Hydrogen sulfide maintains mitochondrial homeostasis and regulates ganoderic acids biosynthesis by SQR under heat stress in Ganoderma lucidum.

Hydrogen sulfide (H2S) has recently been recognized as an important gaseous transmitter with multiple physiological effects in various species. Previous studies have shown that H2S alleviated heat-induced ganoderic acids (GAs) biosynthesis, an important quality index of Ganoderma lucidum. However, a comprehensive understanding of the physiological effects and molecular mechanisms of H2S in G. lucidum remains unexplored. In this study, we found that heat treatment reduced the mitochondrial membrane potential (MMP) and mitochondrial DNA copy number (mtDNAcn) in G. lucidum. Increasing the intracellular H2S concentration through pharmacological and genetic means increased the MMP level, mtDNAcn, oxygen consumption rate level and ATP content under heat treatment, suggesting a role for H2S in mitigating heat-caused mitochondrial damage in G. lucidum. Further results indicated that H2S activates sulfide-quinone oxidoreductase (SQR) and complex III (Com III), thereby maintaining mitochondrial homeostasis under heat stress in G. lucidum. Moreover, SQR also mediated the negative regulation of H2S to GAs biosynthesis under heat stress. Furthermore, SQR might be persulfidated under heat stress in G. lucidum. Thus, our study reveals a novel physiological function and molecular mechanism of H2S signalling under heat stress in G. lucidum with broad implications for research on the environmental response of microorganisms.

Strategies to Increase the Production of Triterpene Acids in Ligzhi or Reishi Medicinal Mushroom (Ganoderma lucidum, Agaricomycetes): A Review.

Ganoderic acids (GAs) are the main active ingredient of Ganoderma lucidum, which has been widely accepted as a medicinal mushroom. Due to the low yield of GAs produced by liquid cultured Ganoderma mycelium and solid cultured fruiting bodies, the commercial production and clinical application of GAs are limited. Therefore, it is important to increase the yield of GA in G. lucidum. A comprehensive literature search was performed with no set data range using the following keywords such as "triterpene," "ganoderic acids," "Ganoderma lucidum," and "Lingzhi" within the main databases including Web of Science, PubMed, and China National Knowledge Infrastructure (CNKI). The data were screened using titles and abstracts and those relevant to the topic were included in the paper and was not limited to studies published in English. Present review focuses on the four aspects: fermentation conditions and substrate, extrinsic elicitor, genetic engineering, and mutagenesis, which play significant roles in increasing triterpene acids production, thus providing an available reference for further research on G. lucidum fermentation.

Enhancing high-efficiency breeding and microbial microdroplet cultivation techniques for Ganoderma lucidum.

Ganoderma lucidum is known for its bioactive compounds, such as polysaccharides and triterpenoids, which are crucial in food and medicine. However, liquid fermentation encounters challenges in terms of strain differentiation and stability. In this research, we employed atmospheric room temperature plasma mutation and a microbial microdroplet culture system to identify strains with enhanced biomass and triterpenoid production. The three mutant strains, YB05, YB09, and YB18, exhibited accelerated growth rates and antagonized the initial strain G0023 more effectively than the controls. Notably, YB18 displayed the fastest growth, with a 17.25% increase in colony radius. Shake flask cultivation demonstrated that, compared with the initial strain, YB05 and YB18 had 26.33% and 17.85% greater biomass, respectively. Moreover, the triterpenoid production of YB05 and YB18 surpassed that of the control by 32.10% and 15.72%, respectively, as confirmed by colorimetric detection. Importantly, these mutant strains remained stable for five generations. This study revealed a comprehensive screening system utilizing atmospheric pressure, room temperature plasma mutation technology and microbial droplet cultivation. This innovative approach offers a promising pathway for obtaining advantageous Ganoderma strains for liquid fermentation. The methodology of atmospheric room temperature plasma mutation and microbial microdroplet culture systems is detailed for better comprehension.

