the mechanisms underlying the effect of putrescine on secondary metabolism in microorganisms, particularly in fungi. G. lucidum is gradually becoming a model organism for studying environmental regulation and metabolism. In this study, a homolog of the gene encoding ODC was cloned in Ganoderma lucidum. We found that the transcript level of the ODC gene and the content of putrescine were significantly decreased in the ODC-silenced strains. The content of ganoderic acid was significantly increased in the ODC-silenced strains. Further studies showed that the effect of the ODC-mediated production of putrescine on ROS might be a factor influencing the biosynthesis of ganoderic acid. Our study reports the role of putrescine in large basidiomycetes, providing a basis for future studies of the physiological functions of putrescine in microbes.

Download Full-text