Generation of Iron-Independent Siderophore-Producing through the Constitutive Expression of .

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