The CBS/H2S signalling pathway regulated by the carbon repressor CreA promotes cellulose utilization in Ganoderma lucidum.

Cellulose is an important abundant renewable resource on Earth, and the microbial cellulose utilization mechanism has attracted extensive attention. Recently, some signalling molecules have been found to regulate cellulose utilization and the discovery of underlying signals has recently attracted extensive attention. In this paper, we found that the hydrogen sulfide (H2S) concentration under cellulose culture condition increased to approximately 2.3-fold compared with that under glucose culture condition in Ganoderma lucidum. Further evidence shown that cellulase activities of G. lucidum were improved by 18.2-27.6% through increasing H2S concentration. Then, we observed that the carbon repressor CreA inhibited H2S biosynthesis in G. lucidum by binding to the promoter of cbs, a key gene for H2S biosynthesis, at "CTGGGG". In our study, we reported for the first time that H2S increased the cellulose utilization in G. lucidum, and analyzed the mechanism of H2S biosynthesis induced by cellulose. This study not only enriches the understanding of the microbial cellulose utilization mechanism but also provides a reference for the analysis of the physiological function of H2S signals.

Glsirt1-mediated deacetylation of GlCAT regulates intracellular ROS levels, affecting ganoderic acid biosynthesis in Ganoderma lucidum.

Lysine acetylation is a reversible, dynamic protein modification regulated by lysine acetyltransferases and deacetylases. However, in Basidiomycetes, the extent of lysine acetylation of nonhistone proteins remains largely unknown. Recently, we identified the deacetylase Glsirt1 as a key regulator of the biosynthesis of ganoderic acid (GA), a key secondary metabolite of Ganoderma lucidum. To gain insight into the characteristics, extent, and biological function of Glsirt1-mediated lysine acetylation in G. lucidum, we aimed to identify additional Glsirt1 substrates via comparison of acetylomes between wild-type (WT) and Glsirt1-silenced mutants. A large amount of Glsirt1-dependent lysine acetylation occurs in G. lucidum according to the results of this omics analysis, involving energy metabolism, protein synthesis, the stress response and other pathways. Our results suggest that GlCAT is a direct target of Glsirt1 and that the deacetylation of GlCAT by Glsirt1 reduces catalase activity, thereby leading to the accumulation of intracellular reactive oxygen species (ROS) and positively regulating the biosynthesis of GA. Our findings provide evidence for the involvement of nonhistone lysine acetylation in the biological processes of G. lucidum and help elucidate the involvement of important ROS signaling molecules in regulating physiological and biochemical processes in this organism. In conclusion, this proteomic analysis reveals a striking breadth of cellular processes affected by lysine acetylation and provides new nodes of intervention in the biosynthesis of secondary metabolites in G. lucidum.

Combined NMR and MS-based metabonomics and real-time PCR analyses reveal dynamic metabolic changes of Ganoderma lucidum during fruiting body growing.

Ganoderma lucidum (G. lucidum) is a rare medicinal fungus with various beneficial properties. One of its main components, ganoderic acids (GAs), are important triterpenoids known for their sedative and analgesic, hepatoprotective, and anti-tumor activities. Understanding the growth and development of the G. lucidum fruiting body is crucial for determining the optimal time to harvest them. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to systematically characterize the metabolites of G. lucidum at seven distinct developmental stages. We also measured the contents of seven kinds of GAs using LC-MS/MS. A total of 49 metabolites were detected in G. lucidum, including amino acids, sugars, organic acids and GAs. During the transition from the bud development period (I) to the budding period (II), we observed a rapid accumulation of glucose, tyrosine, nicotinamide ribotide, inosine and GAs. After the budding period, the contents of most metabolites decreased until the mature period (VII). In addition, the contents of GAs showed an initial raising, followed by a decline during the elongation period, except for GAF, which exhibited a rapid raise during the mature stage. We also detected the expression of several genes involved in GA synthesis, finding that most genes including 16 cytochrome P450 monooxygenase were all down-regulated during periods IV and VII compared to period I. These findings provide valuable insights into the dynamic metabolic profiles of G. lucidum throughout its growth stage, and it is recommended to harvest G. lucidum at period IV.

