| Fungal natural products are considered to be a valuable source of drugs or drug-derived compounds.With the continuous accumulation of genomic sequence data,it has been reported that the number of biosynthetic gene clusters in fungal genomes is much higher than the number of characterized natural products.These biosynthetic gene clusters may not have been structurally characterized due to either the unculturabiliy of the corresponding strain or it being silent under laboratory culture conditions.Therefore,effectively mining fungal-derived biosynthetic gene clusters is a key issue that urgently needs to be solved.Heterologous expression of biosynthetic gene clusters is currently one of the main strategies for activating fungal silent gene clusters.Thus,the development of filamentous fungi heterologous expression platforms is of great significance.Genome reduction is one of the strategies for constructing chasis strains for production of natural products through reducing competition for precursor resources required for natural product synthesis,and increasing the resistance of the heterologous synthesis pathway to homolgous metabolic disturbances.Currently,there is relatively rare research on genome reduction of filamentous fungi,mainly focused on deleting known natural product biosynthetic gene clusters.Another important issue faced in the synthesis of fungal natural products is their low inherent synthesis level,which creates a significant challenge for the biological research and industrial application.Therefore,the development of efficient natural products synthesis strageties is also of great practical significance.As important biocontrol fungi,Trichoderma species have great potential for synthesizing structurally diverse natural products.Among them,Trichoderma reesei exhibited both efficient cellulase secretion abilityand the ability to synthesize polyketide compounds,making it as a potential chasis strain for mining fungal biosynthetic gene clustersThe present thesis used a two-step homologous recombination-based seamless deletion strategy to delete a major polyketide biosynthetic gene cluster in T.reesei to construct a chasis strain with clean metabolite background.In order to achieve the efficient synthesis of polyketide compounds using T.reesei,we explored the efficient synthesis of triacetic acid lactone(TAL)through screening of differential synthases and also compartmentalizationstrategies.The main research work of the thesis includes:1.Biosynthetic gene cluster deletion of T.reeseiGenome reduction is one of the important ways to construct a efficient synthetic chasis strain with a clean background.Firstly,a two-step homologous recombination-based seamless gene deletion strategy using the uridine auxotrophic marker gene pyr4 was established in T.reesei.Deletion of genome fragments of different sizes(8-84.5 kb)was successfully achieved,indicating that this seamless deletion strategy can be used for large-scale genome deletion in T.reesei,laying the foundation for further genome reduction.By deleting the sorbicillinoids biosynthetic gene cluster(sor),a T.reesei QM9414 Δpyr4 Δsor strain was constructed.Growth tests showed that the sor gene cluster deletion strain had no significant difference in growth compared to the wild type when grown on glucose and cellulose,while HPLC analysis showed that the sor gene cluster deletion strain lost the ability to synthesize sorbicillinoid compounds,significantly reducing the metabolite background of T.reesei.Transcriptome analysis showed that the core genes of one terpene(15.3 cluster),three NRPS(10.1 cluster,22.1 cluster,and 31.1 cluster)and one PKS(7.3 cluster)gene cluster had relatively high transcription levels in the sor deletion strain.Further deletion of the 15.3 and 10.3 clusters did not yield homozygous transformants,suggesting that certain specific genes in these two clusters may play important roles in the normal growth and development of T.reesei.2.Synthesis of the polyketide compound TAL using T.reesei as the chasis strain.TAL is an important polyketide platform compound.To achieve the efficient synthesis of TAL using lignocellulosic substrates,T.reesei QM9414 Δpyr4 Δsor with a clean metabolite background was used as the chasis strain.The random integration expression of the type Ⅲ polyketide synthase 2-PS from Gerbera jamesonii was driven by the strong promoter of the cellulase cbhl in T.reesei,resulting in the successful production of TAL with crystalline cellulose Avicel as the carbon source.Through the screening of different TAL synthases and codon optimization of the 2-ps gene,it was found that the expression of codon optimized 2-PS(2PS-CO)signifcatnly increased TAL synthesis by about 1450 times,indicating that the expression level of TAL synthase is a key factor limiting TAL synthesis in T.reesei.The 6-MSAS Y1572F could also efficiently catalyze TAL synthesis with about 700 times more production than unoptimized 2-PS.Optimization of the fermentation conditions of TAL-producing strains showed that the highest TAL yield was achieved at 2.52 g/L using 0.5%peptone as the nitrogen source and 1%Avicel as the carbon source,which was more than 2700 times of the initial TAL synthesis level.This study laid the foundation for the conversion of lignocellulose into the ployketide compound TAL using T.reesei as the chasis cell.Eukaryotes have highly organized organelle structures and each organelle has unique physical and chemical environment,enzymes,and metabolites.In addition to the cytoplasm,the precursor for polyketide synthesis are also present in mitochondria and peroxisomes.Therefore,TAL synthesis was further explored by targeting 2PS-CO to mitochondria and peroxisomes.The results showed that TAL synthesis could be achieved when 2PS-CO was targeted to peroxisomes and mitochondria,and the level of TAL synthesis mediated by mitochondrial-targeted 2PS-CO was higher than that of cytoplasmic-targeted enzyme.These findings provide a foundation for further enhancing TAL synthesis through enzyme compartilization. |
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