Fungal artificial chromosomes, compositions, methods and uses therefor

Abstract

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.

What is claimed is: 
     
         1 . A fungal artificial chromosome (FAC) comprising a bacterial artificial chromosome (BAC) backbone comprising:
 at least one bacterial origin of replication;   a bacterial selectable marker gene;   a fungal selectable marker gene; and   a fungal autonomous replicating element.   
     
     
         2 . The fungal artificial chromosome of  claim 1 , wherein the at least one bacterial origin of replication is selected from the group consisting of a low-copy number bacterial origin of replication, an inducible high-copy number bacterial origin of replication, and a combination thereof. 
     
     
         3 . The fungal artificial chromosome of  claim 2 , 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. 
     
     
         4 . The fungal artificial chromosome of  claim 1 , wherein the bacterial 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. 
     
     
         5 . The fungal artificial chromosome of  claim 1 , wherein the fungal selectable marker gene is selected from the group consisting of pyrG, ptrA, trpC, and a combination thereof. 
     
     
         6 . The fungal artificial chromosome of  claim 1 , wherein the fungal autonomous replicating element is an AMA1 autonomous replicating element. 
     
     
         7 . The fungal artificial chromosome of  claim 1 , wherein the FAC is a plasmid. 
     
     
         8 . The fungal artificial chromosome of  claim 7 , wherein the plasmid replicates extrachromosomally in a bacterial host and in a fungal host. 
     
     
         9 . The fungal artificial chromosome of  claim 8 , wherein the bacterial host is  E. coli  and the fungal host is  Aspergillus.    
     
     
         10 . The fungal artificial chromosome of  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. 
     
     
         11 . The fungal artificial chromosome of  claim 10 , wherein the restriction enzyme that generates non-complementary single-stranded overhangs upon digestion of the FAC is selected from the group consisting of BstXI, I-SceI, BsrI, and I-CeuI. 
     
     
         12 . A fungal artificial chromosome in accordance with  claim 10 , further comprising an insert of up to about 500 kb. 
     
     
         13 . The fungal artificial chromosome of  claim 1 , further comprising an integration site and an integrase gene. 
     
     
         14 . The fungal artificial chromosome of  claim 13 , wherein the integration site is an attP site and the integrase gene is a fungal codon-optimized phi31 integrase gene. 
     
     
         15 . The fungal artificial chromosome of  claim 1 , further comprising two fungal sequences in the same orientation. 
     
     
         16 . The fungal artificial chromosome of  claim 15 , wherein the two fungal sequences are 5′trpC and 3′trpC.