GlPRMT5 inhibits GlPP2C1 via symmetric dimethylation and regulates the biosynthesis of secondary metabolites in Ganoderma lucidum.

PRMT5, a type II arginine methyltransferase, is involved in transcriptional regulation, RNA processing and other biological processes and signal transduction. Secondary metabolites are vital pharmacological compounds in Ganoderma lucidum, and their content is an important indicator for evaluating the quality of G. lucidum. Here, we found that GlPRMT5 negatively regulates the biosynthesis of secondary metabolites. In further in-depth research, GlPP2C1 (a type 2C protein phosphatase) was identified out as an interacting protein of GlPRMT5 by immunoprecipitation-mass spectrometry (IP-MS). Further mass spectrometry detection revealed that GlPRMT5 symmetrically dimethylates the arginine 99 (R99) and arginine 493 (R493) residues of GlPP2C1 to weaken its activity. The symmetrical dimethylation modification of the R99 residue is the key to affecting GlPP2C1 activity. Symmetrical demethylation-modified GlPP2C1 does not affect the interaction with GlPRMT5. In addition, silencing GlPP2C1 clearly reduced GA content, indicating that GlPP2C1 positively regulates the biosynthesis of secondary metabolites in G. lucidum. In summary, this study reveals the molecular mechanism by which GlPRMT5 regulates secondary metabolites, and these studies provide further insights into the target proteins of GlPRMT5 and symmetric dimethylation sites. Furthermore, these studies provide a basis for the mutual regulation between different epigenetic modifications.

4-Coumarate-CoA ligase (4-CL) enhances flavonoid accumulation, lignin synthesis, and fruiting body formation in Ganoderma lucidum.

It is now understood that 4-Coumarate-CoA ligases (4-CL) are pivotal in bridging the phenylpropanoid metabolic pathway and the lignin biosynthesis pathway in plants. However, limited information on 4-CL genes and their functions in fungi is available. In this study, we cloned the 4-CL gene (Gl21040) from Ganoderma lucidum, which spans 2178 bp and consists of 10 exons and 9 introns. We also developed RNA interference and overexpression vectors for Gl21040 to investigate its roles in G. lucidum. Our findings indicated that in the Gl21040 interference transformants, 4-CL enzyme activities decreased by 31 %-57 %, flavonoids contents decreased by 10 %-22 %, lignin contents decreased by 20 %-36 % compared to the wild-type (WT) strain. Conversely, in the Gl21040 overexpression transformants, 4-CL enzyme activity increased by 108 %-143 %, flavonoids contents increased by 8 %-37 %, lignin contents improved by 15 %-17 % compared to the WT strain. Furthermore, primordia formation was delayed by approximately 10 days in the Gl21040-interferenced transformants but occurred 3 days earlier in the Gl21040-overexpressed transformants compared to the WT strain. These results underscored the involvement of the Gl21040 gene in flavonoid synthesis, lignin synthesis, and fruiting body formation in G. lucidum.

Developing Ganoderma lucidum as a next-generation cell factory for food and nutraceuticals.

Ganoderma lucidum holds a colossal reservoir of hydrolytic enzymes and therapeutic compounds and can be a sustainable source of proteins and bioactive compounds. Its metabolic versatility, propelled by its rich genome content, provides excellent biosynthetic machinery for innovation-driven pathway engineering. However, robust regulatory networks and low frequency of homologous recombination are critical bottlenecks that limit the development of molecular tools and precise genetic markers for biomanufacturing innovations in this organism. Modern synthetic biology provides tools that could help to accelerate precise multiple gene targeting and editing and untangling the biosynthetic machinery of G. lucidum. This review provides insight into molecular strategies to unwind the regulatory bottlenecks and transform G. lucidum into efficient cell factories for food and nutraceuticals.

