Genome Mining Of Fungal Natural Herbicide Targeting AHAS

Natural products are an important source of drugs and pesticides.With decades of bioassay-guided isolation of natural products,the discovery of new active compounds has become more difficult.The advancement of genome sequencing and secondary metabolite biosynthesis have made “genome mining” a new growth point in the discovery of new natural products.However,unlike in the traditional bioassay-guided isolation of natural products,the information of biosynthetic gene clusters can be used to predict the structures of compounds,but not the biological activities of compounds.The gene encoding the target protein of the microbial natural product may be colocated on the chromosome with the biosynthetic gene cluster of the compound.Genome mining targeting "self-resistance" genes can help predict the activity of natural products.At present,acetohydroxy acid synthase(AHAS)inhibitors are the largest category in the global herbicide market.AHAS catalyzes the first step of de novo synthesis of branched-chain amino acids in plants and microorganisms,and neither humans nor animals possess AHAS.AHAS is regarded as one of the prime candidates for herbicide targeting.In this study,since self-resistance-gene and the biosynthetic genes were co-located on the fungal chromosome,a self-resistance-gene-directed discovery strategy with AHAS gene as a target was used to search for putative biosynthetic gene clusters in the fungal genome database.The AHAS gene was discovered in a putative polyketide-amino acid gene cluster called fua in the genome of Hypoxylon sp.CO27-5.In addition to a putative gene encoding AHAS,the gene cluster contains three structural genes,encoding a Polyketide Synthase–Nonribosomal Peptide Synthetase(PKS–NRPS),an Enoyl Reductase(ER),a DielsAlderase(DAse).Phylogenetic analysis of AHASs showed that AHASs in fungal secondary metabolite biosynthetic gene clusters are evolutionarily different from AHAS involved in primary metabolism,and form an independent evolutionary direction.Since the strains containing these clusters could not be obtained,the fua gene cluster was synthesized and heterologous expressed in Aspergillus nidulans.Compounds 1 and 2,which had the same molecular weight,similar UV spectrum,and chemical polarity,were produced when PKS-NRPS and ER were expressed.After adding the DAse gene,only compound 1 was produced.The chemical structure of compound 1,a new polyketide-amino acid hybrid named Fujianate,was characterized by high-resolution mass spectrometry and NMR.According to the DAse function and the PKS-NRPS biosynthetic pathway,compound 2 was considered an enantiomer of compound 1.Through the function of the "self-resistance gene",we predicted that Fujianate could inhibit the AHAS involved in primary metabolism,thereby inhibiting the biosynthesis of branched-chain amino acids in plants and microorganisms.The bioassay of inhibition of Arabidopsis thaliana was carried out.The results showed that Fujianate(50 μM)could inhibit the growth of A.thaliana completely,and Fujianate(5 μM)had similar activities with glufosinate(50 μM)to inhibit the growth of A.thaliana.The results of experiments on Cyperus rotundus,one of the world’s top ten malignant weeds,show that Fujianate can completely inhibit its growth in the outdoor soil environment.In terms of mechanism of action,Fujianate can also inhibit the growth of bacteria and fungi.However,due to the different nutritional conditions of microorganisms and plants,the growth environment of pathogenic microorganisms is mostly nutrient-rich.We tested Fujianate in complete media for the inhibition of Escherichia coli CICC 10389,Staphylococcus aureus CICC 10384,Candida albicans CICC 1965,and Pseudomonas aeruginosa CICC 20236,results showed that Fujianate had only moderate inhibitory activity on S.aureus CICC 10384 with MIC 32 μg/m L.Functional studies were carried out on two AHAS in the Hypoxylon sp.CO27-5genome(HCOAHAS is involved in primary metabolism and HCOAHASM is in the fua gene cluster),and only the HCOAHAS was expressed successfully.In vitro catalytic experiments verified the function of HCOAHAS in catalyzing two pyruvates to produce acetolactic acid,and Fujianate had notable inhibitory activity on HCOAHAS.The tertiary structure modeling of HCOAHAS and HCOAHASM were constructed by Alpha Fold 2.Fujianate can bind to the catalytic activity center of AHAS protein binding FAD and Th DP,thereby inhibiting enzyme activity.The amino acid mutation of the Gly384 in HCOAHAS protein to Cys331 directly increases the difficulty of Fujianate binding,which leads to the tolerance of HCOAHASM to Fujianate.For the production of Fujianate by fermentation,the expression plasmids used in the heterologous expression system were modified.Firstly,the independent and autonomous replication AMA1 in the vectors were removed so that the expressed genes could be integrated into the host chromosome for stable expression.The two starch-induced promoters,amy B,and gla A,were replaced with strong constitutive promoters of gpd A and trp C,to eliminate the glucose effect.In this study,the fermentation temperature and time of Aspergillus nidulans expressing Fujianate were studied,and the bioprocess study was carried out on a 50 L fermenter,and the yield was 6.21 mg/m L at 108 h of fermentation.In this study,a new polyketide-amino acid hybrid with herbicide activity was expressed by AHAS-targeted fungal natural product genome mining,and the activities and fermentations of the compound were studied.This study confirmed the high efficiency of fungal self-resistance gene-directed natural product discovery and provided application prospects for the further development of Fujianate as a natural herbicide.