Analysis of the biosynthesis of altertoxins in Alternaria alternata
Creators: name: Gao, Jia
Publisher: name: Karlsruher Institut für Technologie (KIT)
Year: 2022

description: The filamentous fungus Alternaria alternata is one of the most widespread contaminants of food and feed, and a weak plant pathogen. It produces a great diversity of secondary metabolites, many of which are generally recognized as phytotoxins and mycotoxins, with alternariol and its derivatives as prominent examples. A. alternata is a black mold that produces dihydroxynaphthalene (DHN) melanin in its cell walls of spores and hyphae. The DHN-melanin biosynthetic gene cluster in A. alternata contains only three genes: the polyketide synthase-encoding gene pksA, the 1,3,8-trihydroxynaphthalene (T3HN) reductase-encoding gene brm2, and the transcription factor-encoding gene cmrA. The scytalone dehydratase-encoding gene brm1 is found elsewhere in the genome. Compared to the well-characterized DHN-melanin biosynthetic pathway in Aspergillus fumigatus, the pathway for DHN melanin biosynthesis in A. alternata is less well studied. A. alternata also produces several secondary metabolites of the perylene quinone (PQ) family, such as altertoxins (ATX I-III). Some PQs are phytotoxic but exhibit some anticancer activity. On the other hand, ATX II is a powerful mutagen that causes DNA strand-breaks, and is therefore a serious threat to human health. The biosynthesis route of PQs is still unclear. Here, I show that the DHN-melanin biosynthetic pathway shares most of the enzymes with the pathway for PQs. Intriguingly, DHN-melanin was synthesized mainly in spores and aerial hyphae, whereas PQs were formed in substrate hyphae. A. alternata pksA was heterologously expressed in Aspergillus oryzae, and the heptaketide YWA1, the hexaketide AT4HN and the pentaketide T4HN were identified as products of the enzyme reaction. I characterized two α-hydrolyses, AygA and AygB, required for DHN melanin biosynthesis. PQs biosynthesis was independent of the two enzymes. Promoter-reporter assays showed that aygA and aygB are expressed in spores and aerial hyphae, where DHN melanin is synthesized, but not in substrate hyphae, where PQs are formed. These results suggest that T4HN is probably synthesized directly in substrate hyphae and then used to produce PQs. In spores and aerial hyphae, YWA1 and AT4HN are hydrolyzed to T4HN by AygA and AygB, which boosters the concentration of T4HN for DHN melanin biosynthesis. I also identified a new T4HN reductase, Brm3, catalyzing the formation of scytalone from T4HN. Furthermore, Brm1, Brm2 and Brm3 were required to produce PQs. Next, I performed a feeding experiment to determine the intermediate for PQs formation in the DHN-melanin biosynthetic pathway. When different amounts of 1,8-DHN were fed to the pksA-deletion strain, the production of PQs, altertoxin I (ATX I), altertoxin II (ATX II) and alterperylenol (ALP), was recovered. Melanization of the △pksA strain was also recovered in liquid culture in the presence of 1,8-DHN. 1,8-DHN is hence the last common intermediate for melanin and PQs biosynthesis. Several laccase-encoding genes were deleted separately or in combination using the CRISPR/Cas9 knock-out technology. Four laccases, LccB-D and LccF, were required for DHN melanin production, but not for PQs biosynthesis. The dimerization enzyme catalyzing the reaction of 1,8-DHN to PQs remains to be identified. Depletion of the regulator CmrA resulted in a brownish mutant strain, unlike the pale or pinkish pksA-deletion strain. In fact, the genes pksA, aygB, brm2 and lccD were still expressed. The transcription factor CmrA strictly controls the expression of aygA, brm1, brm3, lccB and lccF. PQs were not detected in the cmrA-deletion strain. Promoter-reporter assays showed that pksA is not expressed in substrate hyphae, suggesting that CmrA controls the production of PQs through controlling the expression of pksA and/or the dimerization enzyme-encoding gene. The PQs biosynthesis pathway in A. alternata is an example of how spatial regulation of gene expression of one biosynthetic pathway can lead to different secondary metabolites.