Control of protein to protein interactions of acid decarboxylase

Abstract

This invention provides acid decarboxylase-prion subunit fusion polypeptides, nucleic acid sequences, expression vectors, and host cells expression such fusion polypeptides to produce various amino acids and derivatives of the amino acids such as polyamines.

1 . A product, which is one of the following products I) through IV):
 I) a genetically modified host cell comprising a nucleic acid encoding an acid decarboxylase fusion protein comprising an acid decarboxylase polypeptide joined to a prion subunit fused to the carboxyl end of the acid decarboxylase polypeptide, wherein acid decarboxylase fusion protein has increased activity relative to the acid decarboxylase polypeptide not joined to the prion subunit;   II) an acid decarboxylase fusion protein comprising an acid decarboxylase polypeptide fused to a prion subunit, wherein the fusion protein has improved acid decarboxylase activity in vitro as measured by the production of polyamines at alkaline pH, relative to a counterpart fusion protein lacking the prion subunit;   III) a polynucleotide encoding a fusion protein of II);   IV) an expression vector comprising the polynucleotide of III), operably linked to a promoter.   
     
     
         2 . A product of  claim 1 , which is I) the genetically modified host cell, wherein the prion subunit is at least 50 amino acids in length, at least 75 amino acids in length or at least 100 amino acids in length, but 500 amino acids or fewer in length. 
     
     
         3 . A product of  claim 1 , which is I) the genetically modified host cell of  claim 1 , wherein the prion subunit comprises an amino acid composition having at least 20% glutamine and/or asparagine residues. 
     
     
         4 . A product of  claim 1 , which is I) the genetically modified host cell, where the prion subunit:
 (i) has at least 70%, 75%, 80%, 85%, 90%, or 95% identity to a Sup35, New1, Ure2, or Rnq1 amino acid sequence;   (ii) comprises a Sup35, New1, Ure2, or Rnq1 amino acid sequence;   (iii) has at least 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4;   (iv) comprises SEQ ID NO:3 or SEQ ID NO:4; or   (v) has at least 70%, 75%, 80%, 85%, 90%, or 95% identity to the amino acid sequence of the prion subunit region of any one of SEQ ID NOS: 7, 8, 11, 13, 14, 15, 17, or 19.   
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         5 . A product of  claim 1 , which is I) the genetically modified host cell, wherein the prion subunit is joined at the carboxyl terminus to a BST fragment, λCI fragment, or RecA fragment. 
     
     
         6 . A product of  claim 1 , which is I) the genetically modified host, wherein prion subunit:
 (a) is joined at the C-terminal end to a BST fragment and has at least 70%, 75%, 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO:15, excluding the linker region; or comprises the amino acid sequence to SEQ ID NO:15, excluding the linker region;   (b) is joined at the C-terminal end to a ΔCI fragment and has at least 70%, 75%, 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO:16, excluding the linker region; or comprises the amino acid sequence to SEQ ID NO:16, excluding the linker region;   (c) is joined at the C-terminal end to a RecA fragment and has at least 70%, 75%, 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO:19, excluding the linker region; or comprises the amino acid sequence to SEQ ID NO:19, excluding the linker region; or   (d) is joined at the carboxyl terminus to an amino acid sequence RRFGEASSAF, ASQWPEETFG, or EGVAETNEDF.   
     
     
         9 . (canceled) 
     
     
         7 . A product of  claim 1 , which is I) the genetically modified host cell, wherein the acid decarboxylase is a lysine decarboxylase, ornithine decarboxylase, arginine decarboxylase, or glutamate decarboxylase. 
     
     
         8 . A product of  claim 1 , which is I) the genetically modified host cell of, wherein the acid decarboxylase is a CadA, LdcC, AdiA, SpeA, SpeC, SpeF, GadA, or GadB polypeptide. 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         9 . A product of  claim 8 , wherein the lysine decarboxylase is a CadA lysine decarboxylase or a LdcC lysine decarboxylase. 
     
     
         10 . A product of  claim 8 , wherein the host cell is genetically modified to over express one or more lysine biosynthesis polypeptides. 
     
