1.The high-efficiency expression host— Aspergillus niger is coming!

• 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.
• 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？

• 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.
• 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.
• 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.
• mechanism
• 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.
• The chromosomally encoded protein, ChvE,interacts with the VirA protein to further enhance levels of vir induction in the presence of specific monosaccharides.
• 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.
• 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
• stimulates T-strand production. The VirD2 protein ,assisted by VirD1,stays covalently attached to the 5’ end of the T-strand.
• 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.
• advantages
• 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.
• Reference:
• Caroline B. Michielse .Agrobacterium -mediated transformation as a tool for functional genomics in fungi

3. Targeted gene replacement system

• (1)The targeted gene replacement system mainly adopts the principle of homologous gene recombination. We import homologous gene sequences to the nuclear genome specific receptor gene loci and replace the original genes ,so that we can achieve our purpose of modifying the original genome. In the process of target gene replacement , the construction of vectors is of great importance, which typically includes 2 arms ,respectively in homology with the target gene’s homologous sequences on both sides ,and 1 selection marker gene which is used to eliminate noise bacteria. In our universal system, we choose GlaA5 and GlaA3 as the homologous arms on either side of the target gene site and HPH (hygromycin) as selective marker.
• (2)In our previous detection methods in target gene replacement, we usually extract the genome and conduct the PCR detection, using the homology arms as primiers, which is somewhat complex and time-consuming.
• (3)The noise from NHEJ.The insertion of exogenous gene may include another circumstance that the nonhomologous end, not dependent on DNA homology, force the two DNA end to connect together,which is called non-homologous end joining. This mechanism allows the possibility that displacement vector is randomly inserted into chromosomes, greatly reducing the efficiency of homologous recombination.

What if “colourful” visual gene replacement system?

• Our system use chromeproteins as visual markers for selecting, thus pick out the transformants and distinguish the homologous recombination and non homologous end joining . Taking the Gla site as an example,the genetic recombination target site in host's genome should have the structure of GlaA5-Target site- GlaA3. And our recombination vector is design for the structure of GlaA5- AMILCP -GlaA3-pgPDA(promoter)-CJBLUE –linker- HPH - GlaA3.
• For the first step, the homologous gene replacement will occur between GlaA5 and GlaA3 homology arms, and the whole cassette will be integrated at the Gla site, taking replacement of the original gene, so that the blue and green color will express at the same time . After several generations, homologous recombination will happen between the two GlaA3 sequences, and lead to the loss of HPH and cjBlue, then get the transgenic Aspergillus Niger cells only express amilCP gene which can be easily detected by blue color.The colony will only have blue color, indicating that the selective marker HPH has already been deleted. The blue colonies will be our target transformants. And the chromo protein can be used for our tool to make the target gene replacement system visual!
• For the second step, the AMILCP is replaced by the adaptor element, which works as the loader to connect the genes that we want to be expressed to the vector. When the target genes are imported to the host genome’s GlaA specific site successfully, the blue color will disappear. So that we can distinguish our target transformants!
• Adaptor : a segment of DNA containing NotI and XbaI sites. The two sides of itself are two kinds of restriction enzyme cutting sites, prospectively ApaI and HindIII, meanwhile located between two homology arms:GlaA5 and GlaA3 of the plasmid .So, we could achieve the standardization of sites by connected adapoter to GlaA5 and GlaA3.

Construction of expression vector

• Vectors used in the project for membrane protein expression are modified versions of pSZH, which are originally regulated by GlaA5 promoter. These plasmids can coexist in one cell.
• Considering our filamentous fungi system, it is necessary to use those compatible Ti-plasmids. To stress the advantage of the system, we recruited a suitable promoter, GlaA5 promoter. All proteins in our project are inserted into those reconstructed vectors (pSZH) and under control of Gla5 promoter.
• In pSZH, GlaA5 and GlaA3 is a length of homologous sequence. E1010 is a red chromo protein gene, which we designed to replace target Gla sites. HPH is hygromycin selective marker gene while CJBLUE is visual. OriV and NPTII is necessary section of pSZH.

why continuous?

• There are many efficient expression sites on Aspergillus Niger’s genome. Gla sites is a kind of efficient expression sites. So we designed GlaA5 and GlaA3 sequences as homology arms. Amy sits is another efficient expression site. So we also designed AmyA5 and AmyA3 sequences as homology arms. If we have use our target gene to replace Gla sites, we can also use another target gene to replace Amy sites in the same way again. So two kinds of target genes can efficient express in one kind of Aspergillus Niger. And we can achieve the replacement by other efficient expression sites again and again continuously. So we call our method “continuous”.

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