| Trichoderma reesei(T.reesei)is widely used in the production of various homologous or heterologous proteins due to its high protein secretion,stable genetic traits,rapid growth and reproduction,simple culture conditions and good biosafety.With the increase in efforts on T.reesei research,it is found that under certain conditions,the fungus can considerably secret a kind of secondary metabolites,sorbicillinoids,which have numerous potential applications,such as antioxidants,antibiotics,and anticancer drugs.These valuable compounds have been given a lot of attention for the reason that they can be used as potential sources for new therapeutic agents and drugs,and have potential applications in medicine,pharmacy,and biotechnology.However,it is only known that sorbicillinoids are synthesized from a gene cluster consisting of eight genes,two of which are polyketide synthase genes.The specific production mechanisms are still unknown.In addition,in the future production of sorbicillinoids in the industrial scale,the characteristics of T.reesei high-yield cellulase will increase the difficulty in the downstream separation process.Therefore,it is imperative to study the production mechanism of sorbicillinoids and develop stable strains with high-yield sorbicillinoids and low-yield cellulase.In this paper,we will focus on the following three aspects of sorbicillinoids gene cluster:1.Construction of a genetically-engineered strain Sor3-C30 with high yield of Sorbicillinoids.The Sor3 gene is a synthetic pathway gene in the gene cluster,which encodes a flavin adenine nucleotide-dependent monooxygenase.In this work,we reconstructed a binary vector by single restriction enzyme and obtained engineering strain Sor3-C30 overexpressing Sor3 gene using Agrobacterium-mediated RUT-C30 mononuclear spore technology.The strain was induced by glucose in the Trichoderma minimal media(TMM)medium.The Sorbicillinoids yield of Sor3-C30 is the highest among T.reesei strains reported in the literature.2.When Ypr1 was overexpressed in T.reesei RUT-C30,the yield of Sorbicillinoids increased significantly,and the total cellulase activity decreased notably,indicating that Ypr1 gene is a positive regulator of secondary metabolism of Sorbicillinoids.The Ypr1 gene is a transcription factor of the sorbicillinoids gene cluster,which controls the transcription and expression of the entire gene cluster.In order to study the effect of the Ypr1 gene on the production of sorbicillinoids,T.reesei RUT-C30 was used as starting strain in this work.The T.reesei genetically-engineered strain Ypr1-C30 overexpressing the Ypr1 gene was obtained using Agrobacterium-mediated spore technology for the first time.The yield of sorbicillinoids and the enzyme activities of cellulase in the TMM induced by glucose and cellulose were detected.It was found that the yield of sorbicillinoids increased significantly and the total cellulase activity decreased notably.The production of sorbicillinoids by Ypr1-C30 is highly carbon dependent.3.A screening marker technique was used to mediate multiple genetic transformations of fungi for the first time.The Sor5 gene was overexpressed in T.reesei genetically-engineered strain ZC121 as a starting strain using Agrobacterium-mediated spores.ZC121 is a hygromycin-resistant strain,and Sor5-ZC121 is continuously screened by hygromycin with a correct rate of 40%.For the first time,the same resistance gene was used to achieve secondary Agrobacterium-mediated genetic transformation in T.reesei.On the basis of successful secondary mediation,Agrobacterium carrying different genes was co-cultured with T.reesei RUT-C30 spores to verify whether different genes could be simultaneously introduced into one receptor using only one screening marker.In addition,the experiments indicated that the overexpression of the Sor5 gene in ZC121 strain has little effect on the yield of sorbicillinoids and the enzyme activity of each component of cellulase. |
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