Pathway analysis of host responses to dengue virus serotype 2 infection and inhibition of viral envelope protein by naringenin from Ganoderma lucidum.

Dengue fever cases are spiking over the last two decades. Incessant efforts are still being made to gain deeper insights on this arboviral disease and to identify bioactive antivirals. In this study, bioinformatics analysis was conducted to identify the differentially expressed genes (DEGs) in the expression profiling datasets of dengue virus serotype 2 (DENV2) patients. We found overexpressed genes in dengue patients that can interrupt cell cycle progression and phase transitions of mitosis inside the host to favour the viral replication process. These DEGs were associated with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways such as cell cycle and DNA replication. A protein interaction network consisting of these significant pathways was also constructed using STRING. Futher, the traditional Chinese medicine (TCM) compounds from Ganoderma lucidum were screened to target DENV2 envelope protein, which was crucial for viral fusion activity. Docking, orbital energy, and toxicity prediction analysis revealed that naringenin was the best antiviral candidate. Following molecular dynamics simulations, the predicted binding energy of the protein-naringenin system using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approach was slightly greater than the control system. It is recommended to perform in vitro inhibition of naringenin against DENV2 and use our findings to complement the experimental data obtained.

PRMT5 regulates the polysaccharide content by controlling the splicing of thaumatin-like protein in Ganoderma lucidum.

IMPORTANCE: PRMT5 contributes to secondary metabolite biosynthesis in Ganoderma lucidum. However, the mechanism through which PRMT5 regulates the biosynthesis of secondary metabolites remains unclear. In the current study, PRMT5 silencing led to a significant decrease in the biosynthesis of polysaccharides from G. lucidum through the action of the alternative splicing of TLP. A shorter TLP2 isoform can directly bind to PGI and regulated polysaccharide biosynthesis. These results suggest that PRMT5 enhances PGI activity by regulating TLP binding to PGI. The results of the current study reveal a novel target gene for PRMT5-mediated alternative splicing and provide a reference for the identification of PRMT5 regulatory target genes.

The transcription factor GCN4 contributes to maintaining intracellular amino acid contents under nitrogen-limiting conditions in the mushroom Ganoderma lucidum.

BACKGROUND: Edible mushrooms are delicious in flavour and rich in high-quality protein and amino acids required by humans. A transcription factor, general control nonderepressible 4 (GCN4), can regulate the expression of genes involved in amino acid metabolism in yeast and mammals. A previous study revealed that GCN4 plays a pivotal role in nitrogen utilization and growth in Ganoderma lucidum. However, its regulation is nearly unknown in mushrooms. RESULTS: In this study, we found that the amino acid contents reached 120.51 mg per gram of mycelia in the WT strain under 60 mM asparagine (Asn) conditions, but decreased by 62.96% under 3 mM Asn conditions. Second, silencing of gcn4 resulted in a 54.2% decrease in amino acid contents under 60 mM Asn, especially for the essential and monosodium glutamate-like flavour amino acids. However, these effects were more pronounced under 3 mM Asn. Third, silencing of gcn4 markedly inhibited the expression of amino acid biosynthesis and transport genes. In addition, GCN4 enhanced the tricarboxylic acid cycle (TCA) and glycolytic pathway and inhibited the activity of target of rapamycin complex 1 (TORC1), thus being beneficial for maintaining amino acid homeostasis. CONCLUSION: This study confirmed that GCN4 contributes to maintaining the amino acid contents in mushrooms under low concentrations of nitrogen. In conclusion, our study provides a research basis for GCN4 to regulate amino acid synthesis and improve the nutrient contents of edible mushrooms.

GlMPC activated by GCN4 regulates secondary metabolism under nitrogen limitation conditions in Ganoderma lucidum.

