Bacterial leader sequences for increased expression

Abstract

Compositions and methods for improving expression and/or secretion of a polypeptide of interest in a host cell are provided. Compositions including a coding sequence for a bacterial secretion signal peptide are provided. The compositions of the invention are useful for increasing accumulation of properly processed proteins in the periplasmic space of a host cell, or for increasing secretion of properly processed proteins. In particular, isolated secretion signal peptide-encoding nucleic acid molecules are provided. Additionally, amino acid sequences corresponding to the nucleic acid molecules are encompassed. The present invention provides for isolated nucleic acid molecules including nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24, and the nucleotide sequences set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23, as well as variants and fragments thereof.

1 - 85 . (canceled) 
     
     
         86 . An isolated nucleic acid molecule comprising a secretion signal coding sequence for a secretion polypeptide selected from the group consisting of: a mutant phosphate binding protein (pbp*); a protein disulfide isomerase C (DsbC); a CupA2; a CupB2; a CupC2; a NikA; a tetratricopeptide repeat family protein (ORF5550); a toluene tolerance protein (Ttg2C); a methyl accepting chemotaxis protein (ORF8124); and a TolB secretion polypeptide; wherein said secretion signal coding sequence is obtained from  Pseudomonas , and wherein said secretion signal coding sequence is selected from the group consisting of:
 a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 5, 9, 11, 13, 15, 19, 21, 23 or 49;   b) a nucleic acid molecule comprising a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 1, 5, 9, 11, 13, 15, 19, 21, 23 or 49, wherein said nucleotide sequence encodes a secretion polypeptide;   c) a nucleic acid molecule comprising a nucleotide sequence which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 6, 10, 12, 14, 16, 20, 22, 24 or 50;   d) a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2, 6, 10, 12, 14, 16, 22, 24 or 50, wherein said polypeptide is a secretion polypeptide;   e) a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 96% amino acid sequence identity to the amino acid sequence of SEQ ID NO:20, wherein said polypeptide is a secretion polypeptide;   f) a nucleic acid molecule comprising a nucleotide sequence that hybridizes under stringent conditions over the entire length of the nucleotide sequence of SEQ ID NO: 1, 5, 9, 11, 13, 15, 19, 21, 23 or 49 wherein said stringent conditions comprise a temperature of about 68° C. to about 70° C.; and   g) a nucleic acid molecule comprising a nucleotide sequence that hybridizes under stringent conditions over the entire length to a nucleotide sequence that encodes SEQ ID NO: 2, 6, 10, 12, 14, 16, 20, 22, 24, or 50, wherein said stringent conditions comprise a temperature of about 68° C. to about 70° C.   
     
     
         87 . An isolated nucleic acid molecule of  claim 86 , wherein the nucleic acid molecule comprises a secretion signal coding sequence for a secretion polypeptide having a hydrophobicity score of at least about WS 0.69, wherein said secretion polypeptide having a hydrophobicity score of at least about WS 0.69 is selected from the group consisting of: a CupA2; a CupC2; a tetratricopeptide repeat family protein (ORF5550); a toluene tolerance protein (Ttg2C); a methyl accepting chemotaxis protein (ORF8124) secretion polypeptide; and a TolB secretion polypeptide; and wherein said nucleic acid molecule having a hydrophobicity score of at least about WS 0.69 is selected from the group consisting of:
 a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 9, 13, 19, 21, 23, or 49;   b) a nucleic acid molecule comprising a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 9, 13, 19, 21, 23, or 49, wherein said nucleotide sequence encodes a secretion polypeptide;   c) a nucleic acid molecule comprising a nucleotide sequence which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 10, 14, 20, 22, 24, or 50;   d) a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 10, 14, 22, 24, or 50, wherein said polypeptide is a secretion polypeptide;   e) a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 96% amino acid sequence identity to the amino acid sequence of SEQ ID NO:20, wherein said polypeptide is a secretion polypeptide;   f) a nucleotide sequence that hybridizes under stringent conditions over the entire length of the nucleotide sequence of SEQ ID NO: 9, 13, 19, 21, 23, or 49, wherein said stringent conditions comprise a temperature of about 68° C. to about 70° C.; and   g) a nucleotide sequence that hybridizes under stringent conditions over the entire length to a nucleotide sequence that encodes SEQ ID NO: 10, 14, 20, 22, 24, or 50, wherein said stringent conditions comprise a temperature of about 68° C. to about 70° C.   
     
