Generation of Iron-Independent Siderophore-Producing Agaricus bisporus through the Constitutive Expression of hapX

Abstract

Agaricus bisporus secretes siderophore to uptake environmental iron. Siderophore secretion in A. bisporus was enabled only in the iron-free minimal medium due to iron repression of hapX, a transcriptional activator of siderophore biosynthetic genes. Aiming to produce siderophore using conventional iron-containing complex media, we constructed a recombinant strain of A. bisporus that escapes hapX gene repression. For this, the A. bisporushapX gene was inserted next to the glyceraldehyde 3-phosphate dehydrogenase promoter (pGPD) in a binary vector, pBGgHg, for the constitutive expression of hapX. Transformants of A. bisporus were generated using the binary vector through Agrobacterium tumefaciens-mediated transformation. PCR and Northern blot analyses of the chromosomal DNA of the transformants confirmed the successful integration of pGPD-hapX at different locations with different copy numbers. The stable integration of pGPD-hapX was supported by PCR analysis of chromosomal DNA obtained from the 20 passages of the transformant. The transformants constitutively over-expressed hapX by 3- to 5-fold and sidD, a key gene in the siderophore biosynthetic pathway, by 1.5- to 4-fold in mRNA levels compared to the wild-type strain (without Fe3+), regardless of the presence of iron. Lastly, HPLC analysis of the culture supernatants grown in minimal medium with or without Fe3+ ions presented a peak corresponding to iron-chelating siderophore at a retention time of 5.12 min. The siderophore concentrations of the transformant T2 in the culture supernatant were 9.3-fold (−Fe3+) and 8-fold (+Fe3+) higher than that of the wild-type A. bisporus grown without Fe3+ ions, while no siderophore was detected in the wild-type supernatant grown with Fe3+. The results described here demonstrate the iron-independent production of siderophore by a recombinant strain of A. bisporus, suggesting a new application for mushrooms through molecular biological manipulation.

Establishment of an Efficient Genetic Transformation System in Sanghuangporus baumii

“…This modification resulted in the production of a new binary vector for the genetic transformation of S. baumii. The use of fungal homologous promoters to replace plant promoters is a commonly employed method, as demonstrated in previous studies involving L. edodes [26], G. lucidum [13], and Agaricus bisporus [30], which showed improved transformation efficiency. The gpd promoter region, in particular, has been proven to be highly efficient in directing the expression of heterologous genes in S. cerevisiae [31].…”

Structural Analysis of the A Mating Type Locus and Development of the Mating Type Marker of Agaricus bisporus var. bisporus

“…The heterokaryotic strains, including NH1, Top, and EM, were isolated directly from the fruiting bodies obtained from commercial markets. The mycelial culture was conducted in a compost–dextrose–peptone medium (CDPM) containing 1% glucose, 0.7% malt extract, and 0.5% peptone in compost extract, as previously reported [ 4 ]. The mycelia were cultivated in CDPM at 25 °C.…”

“…In the generation of new strains, the traditional cross-mating between two different monokaryons has been the major technique employed in mushroom breeding. Molecular breeding by modifying genomic sequences to alter the function and expression of genes of interest [ 1 ] or by introducing foreign DNA to the host chromosomal sequence [ 2 , 3 , 4 ] has been attempted with some mushrooms; however, this approach is not applicable to edible mushrooms because the mushrooms generated in this way are essentially genetically modified organisms (GMOs). Nonetheless, a new technology in molecular breeding has emerged recently, namely CRISPR-Cas9 gene-editing technology, which enables targeted modification of the genes or DNA sequences of certain functions [ 5 , 6 , 7 , 8 ].…”

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

“…However, it is a time-consuming process that requires human efforts and financial inputs because obtaining a desirable strain is difficult. Molecular breeding, which involves altering genes of interest in a targeted manner, has been sought to precisely modify some mushroom strains in recent decades 1 – 3 . However, this technique is not feasible for many mushrooms due to the lack of proper molecular biological tools 4 .…”

“…Integrating foreign DNA fragments into chromosomal DNA is also difficult because nonhomologous end-joining (NHEJ) is the main mechanism for DNA damage repair, which instantly repairs double strand breaks (DSBs) and therefore rarely allows homologous recombination (HR) 5 , 6 . Forced chromosomal integration of foreign DNA and selection under antifungal agents or auxotrophic markers have generally been used for the transformation of mushrooms, delivered by Agrobacterium tumefaciens -mediated transformation (ATMT) 3 , 7 – 9 or PEG-mediated transformation 1 , 2 . However, the method is not applicable in commercial strain development due to issues with genetically modified organisms (GMOs).…”