Study On The Functions Of Three TPS Genes In Arthrobotrys Oligospora And Priliminary Construction Of A Ku70Mutant Of Arthrobotrys Oligospora

Nematode-trapping fungus, Arthrobotrys oligospora produces a class of compounds named Arthrosporols as singal molecules that can regulate fungal morphology. Terpene synthase (TPS) and polyketide synthase (PKS) are involved in the biosynthesis of those compounds. This article mainly focused on the functions of TPS in Arthrobotrys oligospora. Bioinformatics analysis of A. oligospora genome, we found seven putative TPS genes in A. oligospora. Among them, two sesquiterpen-e synthase genes (AOL_s00078g383, AOL_s00083g86) and a terpene synthase gene (AOL_s00054g768) are selected as the research targets in this article. With the application of the principles and methods of molecular biology, three mutants with TPS gene knockout were obtained. HPLC analysis of ethyl acetate extracts of PDB fermentation broths of these three mutants and wild type strain showed that three mutants all contain Arthrosporols. This results indicated three TPS genes might not be involved in the biosynthesis of Arthrosporols. But the concentration of Arthrosporol A in the mutant△AOL_s00083g86was higher than that in wild type strain. GC-MS analysis showed that mutant AAOL_s00078g383lacked7-Oxabicyclo [4.1.0] heptane,2,2,6-trimethyl-l-(3-methyl-1,3-butadienyl)-5-m ethylene and the mutant AAOL_s00083g86lacked2-Hydroxy-2,4,4-trimethyl-3-(3-met-hylbuta-1,3-dienyl) cyclohexanone, those two compounds possessed a similar carbon skeleton of sesquiterpenol to that of the sesquiterpenol moiety of Arthrosporols. This result indicated genes AOL_s00078g383, AOL_s00083g86might only be involved in the biosynthesis of sesquiterpenol metabolites, but not be involved in the biosynthesis of PKS/TPS hybrid metabolite Arthrosporols.Three TPS mutants were compared with the wild type strain in aspects of hyphae, conidiation, germination rate, trap number and predatory ability. The results indicated that mutants AAOL_s00083g86grew faster and mutants△AOL_s00083g86and△AOL_s00054g768’s capacity of sporulation have nearly doubled than wild type strain.This article also reports the construction of a Ku70gene mutant of A. oligospora. Ku70plays an important role in DNA double-strand break (DSB) repair in eukaryotes. Druption of Ku70can promote efficiency of homologous recombination and can improve gene targeting efficiency. A Ku70gene knockout strain of A. oligospora was obtained which privode a good foundation for further construction of an efficient engineering strain of A. oligospora.