     
         88 . An isolated nucleic acid molecule of  claim 87 , wherein the nucleic acid molecule comprises a secretion signal coding sequence for a secretion polypeptide having a hydrophobicity score of at least WS 0.69, wherein said secretion polypeptide having a hydrophobicity score of at least WS 0.69 is selected from the group consisting of: a tetratricopeptide repeat family protein (ORF5550); a toluene tolerance protein (Ttg2C); a methyl accepting chemotaxis protein (ORF8124) secretion polypeptide; and a TolB secretion polypeptide; wherein said nucleic acid molecule comprising a secretion signal coding sequence for a secretion polypeptide having a hydrophobicity score of at least WS 0.69 is selected from the group consisting of:
 a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 19, 21, 23 or 49;   b) a nucleic acid molecule comprising a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 19, 21, 23 or 49, wherein said nucleotide sequence encodes a secretion polypeptide;   c) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 20, 22, 24 or 50;   d) a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 22, 24 or 50, wherein said polypeptide is a secretion polypeptide;   e) a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 96% amino acid sequence identity to the amino acid sequence of SEQ ID NO:20, wherein said polypeptide is a secretion polypeptide;   f) a nucleotide sequence that hybridizes under stringent conditions over the entire length of the nucleotide sequence of SEQ ID NO: 19, 21, 23 or 49 wherein said stringent conditions comprise a temperature of about 68° C. to about 70° C.; and   g) a nucleotide sequence that hybridizes under stringent conditions over the entire length to a nucleotide sequence that encodes SEQ ID NO: 20, 22, 24 or 50 wherein said stringent conditions comprise a temperature of about 68° C. to about 70° C.   
     
     
         89 . The isolated nucleic acid molecule of  claim 86 , wherein said nucleic acid molecule has been adjusted to reflect the codon preference of a host organism selected to express the nucleic acid molecule. 
     
     
         90 . A vector comprising a secretion signal coding sequence for a mutant phosphate binding protein (pbp*), a protein disulfide isomerase C (dsbC), a CupA2, a CupB2, a CupC2, a NikA, a tetratricopeptide repeat family protein (ORF5550), a toluene tolerance protein (Ttg2C), a methyl accepting chemotaxis protein (ORF8124), or a TolB secretion polypeptide wherein said secretion signal coding sequence is obtained from  Pseudomonas , and wherein said nucleic acid molecule is selected from the group consisting of:
 a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 5, 9, 11, 13, 15, 19, 21, 23 or 49;   b) a nucleic acid molecule comprising a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 1, 5, 9, 11, 13, 15, 19, 21, 23 or 49, wherein said nucleotide sequence encodes a secretion polypeptide;   c) a nucleic acid molecule comprising a nucleotide sequence which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 6, 10, 12, 14, 16, 20, 22, 24 or 50;   d) a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2, 6, 10, 12, 14, 16, 22, 24 or 50; wherein said polypeptide is a secretion polypeptide;   e) a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 96% amino acid sequence identity to the amino acid sequence of SEQ ID NO:20, wherein said polypeptide is a secretion polypeptide;   f) a nucleic acid molecule comprising a nucleotide sequence that hybridizes under stringent conditions over the entire length of the nucleotide sequence of SEQ ID NO: 1, 5, 9, 11, 13, 15, 19, 21, 23 or 49 wherein said stringent conditions comprise a temperature of about 68° C. to about 70° C.; and   g) a nucleic acid molecule comprising a nucleotide sequence that hybridizes under stringent conditions over the entire length to a nucleotide sequence that encodes SEQ ID NO: 2, 6, 10, 12, 14, 16, 20, 22, 24 or 50; wherein said stringent conditions comprise a temperature of about 68° C. to about 70° C.   
     
