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Aspergillus Niger is an important fermentation microbial ,which is widely used in industrial enzymes'production and organic acid fermentation.It can produce more than 30 kinds of enzyme product such as amylase, acid protease, cellulase, pectinase and glucose oxidase. In fact, Aspergillus Niger has been applied in the field of food production for a long time.Because of its excellent ability of secreting proteins, Aspergillus Niger is developed for universal heterologous protein expression host.
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As receptor bacteria of genetic engineering ,Aspergillus Niger has unique advantages upon bacteria and yeast .It's able to do majority kinds of processing after translation , and the glycosylation system is similar to that of higher eukaryotes.It's well known for high protein secretion capacity and recognized as the safe strain for heterologous protein production,in that the fermentation and post-processing technologies are mature.In addition,its genome sequences has already been published.
2.Why AMT？
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Agrobacterium-mediated fungal transformation(AMT)is a potential tool for performing targeted and random mutagenesis .This method is commonly used for plant-cell transformations and recently widely applied to various fungus. In the AMT system,A.Tumefaciens is able to transfer T-DNA to a wide variety of fungi ,and it has especial high efficiency.
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The gram-negative bacterium A. tumefaciens is a plant pathogen, which causes crown gall tumors.A. tumefaciens induces this tumorous growth by transferring a part of its DNA (T-DNA) ,which is located on its 200-kbp tumor-inducing (Ti) plasmid to the host. After integration into the host genome, genes that are naturally located on this T-DNA and encode enzymes for the production of metabolites and regulators for the plant growth. But another segment,the virulence region, which is composed of a large number of vir genes,is necessary for the tumorigenicity .So the binary vector system is used,in which the T-DNA and the virulence region are placed on two separate plasmids.
Structure of A. tumefaciens' plasmid
- The gram-negative bacterium A. tumefaciens is a plant pathogen, which causes crown gall tumors.A. tumefaciens induces this tumorous growth by transferring a part of its DNA (T-DNA) which is located on its 200-kbp tumor-inducing (Ti) plasmid to the host. After integration into the host genome, genes that are naturally located on this T-DNA and encode enzymes for the production of plant growth regulators are expressed.
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And another segment,the virulence region, which is composed of a large number of vir genes,is necessary for the tumorigenicity.Proteins encoded by the virulence region are involved in the formation, transport and possibly also integration of the T-DNA.And the T-region of the Ti plasmid is surrounded by a 24-bp border repeat, which is the cis-acting signal for the DNA delivery system to plant cells.Otherwise,all the sequences of the natural T-DNA can be deleted and replaced by other DNA sequences without a negative effect.So the binary vector system is used,in which the T-DNA and the virulence region are placed on two separate plasmids.
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mechanism
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Phenolic compounds such as acetosyringone are used to induce the vir genes that encode the T-DNA transfer machinery of A. Tumefaciens.VirA, an inner membrane protein, senses acetosyringone and responds by autophosphorylation.
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The chromosomally encoded protein, ChvE,interacts with the VirA protein to further enhance levels of vir induction in the presence of specific monosaccharides.
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The activated VirG, which has DNA-binding properties, then acts as a transcriptional activator of itself and other virulence genes after VirA transfers phosphoryl group to it.
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For the generation of a single-stranded DNA copy of the T-DNA,the virC and virD operons are needed.VirC1 can bind the25-bp “overdrive” sequence and thereby
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stimulates T-strand production. The VirD2 protein ,assisted by VirD1,stays covalently attached to the 5’ end of the T-strand.
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The next step in T-DNA transfer is piloting the T-strand through the bacterial membrane and cell wall.The VirB proteins form a transport pore and a structure on the surface,and the virulence proteins VirE2, VirE3, and VirF are also exported .VirE2 is a single-stranded DNA-binding protein and is thought to coat the T-strand in the host to protect it against nucleases and to keep the T-strand in an unfolded state to facilitate transport. Once inside the nucleus, the T-DNA stably integrates into the genome.
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advantages
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It has been shown to have several advantages over conventional transformation methods.AMT generates a high percentage of transformants with a single-copy integrated DNA, which facilitates the isolation of tagged genes,and the T-DNA is an efficient substrate for homologous recombination. Above all,AMT is well suited to perform insertional mutagenesis in fungi.
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Reference:
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Caroline B. Michielse .Agrobacterium -mediated transformation as a tool for functional genomics in fungi
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