Overexpression of the Squalene Epoxidase Gene Alone and in Combination with the 3-Hydroxy-3-methylglutaryl Coenzyme A Gene Increases Ganoderic Acid Production in <i>Ganoderma lingzhi</i>

Zhang, De-Huai; Lei, Jiang; Li, Na; Yu, Xuya; Zhao, Peng; Li, Tao; Xu, Junwei

doi:10.1021/acs.jafc.7b00629

Cited by 46 publications

(43 citation statements)

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“…Genetic transformation systems, including Agrobacterium tumefacien ‐mediated and polyethylene glycol (PEG)‐mediated transformations, have been developed in G. lucidum (Shi et al , ; Xu et al , ; Xu and Zhong, ) and have been successfully used to enhance the production of bioactive compounds, such as ganoderic acids and polysaccharides (Zhou et al , ; Li et al , ; Zhang et al , , ; Fei et al , ; Xu et al , ). Gene silencing systems have also been used for the partial suppression of genes of interest in G. lucidum (Mu et al , ; Liu et al , ; Zhu et al , ).…”

Section: Introductionmentioning

confidence: 99%

Dual sgRNA‐directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system

Liu

Sun

You

et al. 2020

Microbial Biotechnology

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Ganoderma lucidum is an important medicinal mushroom in traditional Chinese medicine. However, the lack of adequate genetic tools has hindered molecular genetic research in and the genetic modification of this species. Here, we report that the presence of an intron is necessary for the efficient expression of the heterologous phosphinothricin‐resistance and green fluorescent protein genes in G. lucidum. Moreover, we improved the CRISPR/Cas9‐mediated gene disruption frequency in G. lucidum by adding an intron upstream of the Cas9 gene. Our results showed that the disruption frequency of the orotidine 5’‐monophosphate decarboxylase gene (ura3) in transformants containing the glyceraldehyde‐3‐phosphate dehydrogenase gene intron in the Cas9 plasmid is 14–18 in 107 protoplasts, which is 10.6 times higher than that in transformants without any intron sequence. Furthermore, genomic fragment deletions in the ura3 and GL17624 genes were achieved via a dual sgRNA‐directed CRISPR/Cas9 system in G. lucidum. We achieved a ura3 deletion frequency of 36.7% in G. lucidum. The developed method provides a powerful platform to generate gene deletion mutants and will facilitate functional genomic studies in G. lucidum.

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Section: Introductionmentioning

confidence: 99%

Dual sgRNA‐directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system

Liu

Sun

You

et al. 2020

Microbial Biotechnology

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“…A single copy of the latter gene is present in the genome of H. carpetanum which we assume to be responsible for ergosterol biosynthesis. Recently, the LS in Ganoderma lingzhi, besides its role in ergosterol biosynthesis, has been shown to provide the intermediate for the biosynthesis of ganoderic acids (Zhang et al, 2017). Another related gene tagged as oxidosqualene-clavarinone cyclase (occ) was revealed to encode the core cyclase for the clavaric acid biosynthetic pathway in the basidiomycete Hypholoma sublateritium (Godio et al, 2007;Godio and Martín, 2009).…”

Section: Discussionmentioning

confidence: 99%

Enfumafungin synthase represents a novel lineage of fungal triterpene cyclases

Kuhnert

et al. 2018

Environmental Microbiology

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Summary Enfumafungin is a glycosylated fernene-type triterpenoid produced by the fungus Hormonema carpetanum. Its potent antifungal activity, mediated by its interaction with β−1,3-glucan synthase and the fungal cell wall, has led to its development into the semi-synthetic clinical candidate, ibrexafungerp (=SCY-078). We report on the preliminary identification of the enfumafungin biosynthetic gene cluster (BGC) based on genome sequencing, phylogenetic reconstruction, gene disruption, and cDNA sequencing studies. Enfumafungin synthase (efuA) consists of a terpene cyclase domain (TC) fused to a glycosyltransferase (GT) domain and thus represents a novel multifunctional enzyme. Moreover, the TC domain bears a phylogenetic relationship to bacterial squalene–hopene cyclases (SHC) and includes a typical DXDD motif within the active centre suggesting that efuA evolved from SHCs. Phylogenetic reconstruction of the GT domain indicated that this portion of the fusion gene originated from fungal sterol GTs. Eleven genes flanking efuA are putatively involved in the biosynthesis, regulation, transport and self-resistance of enfumafungin and include an acetyltransferase, three P450 monooxygenases, a dehydrogenase, a desaturase and a reductase. A hypothetical scheme for enfumafungin assembly is proposed in which the E-ring is oxidatively cleaved to yield the four-ring system of enfumafungin. EfuA represents the first member of a widespread lineage of fungal SHCs.

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“…Ganoderma lucidum CGMCC 5.616 was bought from the China General Microbiological Fermentation Center and is conspecific with Ganoderma lingzhi (Zhang et al ., ,). The VHb‐expressing G. lingzhi was constructed in a previous study (Li et al ., ).…”

Section: Methodsmentioning

confidence: 99%

“…Ganoderic acids are synthesized via the mevalonate/isoprenoid (MVA) pathway in Ganoderma species (Xu et al ., ). Lanosterol, an important intermediate, is converted to different individual GAs through a series of modifications, including oxidation, reduction and acylation reactions, in Ganoderma lucidum (Xu et al ., ; Chen et al ., ; Zhang et al ., ,). Some key genes involved in the upstream cyclization pathway of the lanostane skeleton, including 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGR), farnesyl‐diphosphate synthase (FPS) and squalene synthase (SQS), have been cloned from G. lucidum (Shi et al ., ; Zhou et al ., ).…”

Section: Introductionmentioning

confidence: 99%

“…Due to their important pharmacological activities, many strategies have been used to increase the production of GAs during mycelia fermentation. Progress has been achieved in the last several decades in enhancing GA production in G. lucidum by the manipulation of fermentation strategies (Xu et al ., ; Upadhyay et al ., ; Wang et al ., ), addition of metal ions and elicitors (Ren et al ., ; Xu and Zhong, ; You et al ., , ; Zhang et al ., ; Gu et al ., ) and genetic engineering (Xu et al ., ; Xu and Zhong, ; Li et al ., ,,; Zhang et al ., ,). However, further improvement of GA production is necessary to meet the demand of increasing preclinical studies and commercial applications.…”

Section: Introductionmentioning

confidence: 99%

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

Yue

et al. 2019

Microbial Biotechnology

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Summary Ganoderic 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.

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Overexpression of the Squalene Epoxidase Gene Alone and in Combination with the 3-Hydroxy-3-methylglutaryl Coenzyme A Gene Increases Ganoderic Acid Production in Ganoderma lingzhi

Cited by 46 publications

References 46 publications

Dual sgRNA‐directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system

Dual sgRNA‐directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system

Enfumafungin synthase represents a novel lineage of fungal triterpene cyclases

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

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