     
         91 . The vector of  claim 90 , wherein said nucleic acid molecule has been adjusted to reflect the codon preference of a host organism selected to express the nucleic acid molecule. 
     
     
         92 . The vector of  claim 90 , wherein the secretion signal coding sequence is operably linked to a sequence encoding a protein or polypeptide of interest. 
     
     
         93 . The vector of  claim 92 , wherein the protein or polypeptide of interest is derived from a protein or polypeptide that is not native to a host organism in which the protein or polypeptide of interest is expressed. 
     
     
         94 . The vector of  claim 92 , wherein the protein or polypeptide of interest is from an organism that is not a Pseudomonad. 
     
     
         95 . The vector of  claim 90 , wherein the protein or polypeptide of interest is derived from a eukaryotic organism. 
     
     
         96 . The vector of  claim 95 , wherein the protein or polypeptide of interest is derived from a mammalian organism. 
     
     
         97 . The vector of  claim 90 , further comprising a linkage sequence between the signal polypeptide sequence and the protein or polypeptide of interest sequence. 
     
     
         98 . The vector of  claim 97 , wherein the linkage sequence is cleavable by a signal peptidase. 
     
     
         99 . The vector of  claim 90 , wherein the vector further comprises a promoter. 
     
     
         100 . The vector of  claim 99 , wherein the promoter is native to a bacterial host cell. 
     
     
         101 . The vector of  claim 99 , wherein the promoter is not native to a bacterial host cell. 
     
     
         102 . The vector of  claim 100 , wherein the promoter is native to  E. coli.    
     
     
         103 . The vector of  claim 99 , wherein the promoter is an inducible promoter. 
     
     
         104 . The vector of  claim 100 , wherein the promoter is a lac promoter or a derivative of a lac promoter. 
     
     
         105 . A recombinant cell comprising a secretion signal coding sequence for a mutant phosphate binding protein (pbp*), a protein disulfide isomerase C (dsbC), a CupA2, a CupB2, a CupC2, a NikA, a tetratricopeptide repeat family protein (ORF5550), a toluene tolerance protein (Ttg2C), a methyl accepting chemotaxis protein (ORF8124) or a TolB secretion polypeptide, wherein said secretion signal coding sequence is obtained from  Pseudomonas , and wherein said secretion signal coding sequence is selected from the group consisting of:
 a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 5, 9, 11, 13, 15, 19, 21, 23 or 49;   b) a nucleic acid molecule comprising a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 1, 5, 9, 11, 13, 15, 19, 21, 23 or 49, wherein said nucleotide sequence encodes a secretion polypeptide;   c) a nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 6, 10, 12, 14, 16, 20, 22, 24 or 50;   d) a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2, 6, 10, 12, 14, 16, 22, 24 or 50; wherein said polypeptide is a secretion polypeptide;   e) a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 96% amino acid sequence identity to the amino acid sequence of SEQ ID NO:20, wherein said polypeptide is a secretion polypeptide;   f) a nucleotide sequence that hybridizes under stringent conditions over the entire length of the nucleotide sequence of SEQ ID NO: 1, 5, 9, 11, 13, 15, 19, 21, 23 or 49 wherein said stringent conditions comprise a temperature of about 68° C. to about 70° C.; and   g) a nucleotide sequence that hybridizes under stringent conditions over the entire length to a nucleotide sequence that encodes SEQ ID NO: 2, 6, 10, 12, 14, 16, 20, 22, 24 or 50; wherein said stringent conditions comprise a temperature of about 68° C. to about 70° C.   
     
     
         106 . The cell of  claim 105 , wherein the secretion signal coding sequence is in an expression vector. 
     
     
         107 . The cell of  claim 105 , wherein the secretion signal coding sequence is operably linked to a sequence encoding a protein or polypeptide of interest. 
     
     
         108 . The cell of  claim 107 , wherein the cell expresses the protein or polypeptide of interest operably linked to the secretion signal polypeptide. 
     
     
         109 . The cell of  claim 108 , wherein the protein or polypeptide is expressed in a periplasmic compartment of the cell. 
     
