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. 2024 May 30:15:1410368.
doi: 10.3389/fmicb.2024.1410368. eCollection 2024.

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

Ding-Xi Zhou #  1 , Xiang-Ming Kong #  2 , Xiong-Min Huang  1 , Na Li  3 , Na Feng  4 , Jun-Wei Xu  1
Affiliations

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

Ding-Xi Zhou et al. Front Microbiol. .

Abstract

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.

Keywords: Ganoderma; breeding; ganoderic acids; genetic engineering; mono–mono crossing.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Chemical structures of individual ganoderic acid-P, ganoderic acid-S, ganoderic acid-T, and ganoderic acid-Me.
Figure 2
Figure 2
Isolation and identification of monokaryotic G. lucidum strains. (A) Isolation of the monokaryotic strain G. 260124 and G. 260125 from the dikaryotic CGMCC 5.0026 strain by protoplast formation and regeneration. (B) Identification of the monokaryotic strains G. 260124 and G. 260125 by morphological observation and SNP-PCR. Clamp connections and two SNPs are shown.
Figure 3
Figure 3
Generation and verification of the transgenic G. lucidum strain G. 260125-sqs and G. 260124-vgb. Structures of the plasmids pJW-EXP-SQS (A) and pJW-EXP-In-Op-vgb (E). Selection of the transgenic strains G. 260124-vgb (B) and G. 260125-sqs (F) on a CYM selection plate. Identification and characterization of the transgenic strains G. G. 260124-vgb (C) and G. 260125-sqs (G) by genome PCR. (D) Transcriptional levels of the sqs in the CGMCC 5.0026 (blank) and the G. 260125-sqs (gray) strains. (H) CO-difference spectra analysis of the CGMCC 5.0026 (dotted line) and G. 260124-vgb (solid line) strains. Lane P, the plasmid positive control; Lane N, negative control; Lanes 1, 2, 3, the transgenic strains; Lane M, DNA marker DL2000.
Figure 4
Figure 4
Generation and verification of the dikaryotic G. lucidum strain sqs-vgb by mating mycelia of the G. 260125-sqs and G. 260124-vgb strains. (A) Cross mating of two monokaryon G. 260125-sqs and G. 260124-vgb, and regeneration of protoplasts from the mycelia from the contact area. Identification of the dikaryotic strain sqs-vgb by morphological observation (B) and SNP-PCR (C). The integration of sqs (D) and vgb (E) in the genome of the selected sqs-vgb strain was confirmed via PCR. Clamp connections and two SNPs are shown. Lane P, the plasmid positive control; Lane N, negative control; Lanes 1, 2, 3, 4, 5, and 6, the selected sqs-vgb strains; Lane M, DNA marker DL2000.
Figure 5
Figure 5
Growth of the CGMCC 5.0026 and sqs-vgb strains (A) and contents of individual GA-P (B), GA-T (C), GA-S (D), and GA-Me (E) at three different developmental stages of the CGMCC 5.0026 (blank) and sqs-vgb strains (filled). *A significantly different from the CGMCC 5.00296 strain.
Figure 6
Figure 6
Intermediate accumulations of and gene expression levels of GA biosynthetic genes at three different developmental stages of the CGMCC 5.0026 and sqs-vgb strains. Accumulation of squalene (A) and lanosterol (B) in the CGMCC 5.0026 (blank) and sqs-vbg (filled) strains. Transcriptional levels of hmgr, sqs and ls at three developmental stages of the CGMCC 5.0026 and sqs-vgb (C). The expression of genes in the CGMCC 5.0026 strain was defined as 1.0, and the expression levels in the sqs-vgb strain are shown as fold changes compared to the reference. *A significantly different from the CGMCC 5.00296 strain.
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