     
         11 . A product of  claim 1 , which is I) the genetically modified host cell, wherein the nucleic acid encoding the acid decarboxylase fusion protein is encoded by an expression vector introduced into the cell, wherein the expression vector comprises the nucleic acid encoding the acid decarboxylase fusion protein operably linked to a promoter, and/or the nucleic acid encoding the acid decarboxylase fusion protein is integrated into the host chromosome. 
     
     
         17 . (canceled) 
     
     
         12 . A product of  claim 1 , which is I) the genetically modified host cell, wherein the nucleic acid encoding the acid decarboxylase fusion polypeptide comprises the nucleic acid sequence of any one of SEQ ID NOS:1, 5, 6, 9, 10, 16, 18, or 20. 
     
     
         13 . A product of  claim 1 , which is I) the genetically modified host cell, wherein the host cell is a bacterium. 
     
     
         14 . A product of  claim 13 , wherein the host cell is from the genus  Escherichia  or  Hafnia.    
     
     
         15 . A product of  claim 14 , wherein the host cell is  Escherichia coli  or  Hafnia  alvei. 
     
     
         16 . A method, which is one of the following methods I) through III):
 (I) a method for producing an acid decarboxylase fusion protein comprising cultivating a host cell of  claim 1  I) under conditions in which the acid decarboxylase fusion protein is expressed;   (II) a method of producing an amino acid or an amino acid derivative, the method comprising culturing a host cell of  claim 1  I) under conditions in which the acid decarboxylase fusion protein is expressed;   (III) a method of improving acid decarboxylase activity in vitro under alkaline pH comprising fusing a prion subunit to the carboxyl terminus of an acid decarboxylase, and subjecting the recombinant protein to alkaline pH.   
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         17 . A product of  claim 1 , which is II) the fusion protein, wherein the prion subunit:
 (i) is 30 amino acids in length, at least 50 amino acids in length, at least 75 amino acids in length or at least 100 amino acids in length, but 1200 amino acids or fewer in length;   (ii) comprises an amino acid composition having at least 20% glutamine and/or asparagine residues;   (iii) has at least 70%, 75%, 80%, 85%, 90%, or 95% identity to a Sup35, New1, Ure2, or Rnq1 amino acid sequence;   (iv) comprises a Sup35, New1, Ure2, or Rnq1 amino acid sequence;   (v) has at least 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4;   (vi) comprises SEQ ID NO: 3 or SEQ ID NO: 4; or   (vii) has at least 70%, 75%, 80%, 85%, 90%, or 95% identity to the amino acid sequence of the prion subunit region of any one of SEQ ID NOS: 7, 8, 11, 13, 14, 15, 17, or 19.   
     
     
         27 . (canceled) 
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . (canceled) 
     
     
         18 . A product of  claim 1 , which is II) the fusion protein wherein the prion subunit:
 (i) is joined to the C-terminus of the acid decarboxylase;   (ii) is joined at the carboxyl terminus to a BST fragment, ΔCI fragment, or RecA fragment;   (iii) is joined the carboxyl terminus to an amino acid sequence RRFGEASSAF, ASQWPEETFG, or EGVAETNEDF;   (iv) is joined at the C-terminal end to a BST fragment and has at least 70%, 75%, 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 15, excluding the linker region; or comprises the amino acid sequence to SEQ ID NO: 15, excluding the linker region;   (v) is joined at the C-terminal end to a ΔCI fragment and has at least 70%, 75%, 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 16, excluding the linker region; or comprises the amino acid sequence to SEQ ID NO: 16, excluding the linker region; or   (vi) is joined at the C-terminal end to a RecA fragment and has at least 70%, 75%, 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 19, excluding the linker region; or comprises the amino acid sequence to SEQ ID NO: 19, excluding the linker region.   
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . (canceled) 
     
     
         35 . (canceled) 
     
     
         19 . A product of  claim 1 , which is II) the fusion protein, wherein the acid decarboxylase is a CadA, LdcC, AdiA, SpeA, SpeC, SpeF, GadA, or GadB polypeptide. 
     
     
         37 . (canceled) 
     
     
         38 . (canceled) 
     
     
         39 . (canceled) 
     
     
         40 . (canceled) 
     
     
         20 . A product of  claim 1 , which is II) the fusion protein, wherein the fusion protein is immobilized to a solid support. 
     
     
         42 . (canceled) 
     
     
         43 . (canceled)