Signal sequence trapping

Abstract

The present invention allows the screening of previously established genebanks or libraries by proxy for genes encoding secreted, partially secreted, or cell surface-displayed polypeptides of industrial interest, such as enzymes, receptors, cytokines, peptide hormones etc. that would not likely have been isolated using conventional screening assays. A method for isolating genes encoding secreted, partially secreted, or cell surface displayed polypeptides from existing gene libraries is described in which the endogenous secretion signal sequences are detected using an in vitro polynucleotide insertion reaction where the inserted polynucleotide comprises a promoter-less and secretion signal-less secretion reporter.

49 . A method for identifying the complete coding sequence of a gene of interest from a gene library, wherein the gene encodes a polypeptide carrying a signal sequence for secretion or partial secretion, the method comprising the steps of:
 (a) providing a genomic DNA library or a cDNA library;   (b) inserting by in vitro transposition into a gene in said library a transposon comprising a polynucleotide encoding a promoterless and secretion signal-less secretion reporter; wherein there is a continuous open reading frame between the transposon and the polynucleotide encoding the secretion reporter;   (c) introducing the library comprising the inserted transposon into a fungal host cell;   (d) screening for and selecting a fungal host cell that secretes or partially secretes the secretion reporter;   (e) identifying the coding sequence of the gene of interest into which the transposon was inserted in the selected fungal host cell, by sequencing DNA flanking the inserted transposon; and   (f)identifying the complete coding sequence of the gene of interest identified in step (e) by sequencing.   
     
     
         50 . The method of  claim 49 , wherein the complete coding sequence of the gene of interest in step (f)is isolated from the library of step (a). 
     
     
         51 . The method of  claim 49 , wherein the genomic DNA library or the cDNA library is normalized. 
     
     
         52 . The method of  claim 49 , wherein the transposon comprises an origin of replication which is functional in the host cell. 
     
     
         53 . The method of  claim 49 , wherein the secretion reporter is a protein which, when secreted from the host cell, allows said cell to grow in the presence of a substance which otherwise inhibits growth of said cell. 
     
     
         54 . The method of  claim 53 , wherein the secretion reporter is a β-lactamase or an invertase. 
     
     
         55 . The method of  claim 49 , wherein the polynucleotide of the DNA-fragment of step (b) encodes a secretion reporter carrying an N-terminal peptide linker which comprises a specific target site for proteolytic cleavage. 
     
     
         56 . method of  claim 49 , wherein the sequencing step of step (e) is performed using at least one primer directed to the transposon, or using at least one primer directed to a vector in which the DNA library or cDNA library is cloned. 
     
     
         57 . The method of  claim 49 , further comprising isolating the complete coding sequence of the gene of interest by utilizing the DNA sequence information obtained in the sequencing step of step (e). 
     
     
         58 . The method of  claim 49 , further comprising constructing an expression system which comprises the complete coding sequence of the gene of interest identified in step (f). 
     
     
         59 . The method of  claim 49 , wherein the fungal host cell is an  Acremonium, Aspergillus, Aureobasidium, Candida, Cryptococcus, Filibasidium, Fusarium, Humicola, Kluyveromyces, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Pichia, Piromyces, Saccharomyces, Schizosaccharomyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma,  or  Yarrowia  cell. 
     
     
         60 . A method for identifying the complete coding sequence of a gene of interest from a gene library, wherein the gene encodes a polypeptide carrying a signal sequence for secretion or partial secretion, the method comprising the steps of:
 (a) providing a genomic DNA library or a cDNA library;   (b) inserting by in vitro transposition into a gene in said library a transposon comprising a polynucleotide encoding a promoterless and secretion signal-less secretion reporter; wherein there is a continuous open reading frame between the transposon and the polynucleotide encoding the secretion reporter;   (c) introducing the library comprising the inserted transposon into a bacterial host cell;   (d) screening for and selecting a bacterial host cell that secretes or partially secretes the secretion reporter;   (e) identifying the coding sequence of the gene of interest into which the transposon was inserted in the selected bacterial host cell, by sequencing DNA flanking the inserted transposon; and   (f) identifying the complete coding sequence of the gene of interest identified in step (e) by sequencing.   
     
     
         61 . The method of  claim 60 , wherein the complete coding sequence of the gene of interest in step (f)is isolated from the library of step (a). 
     
     
         62 . The method of  claim 60 , wherein the genomic DNA library or the cDNA library is normalized. 
     
     
         63 . The method of  claim 60 , wherein the transposon comprises an origin of replication which is functional in the host cell. 
     
     
         64 . The method of  claim 60 , wherein the secretion reporter is a protein which, when secreted from the host cell, allows said cell to grow in the presence of a substance which otherwise inhibits growth of said cell. 
     
     
         65 . The method of  claim 64 , wherein the secretion reporter is a β-lactamase or an invertase. 
     
     
         66 . The method of  claim 60 , wherein the polynucleotide of the DNA-fragment of step (b) encodes a secretion reporter carrying an N-terminal peptide linker which comprises a specific target site for proteolytic cleavage. 
     
     
         67 . The method of  claim 60 , wherein the sequencing step of step (e) is performed using at least one primer directed to the transposon, or using at least one primer directed to a vector in which the DNA library or cDNA library is cloned. 
     
     
         68 . The method of  claim 60 , further comprising isolating the complete coding sequence of the gene of interest by utilizing the DNA sequence information obtained in the sequencing step of step (e). 
     
     
         69 . The method of  claim 60 , further comprising constructing an expression system which comprises the complete coding sequence of the gene of interest identified in step (f). 
     
     
         70 . The method of  claim 60 , wherein the bacterial host cell is a  Bacillus, Enterococcus, Escherichia,  Lactococcus, or  Streptomyces  cell. 
     
     
         71 . The method of  claim 60 , wherein the bacterial host cell is a  Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Enterococcus faecalis, Escherichia coli, Lactococcus lactis, Streptomyces coelicor,  or  Streptomyces griseus  cell.