| Nematode-trapping fungus, Arthrobotrys oligospora produces a class of compounds named arthrosporols as sigal molecules that can regulate fungal morphology. Polyketide synthase (PKS),Terpene synthase (TPS),Transferase, Dehydrogenase, Hydroxylase and Isomerase are involved in the biosynthesis of those compounds. By bioinformatics analysis and similar structures of the compounds biosynthetic genes blast analysis,14 genes in the genome of A.oligosporawere screened out to be involved in the biosynthesis of arthrosporols, including AOL_s00080g121(TPS), AOL_s00215g22(P450), AOL_s00215g274(Dehydrogenase), AOL_s00215g259(2-polyprenyl-6-methoxyphenol hydroxylase), AOL_s00215g275 (Hypothetical protein),AOL_s00215g276 (Polyprenyltransferase), AOL_s00215g277 (Isomerase), AOL_s00215g278 (P450). AOL_s00215g279(Dehydrogenase),AOL_s00215g280(P450),AOL_s00215g281(Amidohydrol ase), AOL_s00215g282(P450), AOL_s00215g283(PKS) and AOL_s00215g926(PKS). These 14 genes were selected as the research targets in this paper, a combination of genetic manipulation, chemical analyses and phenotype comparison was applied to explore the roles of these biosynthetic genes in the synthesis of arthrosporols as well as fungi growth and predation.The main results were described as follows:1. Through in-Fusion ligation-mediated method for constructing 15 gene knockout plasmid vectors and CaCl2-PEG mediated transformation method was used to transform the A. oligspora protoplast, we get 14 genes mutant strains, we also build a large fragment of a mutant strain AOL_s00215g277-283.2. HPLC and GC-MS analysis of ethyl acetate extracts of PDB broths of these fourteen mutant strains and wild type strain showed that disruption of ten genes 215g274.215g276,215g277,215g278,215g279,215g280,215g281.215g282,215g283,215g926 led to a total loss of arthrosporol A. however, another three mutant strains still can detected arthrosporol A, showed that ten genes 274,276,277,278.279,280,281,282,283,926 in the 215g cluster are involved in the biosynthesis of arthrosporol A, while the genes 22 and 275 in this cluster and 80g121 in another cluster are not involved in the biosynthesis of arthrosporol A.3. By comparison,215g281,215g282,215g276 mutant strains with peculiar peaks with the standards, three intermediates 6-methylsalicylic acid, m-cresol, and toluquinol respectively in the biosynthesis of arthrosporols were isolated and identified. Four steps in the biosynthesis of arthrosporol were deduced. The first step is theformation of 6-methylsalicylic acid with gene 215g283, the second step is formed m-cresol by decarboxylation of 6-methylsalicylic acid with gene 215g281, the third step is converted into Toluquinol by hydroxy of m-cresolwith the cytochrome P450 encoded by 215g282, the fourth step is conversion of toluquinol into one key TPS/PKS intermediate with gene 215g276, the product structure needs to be isolated and characterized.4. By comparison phenotypes of three mutant strains △80g121, △215g22, △215g283 with the wildtype in mycelium morphology, mycelium growth rate, sporulation, spore germination rate, the number of three-dimensional trap formations, and nematode-trapping capacity, interestingly, the 215g283 mutant strain displayed significant increases in the trap formation and the nematicidal activity by 10 and 2 times, respectively, higher than the wild-type strain.5. By RNA-Seq sequencing and analysi of △215g22, △215g283 mutant and wild-type strains fermented mycelias shows, in the △215g22 mutant strains the expressions of 30 genes were found down-regulated, while 17 genes were up-regulated compared to wild type. In the △215g283 mutant strains the expressions of 187 genes were found down-regulated, while 147 genes were up-regulated compared to wild type. Furthermore, the disruption of gene 215g283 causing genes 215g274,215g276,215g277,215g278,215g279,215g280,215g282 up-regulated,these genes are all involved in the biosynthesis of arthrosporol. in addition to,it also caused gene 215g286 down-regulated, the gene is located upstream of the 283, it is likely to be involved in the regulation of the biosynthesis of arthrosporol.Innovations in this paper:1. A combination of genetic manipulation and chemical analyses was applied to characterize the function of 15 biosynthetic genes. The cluster 215g was found to be involved in the biosynthesis of morphological regulatory PKS and TPS hybrid arthrosporols. The genes 283,281,282, and 276 are involved in the first four-step reaction in the biosynthesis of arthrosporol, respectively. It seems that a TPS derived farnesyl was transferred to a PKS derived Toluquinol to form a key TPS-PKS hybrid intermediate for the biosynthesis of the arthrosporols.2. Disruption of biosynthesis gene 215g283 led to not onlythe significant reduction in the production of secondary metabolites but also significant increases in the trap formation and the nematicidal activity.3. Disruption of two polyketide synthase (PKS) genes 215g283 and 215g926, showed that they are all involved in the biosynthesis of arthrosporol,215g283 was involved in the production of 6-methylsalicylic acid, however, the function of 215g926 still unknown. |
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