Applied Microbiology: Open Access
Open Access
ISSN: 2471-9315
+44 1300 500008
ISSN: 2471-9315
+44 1300 500008
Mini Review - (2023)Volume 9, Issue 4
Ganoderma lucidum, a kind of basidiomycete, can produce various valuable metabolites with bioactive. For a long time, the shortage of the genome and gene manipulation methods limited the analysis of gene functions, which delayed the elucidation of biosynthesis pathways of those products in G. lucidum. As G. lucidum was sequenced and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) system was established, gene modification and gene function analysis was feasible. In this review, we summarized the establishment of CRISPR/Cas9 system and its biotechnological application in G. lucidum. Besides, we discuss challenges to the current CRISPR/Cas9 system and give perspectives on the biotechnology research in G. lucidum.
Ganoderma lucidum, CRISPR/Cas9, Gene modification, Gene function analysis
G. lucidum can produce various bioactive products (ganoderic acids, polysaccharides, sterols, etc.), which have limitless potential in the food and biological medicine [1]. While low yield and unclear biosynthesis pathways of these metabolites limited their development and application [2]. As the G. lucidum lacked the ability to homologous recombination, the gene disruption and complementation strategies were shortage before the establishment of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) system, which limited the research for genes function and biosynthesis of valuable metabolites [3,4].
As the genome of G. lucidum was sequenced in 2012, increasing candidate biosynthesis genes of valuable metabolites were analyzed in heterologous hosts, especially in yeast [4-7]. For example, Wang, et al. selected the CYP5150l8 gene from 82 cytochrome P450 monooxygenase (CYP450) genes by using Saccharomyces cerevisiae as a host, and the gene was proved to catalyze the lanosterol at C-26 to produce an anti-tumor Ganoderic Acid (GA), 3-Hydroxylanosta-8, 24-Dien-26 Oic Acid (HLDOA) by a three-step oxidation [6]. By similar method, the CYP5139G1 was proved to oxidate the HLDOA at C-28 to produce a new GA 3,28-Dihydroxy-Lanosta-8,24-Dien-26-Oic Acid (DHLDOA) [6]. However, as the genetic background in S. cerevisiae was simpler than that of G. lucidum, whether the CYP5150L8 and the CYP5139G1 had same function was unknown in G. lucidum.
The establishment of the CRISPR/Cas9 assisted functional gene disruption and in situ complementation promoted gene function exploration in G. lucidum [3,8]. In 2017, Qin, et al. firstly disrupted ura3 marker gene in G. lucidum by transferring the in vitro transcribed guide RNA (gRNA) into the cells expressing Cas9 constitutively [9]. As only one appropriate selection marker in G. lucidum, the CRISPR/Cas9 system with in vitro transcribed gRNA was not suitable for functional genes manipulation [9]. Wang, et al. established an efficient CRISPR/Cas9 system, which achieved functional genes (CYP5150l8 and CYP505d13) disruption and precise repair by HR [4]. And then a CRISPR/ Cas9 assisted functional genes in situ complementation strategy was developed in G. lucidum in 2022 [3]. The CYP5150L8 and Gl26097 was proved to play a crucial role in the biosynthesis of Ganoderic Acids (GAs), which was a precedent for exploring secondary metabolites synthesis pathway in G. lucidum.
In this review, we summarize the establishment of CRISPR/ Cas9 and its biotechnological application in G. lucidum. What’s more, the potential challenges of CRISPR/Cas9 are discussed and its biotechnological application was prospected.
The establishment of CRISPR-Cas9 system in G. lucidum
CRISPR-Cas9 system with in vitro transcribed sgRNA: For in vitro transcription of sgRNA, a costly commercial kit, for example MEGAscript T7 Kit (Ambion, Austin,TX, USA), was usually used to synthesize sgRNA in vitro. Then the in vitro transcribed sgRNA was transformed to cells expressing Cas9. As lacking the ability to Homologous Recombination (HR), there was still a challenge for gene disruption in G. lucidum before 2017, when Qin, et al. disrupted the ura3 gene by transforming the in vitro transcribed ura3 sgRNA into G. lucidum protoplasts expressing the optimized Cas9 [9]. Then Liu et al. designed a dual sgRNA-mediated CRISPR/cas9 system, in which two sgRNAs were delivered into cells with an intron upstream of the Cas9 gene and achieved the ura3 disruption with efficiency of 36.7% [10]. What’s more, Tu, et al. further improved the dual sgRNA assisted ura3 disruption efficiency (exceeding 90%) by impairing nonhomologous end joining pathway (inactivating ku70 gene) [11]. Besides, Eom, et al. firstly used the Cas9-gRNA Ribnnucleoprotein (RNP) complex to edit the pyrG gene in G. lucidum in 2023 [12]. The RNP procedure was more complex, apart from in vitro transcribed sgRNA, Cas9 was expressed in E. coli and purified by a troublesome process.