IMPORTANCE: Mitochondrial pyruvate carrier (MPC) is a pyruvate transporter that plays a crucial role in regulating the carbon metabolic flow and is considered an essential mechanism for microorganisms to adapt to environmental changes. However, it remains unclear how MPC responds to environmental stress in organisms. General control non-derepressible 4 (GCN4), a key regulator of nitrogen metabolism, plays a pivotal role in the growth and development of fungi. In this study, we report that GCN4 can directly bind to the promoter region and activate the expression of GlMPC, thereby regulating the tricarboxylic acid cycle and secondary metabolism under nitrogen limitation conditions in Ganoderma lucidum. These findings provide significant insights into the regulation of carbon and nitrogen metabolism in fungi, highlighting the critical role of GCN4 in coordinating metabolic adaptation to environmental stresses.

Increased production and anti-senescence activity of exopolysaccharides in Ganoderma lingzhi by co-overexpression of ß-1,3-glucan synthase and UDP-glucose pyrophosphorylase.

A ß-1,3-glucan synthase gene (gls) was cloned and overexpressed in Ganoderma lingzhi. The content of intracellular polysaccharides (IPS) in G. lingzhi overexpressing gls was 22.36 mg/100 mg dry weight (DW), 19 % higher than those in the wild-type (WT) strain. Overexpression of gls did not affect the expression of the phosphoglucomutase gene and the UDP-glucose pyrophosphorylase gene (ugp) in the polysaccharide biosynthesis. The gls and ugp were then simultaneously overexpressed in G. lingzhi for the first time. The combined overexpression of these two genes increased the IPS content and exopolysaccharides (EPS) production to a greater extent than the overexpression of gls independently. The maximum IPS content of the overexpressed strain was 24.61 mg/100 mg, and the maximum EPS production was 1.55 g/L, 1.31- and 1.50-fold higher than that in the WT strain, respectively. Moreover, the major EPS fractions from the overexpression strain contained more glucose (86.7 % and 72.5 %) than those from the WT strain (78.2 % and 62.9 %). Furthermore, the major fraction G+U-0.1 from the overexpression strain exhibited stronger antioxidant and anti-senescence activities than the WT-0.1 fraction from the WT strain. These findings will aid in the hyperproduction and application of Ganoderma polysaccharides and facilitate our understanding of mushroom polysaccharide biosynthesis.

The Cas9-gRNA ribonucleoprotein complex-mediated editing of pyrG in Ganoderma lucidum and unexpected insertion of contaminated DNA fragments.

Gene editing is a promising alternative to traditional breeding for the generation of new mushroom strains. However, the current approach frequently uses Cas9-plasmid DNA to facilitate mushroom gene editing, which can leave residual foreign DNA in the chromosomal DNA raising concerns regarding genetically modified organisms. In this study, we successfully edited pyrG of Ganoderma lucidum using a preassembled Cas9-gRNA ribonucleoprotein complex, which primarily induced a double-strand break (DSB) at the fourth position prior to the protospacer adjacent motif. Of the 66 edited transformants, 42 had deletions ranging from a single base to large deletions of up to 796 bp, with 30 being a single base deletion. Interestingly, the remaining 24 contained inserted sequences with variable sizes at the DSB site that originated from the fragmented host mitochondrial DNA, E. coli chromosomal DNA, and the Cas9 expression vector DNA. The latter two were thought to be contaminated DNAs that were not removed during the purification process of the Cas9 protein. Despite this unexpected finding, the study demonstrated that editing G. lucidum genes using the Cas9-gRNA complex is achievable with comparable efficiency to the plasmid-mediated editing system.

Neuroprotective, neurogenic, and anticholinergic evidence of Ganoderma lucidum cognitive effects: Crucial knowledge is still lacking.