     
         110 . The cell of  claim 108 , wherein an enzyme in the cell cleaves the secretion signal polypeptide from the protein or polypeptide of interest. 
     
     
         111 . The cell of  claim 105 , wherein the cell is obtained from a bacterial host. 
     
     
         112 . The cell of  claim 111 , wherein the host is a Pseudomonad. 
     
     
         113 . The cell of  claim 112 , wherein the host is a  P. fluorescens.    
     
     
         114 . The cell of  claim 111 , wherein the host is an  E. coli.    
     
     
         115 . An isolated polypeptide comprising a mutant phosphate binding protein (pbp*), a protein disulfide isomerase C (dsbC), a CupA2, a CupB2, a CupC2, a NikA, a tetratricopeptide repeat family protein (ORF5550), a toluene tolerance protein (Ttg2C), a methyl accepting chemotaxis protein (ORF8124) or a TolB secretion polypeptide, wherein said secretion signal polypeptide is obtained from  Pseudomonas  and wherein said polypeptide is selected from the group consisting of:
 a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 6, 10, 12, 14, 16, 20, 22, 24 or 50;   b) a polypeptide encoded by the nucleotide sequence SEQ ID NO: 1, 5, 9, 11, 13, 15, 19, 21, 23 or 49;   c) a polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2, 6, 10, 12, 14, 16, 20, 22, 24 or 50, wherein said polypeptide is a secretion signal polypeptide;   d) a polypeptide that is encoded by a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 1, 5, 9, 11, 13, 15, 19, 21, 23 or 49, wherein said polypeptide is a secretion signal polypeptide; and,   e) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions over the entire length of the nucleotide sequence of SEQ ID NO: 1, 5, 9, 11, 13, 15, 19, 21, 23 or 49, wherein said stringent conditions comprise a temperature of about 68° C. to about 70° C.   
     
     
         116 . The polypeptide of  claim 115 , wherein the secretion signal polypeptide is operably linked to a protein or polypeptide of interest. 
     
     
         117 . The polypeptide of  claim 116 , wherein the protein or polypeptide of interest is obtained from an organism that is not a  P. fluorescens  organism. 
     
     
         118 . An expression system for expression of a polypeptide or protein of interest comprising:
 a) a host cell; and   b) a vector comprising a nucleic acid molecule encoding the protein or polypeptide of interest operably linked to a secretion signal coding sequence for a mutant phosphate binding protein (pbp*), a protein disulfide isomerase C (dsbC), a CupA2, a CupB2, a CupC2, a NikA, a tetratricopeptide repeat family protein (ORF5550), a toluene tolerance protein (Ttg2C), a methyl accepting chemotaxis protein (ORF8124) or a TolB secretion signal polypeptide, wherein said secretion signal coding sequence is obtained from  Pseudomonas , and wherein said secretion signal coding sequence is selected from the group consisting of:   a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 5, 9, 11, 13, 15, 19, 21, 23 or 49;   b) a nucleic acid molecule comprising a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 1, 5, 9, 11, 13, 15, 19, 21, 23 or 49, wherein said nucleotide sequence encodes a secretion polypeptide;   c) a nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 6, 10, 12, 14, 16, 20, 22, 24 or 50;   d) a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2, 6, 10, 12, 14, 16, 22, 24 or 50, wherein said polypeptide is a secretion polypeptide;   e) a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 96% amino acid sequence identity to the amino acid sequence of SEQ ID NO:20, wherein said polypeptide is a secretion polypeptide;   f) a nucleotide sequence that hybridizes under stringent conditions over the entire length of the nucleotide sequence of SEQ ID NO: 1, 5, 9, 11, 13, 15, 19, 21, 23 or 49 wherein said stringent conditions comprise a temperature of about 68° C. to about 70° C.; and   g) a nucleotide sequence that hybridizes under stringent conditions over the entire length to a nucleotide sequence that encodes SEQ ID NO: 2, 6, 10, 12, 14, 16, 20, 22, 24 or 50 wherein said stringent conditions comprise a temperature of about 68° C. to about 70° C.   
     
