Fungal artificial chromosomes, compositions, methods and uses therefor
Fungal artificial chromosome (FAC) vectors are disclosed. A vector can be replicated in a bacterial or a fungal host, and can comprise an insert of heterologous DNA up to about 500 kilobases. A vector can be used for cloning and expressing a secondary metabolite (SM) gene cluster. An insert sequence can be modified by homologous recombination. A vector can be a plasmid comprising bacterial and fungal origins of replication, as well as bacterial and fungal selection marker genes. Also disclosed are vectors that can be integrated into a fungal genome, and dual function vectors which can be replicated in a bacterial or a fungal host and can also be integrated into a fungal genome. Also disclosed are methods of generating plasmid libraries including vectors comprising intact SM gene clusters.
1. A fungal artificial chromosome (FAC) comprising a bacterial artificial chromosome (BAC) vector comprising:
a low-copy number bacterial origin of replication;
an inducible high-copy number bacterial origin of replication;
an E. coli selectable marker gene;
an Aspergillus selectable marker gene; and
an AMA1 filamentous fungal autonomous replicating element,
wherein the FAC is a plasmid that replicates extrachromosomally in an E. coli host and in an Aspergillus fungal host.
2. A fungal artificial chromosome in accordance with claim 1 , further comprising a pair of recognition sites in a head-to-head orientation for a restriction enzyme that generates non-complementary single-stranded overhangs upon digestion of the FAC.
3. A fungal artificial chromosome in accordance with claim 2 , wherein the restriction enzyme that generates non-complementary single-stranded overhangs upon digestion of the FAC is selected from the group consisting of BstXI and I-SceI.
4. A fungal artificial chromosome in accordance with claim 1 , wherein the low-copy number bacterial origin of replication is an oriS and the inducible high-copy number bacterial origin of replication is an oriV.
5. A fungal artificial chromosome in accordance with claim 1 , wherein the E. coli selectable marker gene is selected from the group consisting of a chloramphenicol resistance gene (camR), kanR, ampR, genR, tetA, strepR, galK, and a combination thereof.
6. A fungal artificial chromosome in accordance with claim 1 , wherein the Aspergillus selectable marker gene is selected from the group consisting of pyrG, ptrA, trpC, and a combination thereof.
7. A fungal artificial chromosome in accordance with claim 1 , further comprising an insert of at least 20 kb.
8. A fungal artificial chromosome in accordance with claim 1 , further comprising an insert of at least 100 kb.
9. A fungal artificial chromosome in accordance with claim 1 , further comprising an insert comprising at least one secondary metabolite (SM) gene cluster.
10. A fungal artificial chromosome in accordance with claim 1 , further comprising an integration site and an integrase gene.
11. An Aspergillus fungus comprising the fungal artificial chromosome of claim 1 .
12. An Aspergillus fungus in accordance with claim 11 , wherein the fungal artificial chromosome comprises at least one secondary metabolite (SM) gene cluster that is heterologous to the fungus.
13. A method of unbiased FAC library construction, comprising:
providing high molecular weight (HMW) genomic DNA from a filamentous fungus;
mechanically shearing the HMW genomic DNA into fragments of 100 kb-300 kb in length;
generating blunt ends on the DNA fragments;
ligating BstXI linkers to the blunt ends, thereby generating linker-ligated DNA fragments;
purifying the linker-ligated DNA fragments by pulse field gel electrophoresis; and
ligating the purified and linker-ligated DNA fragments into a BstXI-cut fungal artificial chromosome (FAC) of claim 1 .
14. A method in accordance with claim 13 , further comprising transforming a host microorganism with the ligated BstXI-cut FAC, wherein the host microorganism is an E. coli.
15. A method of inserting a DNA sequence into a targeted location in a secondary metabolite (SM) gene cluster, comprising:
providing a fungal artificial chromosome (FAC) comprising a secondary metabolite (SM) gene cluster in accordance with claim 9 ;
providing an insertion DNA comprising a) a first sequence homologous to a sequence flanking a first side of the targeted location, b) a sequence to be inserted, c) a second sequence homologous to a sequence flanking a second side of the targeted location and d) a bacterial selectable marker;
transforming the FAC and the insertion DNA into an E. coli strain that expresses Red/ET recombinase enzymes; and
selecting a transformed E. coli cell that comprises the bacterial selectable marker.
16. A method of deleting a targeted DNA sequence from a secondary metabolite (SM) gene cluster, comprising:
providing a fungal artificial chromosome (FAC) comprising a secondary metabolite (SM) gene cluster in accordance with claim 9 ;
providing a deletion DNA comprising a) a first sequence homologous to a sequence flanking a first side of the targeted DNA sequence, b) a second sequence homologous to a sequence flanking a second side of the targeted DNA sequence, and c) a bacterial selectable marker;
transforming the FAC and the insertion DNA into an E. coli strain that expresses Red/ET recombinase enzymes; and
selecting a transformed E. coli cell that comprises the bacterial selectable marker.
17. A fungal artificial chromosome in accordance with claim 1 , wherein:
the low-copy number bacterial origin of replication is an oriS;
the inducible high-copy number bacterial origin of replication is an oriV;
the E. coli selectable marker gene is selected from the group consisting of a chloramphenicol resistance gene (camR), kanR, ampR, genR, tetA, strepR, galK, and a combination thereof; and
the Aspergillus selectable marker gene is selected from the group consisting of pyrG, ptrA, trpC, and a combination thereof.
18. A fungal artificial chromosome in accordance with claim 17 , further comprising a pair of recognition sites in a head-to-head orientation for a restriction enzyme that generates non-complementary single-stranded overhangs upon digestion of the plasmid.
19. A fungal artificial chromosome comprising a nucleic acid sequence as set forth in SEQ ID NO:7, SEQ ID NO:11, or SEQ ID NO:14.