Ganoderma lucidum is a mushroom that has been widely used for centuries in Asian countries for its antiaging properties. It is popularly known as "Ling Zhi," "Reishi," and "Youngzhi," and because of its benefits, it is known as the "immortality mushroom." Pharmacological assays have revealed that G. lucidum ameliorates cognitive impairments through inhibition of ß-amyloid and neurofibrillary tangle formation, antioxidant effect, reduction of inflammatory cytokine release and apoptosis, genic expression modulation, among other activities. Chemical investigations on G. lucidum have revealed the presence of metabolites such as triterpenes, which are the most explored in this field, as well as flavonoids, steroids, benzofurans, and alkaloids; in the literature, these have also been reported to have mnemonic activity. These properties of the mushroom make it a potential source of new drugs to prevent or reverse memory disorders, as actual medications are able to only alleviate some symptoms but are unable to stop the progress of cognitive impairments, with no impact on social, familiar, and personal relevance. In this review, we discuss the cognitive findings of G. lucidum reported in the literature, converging the proposed mechanisms through the several pathways that underlie memory and cognition processes. In addition, we highlight the gaps that deserve particular attention to support future studies.

Transcriptome analysis reveals insight into the protective effect of N-acetylcysteine against cadmium toxicity in Ganoderma lucidum (Polyporales: Polyporaceae).

Ganoderma lucidum is widely cultivated and used as traditional medicine in China and other Asian countries. As a member of macrofungi, Ganoderma lucidum is also prone to bioaccumulation of cadmium and other heavy metals in a polluted environment, which affects the growth and production of Ganoderma lucidum, as well as human health. N-Acetyl-L-cysteine (NAC) is considered a general antioxidant and free radical scavenger that is involved in the regulation of various stress responses in plants and animals. However, whether NAC could regulate cadmium stress responses in macrofungi, particularly edible fungi, is still unknown. In this work, we found that the exogenous NAC could alleviate Cd-induced growth inhibition and reduce the cadmium accumulation in Ganoderma lucidum. The application of the NAC cloud also inhibit cadmium-induced H2O2 production in the mycelia. By using transcriptome analysis, 2920 and 1046 differentially expressed unigenes were identified in "Cd100 vs CK" and "NAC_Cd100 vs Cd100," respectively. These differential unigenes were classified into a set of functional categories and pathways, which indicated that various biological pathways may play critical roles in the protective effect of NAC against Cd­induced toxicity in Ganoderma lucidum. Furthermore, it suggested that the ATP-binding cassette transporter, ZIP transporter, heat shock protein, glutathione transferases, and Cytochrome P450 genes contributed to the increased tolerance to cadmium stress after NAC application in Ganoderma lucidum. These results provide new insight into the physiological and molecular response of Ganoderma lucidum to cadmium stress and the protective role of NAC against cadmium toxicity.

Effects of glutamate oxaloacetate transaminase on reactive oxygen species in Ganoderma lucidum.

Nitrogen metabolism can regulate mycelial growth and secondary metabolism in Ganoderma lucidum. As an important enzyme in intracellular amino acid metabolism, glutamate oxaloacetate transaminase (GOT) has many physiological functions in animals and plants, but its function in fungi has been less studied. In the present study, two GOT isoenzymes were found in G. lucidum; one is located in the mitochondria (GOT1), and the other is located in the cytoplasm (GOT2). The reactive oxygen species (ROS) level was increased in got1 silenced strains and was approximately 1.5-fold higher than that in the wild-type (WT) strain, while silencing got2 did not affect the ROS level. To explore how GOT affects ROS in G. lucidum, experiments related to the generation and elimination of intracellular ROS were conducted. First, compared with that in the WT strain, the glutamate content, one of the substrates of GOT, decreased when got1 or got2 was knocked down, and the glutathione (l-γ-glutamyl-l-cysteinylglycine) (GSH) content decreased by approximately 38.6%, 19.3%, and 40.1% in got1 silenced strains, got2 silenced strains, and got1/2 co-silenced strains respectively. Second, GOT also affects glucose metabolism. The pyruvate (PA), acetyl-CoA and α-ketoglutarate (α-KG) contents decreased in got1 and got2 silenced strains, and the transcription levels of most genes involved in the glycolytic pathway and the tricarboxylic acid cycle increased. The NADH content was increased in got1 silenced strains and got2 silenced strains, and the NAD+/NADH ratio was decreased, which might result in mitochondrial ROS production. Compared with the WT strain, the mitochondrial ROS level was approximately 1.5-fold higher in the got1 silenced strains. In addition, silencing of got1 or got2 resulted in a decrease in antioxidant enzymes, including superoxide dismutase, catalase, glutathione reductase, and ascorbate peroxidase. Finally, ganoderic acid (GA) was increased by approximately 40% in got1 silenced strains compared with the WT strain, while silencing of got2 resulted in a 10% increase in GA biosynthesis. These findings provide new insights into the effect of GOT on ROS and secondary metabolism in fungi. KEY POINTS: • GOT plays important roles in ROS level in Ganoderma lucidum. • Silencing of got1 resulted in decrease in GSH content and antioxidant enzymes activities, but an increase in mitochondrial ROS level in G. lucidum. • Silencing of got1 and got2 resulted in an increase in ganoderic acid biosynthesis in G. lucidum.