CRISPR-Cas9 system with in vivo transcribed sgRNA: For the transcription of in vivo sgRNA, polymerase III (pol III) type promoters, such as u3 and u6 promoter, were usually used. In 2020, Wang, et al. firstly identified suitable u6 promoters to express sgRNA in vivo, and established a CRISPR/Cas9 system with in vivo expressed sgRNA, which achieved functional gene disruption and precise editing by HR [3]. Firstly, the effect of two u6 promoters and self-cleaving ribozyme HDV on disruption efficiency of ura3 marker gene was tested, the combination of u6-3 promoter and HDV achieved highest disruption efficiency (32.0%). And then optimized sgRNA scaffold (u6-3 promotersgRNA- HDV) was used to disrupt functional genes (CYP5150L8 and CYP505D13) successfully. What’s more, the HR of CYP5150l8 was achieved by transforming the CRIPSR/Cas9 plasmid and a 2kb repair donor containing 1kb upstream and downstream sequence close to Cas9 cutting site simultaneously. Moreover, Wang, et al. developed a novel CRISPR/Cas9 assisted in situ complementation of functional genes (CYP5150l8 and gl26097)[4]. Based on mutants of CYP5150l8 and gl26097 by CRISPR/Cas9 system in our previous work, sgRNA plasmids disrupting mutant genes and in situ complementation donors were designed, and then were delivered to disruptants [3,13]. After confirmation by sequencing, in situ complemented strains of two mutants were obtained. What’s more, compared to the ex situ complemented ones, the in situ complementation gl26097 mutant can restored the cell growth better. This was the first research report of in-situ complementation and helpful to study gene functions and metabolites biosynthesis pathway in G. lucidum.
Biotechnological application of CRISPR-Cas9 in G. lucidum
Based on the established CRISPR/Cas9 system, genes function was investigated by several research [3,4]. In our previous work, CYP5150L8 of G. lucidum was proved to catalyze the lanosterol at C-26 by a three-step oxidation and then produced a novel GA (HLDOA) in Saccharomyces cerevisiae [6]. The function of CYP5150L8 was confirmed in G. lucidum, its disruption significantly reduced the yield of four individual GAs and its in situ complementation restored these GAs content noticeably [3,4]. Eventually, the CYP5150L8 was proved to play a crucial role in the biosynthesis of GAs from lanosterol in G. lucidum. Similarly, Li, et al. disrupted the gl26097/glcrz2 gene by using the optimized CRISPR/Cas9 system in G. lucidum [3,13]. The deletion of gl26097/glcrz2 reduced GAs biosynthesis and cell growth severely [13]. Restoration of GAs production and cell growth was confirmed in the gl26097/glcrz2 in-situ complemented strains [4]. Therefore, the gl26097/glcrz2 may be involved in the GAs biosynthesis and cell growth.
For the CRISPR-Cas9 system with in vitro transcribed sgRNA, the sgRNA was usually synthesized by a costly kit [9]. Besides, tedious preparing process of sgRNA and Cas9 protein was timeconsuming and risk of contamination. What’s more, as marker genes (ura3 and pyrG, etc.) mutants had easy-to-select phenotype and disruptants of functional genes usually had not, it was a challenge to disrupt functional genes by using the CRISPR/Cas9 system with in vitro sgRNA. Compared to in vitro one, the procedure of CRISPR/Cas9 system with in vivo transcribed sgRNA was simpler and cost-efficient [3]. What’s more, the in vivo system can be utilized to disrupt functional genes and achieve precise gene editing by stimulating HR [3]. However, as multiple genes disruption strategies and base editor systems (cytosine base editor and adenine base editor) were still lacked, the promotion of current CRISPR system was still to be done.
As for the elucidation of valuable metabolites, such as ganoderic acids, it was just a beginning. Many candidate genes were still to be analyzed by CRISPR/Cas9 assisted gene disruption and in situ complementation. Up to now, the CYP5139G1 can catalyze the HLDOA at C-28 to produce a new GA, 3,28-Dihydroxy- Lanosta-8,24-Dien-26-Oic Acid (DHLDOA), in yeast [7]. CYP512W2 was proved to be responsible for the conversion of HLDOA to type II GAs (GA-Y and GA-Jb) in baker’s yeast [8]. The CYP512U6 can hydroxylate GA-DM and GA-TR at the C-23 to produce hainanic acid A and GA-Jc in vitro, respectively [14]. However, the functions of these CYP genes were still not tested in G. lucidum. Compared to yeast, the genetic background of G. lucidum was more complex, it was a challenge to study the GAs biosynthesis pathway in G. lucidum.
In conclusion, two kinds of CRISPR/Cas9 systems (in vitro transcribed sgRNA and in vivo transcribed sgRNA) were established and utilized for the biotechnological application in G. lucidum. In future, increasing gene function will be investigated. The biosynthesis pathway of various valuable metabolites will be gradually elucidated, which is crucial for the healthcare industry.
No ethical approval was required as this is a review article with no original research data.
All authors have no competing interests.
Ping-An Wang conceived the work and drafted the manuscript.
Citation: Wang P (2023) The Application of CRISPR/Cas9 System in the Basidiomycete Ganoderma lucidum. Appli Microbiol Open Access. 9:270.
Received: 11-Sep-2023, Manuscript No. AMOA-23-26715; Editor assigned: 13-Sep-2023, Pre QC No. AMOA-23-26715(PQ); Reviewed: 02-Oct-2023, QC No. AMOA-23-26715; Revised: 10-Oct-2023, Manuscript No. AMOA-23-26715(R); Published: 18-Oct-2023 , DOI: 10.35284/2471-9315.23.9.270
Copyright: © 2023 Wang P. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.