Mitochondrial pyruvate carrier influences ganoderic acid biosynthesis in Ganoderma lucidum.

Mitochondrial pyruvate carriers (MPCs), located in the inner membrane of mitochondria, are essential carriers for pyruvate to enter mitochondria. MPCs regulate a wide range of intracellular metabolic processes, such as glycolysis, the tricarboxylic acid cycle (TCA cycle), fatty acid metabolism, and amino acid metabolism. However, the metabolic regulation of MPCs in macrofungi is poorly studied. We studied the role of MPCs in Ganoderma lucidum (GlMPC) on ganoderic acid (GA) biosynthesis regulation in G. lucidum. In this study, we found that the mitochondrial/cytoplasmic ratio of pyruvate was downregulated about 75% in GlMPC1- and GlMPC2-silenced transformants compared with wild type (WT). In addition, the GA content was 17.72 mg/g and increased by approximately 50% in GlMPC1- and GlMPC2-silenced transformants compared with WT. By assaying the expression levels of three key enzymes and the enzyme activities of isocitrate dehydrogenase (IDH) and α-ketoglutarate dehydrogenase (α-KGDH) of the TCA cycle in GlMPC1- and GlMPC2-silenced transformants, it was found that the decrease in GlMPCs activity did not significantly downregulate the TCA cycle rate, and the enzyme activity of IDH increased by 44% compared with WT. We then verified that fatty acid ß-oxidation (FAO) supplements the TCA cycle by detecting the expression levels of key enzymes involved in FAO. The results showed that compared with WT, the GA content was 1.14 mg/g and reduced by approximately 40% in co-silenced transformants. KEY POINTS: • GlMPCs affects the distribution of pyruvate between mitochondria and the cytoplasm. • Acetyl-CoA produced by FAO maintains the TCA cycle. • Acetyl-CoA produced by FAO promotes the accumulation of GA.

Biosynthesis of mushroom-derived type II ganoderic acids by engineered yeast.

Type II ganoderic acids (GAs) produced by the traditional medicinal mushroom Ganoderma are a group of triterpenoids with superior biological activities. However, challenges in the genetic manipulation of the native producer, low level of accumulation in the farmed mushroom, the vulnerabilities of the farming-based supply chain, and the elusive biosynthetic pathway have hindered the efficient production of type II GAs. Here, we assemble the genome of type II GAs accumulating G. lucidum accession, screen cytochrome P450 enzymes (CYPs) identified from G. lucidum in baker's yeast, identify key missing CYPs involved in type II GAs biosynthesis, and investigate the catalytic reaction sequence of a promiscuous CYP. Then, we engineer baker's yeast for bioproduciton of GA-Y (3) and GA-Jb (4) and achieve their production at higher level than those from the farmed mushroom. Our findings facilitate the further deconvolution of the complex GA biosynthetic network and the development of microbial cell factories for producing GAs at commercial scale.