     
         119 . The expression system of  claim 118 , wherein the host cell expresses the protein or polypeptide of interest operably linked to the secretion signal polypeptide. 
     
     
         120 . The expression system of  claim 119 , wherein the protein or polypeptide of interest is expressed in a periplasmic compartment of the cell. 
     
     
         121 . The expression system of  claim 119 , wherein an enzyme in the cell cleaves the signal polypeptide from the protein or polypeptide of interest. 
     
     
         122 . The expression system of  claim 119 , wherein the cell is obtained from a bacterial host. 
     
     
         123 . The expression system of  claim 118 , wherein the host is a Pseudomonad. 
     
     
         124 . The expression system of  claim 123 , wherein the host is  P. fluorescens.    
     
     
         125 . The expression system of  claim 122 , wherein the host is  E. coli.    
     
     
         126 . The expression system of  claim 118 , further comprising a fermentation medium. 
     
     
         127 . The expression system of  claim 126 , wherein the fermentation medium comprises a chemical inducer. 
     
     
         128 . A method for the expression of a recombinant protein in a host cell comprising providing a host cell comprising a vector encoding a protein or polypeptide of interest operably linked to a secretion signal coding sequence encoding a mutant phosphate binding protein (pbp*), a protein disulfide isomerase C (dsbC), a CupA2, a CupB2, a CupC2, a NikA, a tetratricopeptide repeat family protein (ORF5550), a toluene tolerance protein (Ttg2C), a methyl accepting chemotaxis protein (ORF8124) or a TolB secretion signal polypeptide, wherein said secretion signal coding sequence is obtained from  Pseudomonas , and wherein said secretion signal coding sequence is selected from the group consisting of:
 a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 5, 9, 11, 13, 15, 19, 21, 23 or 49;   b) a nucleic acid molecule comprising a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 1, 5, 9, 11, 13, 15, 19, 21, 23 or 49, wherein said nucleotide sequence encodes a secretion polypeptide;   c) a nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 6, 10, 12 14, 16, 20, 22, 24 or 50;   d) a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2, 6, 10, 12, 14, 16, 22, 24 or 50, wherein said polypeptide is a secretion polypeptide;   e) a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 96% amino acid sequence identity to the amino acid sequence of SEQ ID NO:20, wherein said polypeptide is a secretion polypeptide;   f) a nucleotide sequence that hybridizes under stringent conditions over the entire length of the nucleotide sequence of SEQ ID NO: 1, 5, 9, 11, 13, 15, 19, 21, 23 or 49 wherein said stringent conditions comprise a temperature of about 68° C. to about 70° C.; and   g) a nucleotide sequence that hybridizes under stringent conditions over the entire length to a nucleotide sequence that encodes SEQ ID NO: 2, 6, 10, 12, 14, 16, 20, 22, 24 or 50 wherein said stringent conditions comprise a temperature of about 68° C. to about 70° C.   
     
     
         129 . The method of  claim 128 , wherein the cell is grown to a high cell density. 
     
     
         130 . The method of  claim 129 , wherein the cell is grown to a cell density of at least 20 g/L. 
     
     
         131 . The method of  claim 128 , further comprising purifying the recombinant protein. 
     
     
         132 . The method of  claim 131 , wherein the recombinant protein is purified by affinity chromatography. 
     
     
         133 . The method of  claim 128 , wherein the operable linkage of the protein or polypeptide of interest and the secretion signal polypeptide is cleavable by an enzyme native to the host cell. 
     
     
         134 . The method of  claim 133 , wherein the secretion signal polypeptide is cleaved from the protein or polypeptide of interest during expression. 
     
     
         135 . The method of  claim 128 , wherein the protein or polypeptide of interest is not native to the organism from which the host cell is derived. 
     
     
         136 . The method of  claim 128 , wherein the protein or polypeptide of interest is obtained from an organism that is not a Pseudomonad. 
     
     
         137 . The method of  claim 128 , wherein the protein or polypeptide of interest is obtained from a eukaryotic organism. 
     
     
         138 . The method of  claim 128 , wherein said vector further comprises a nucleic acid sequence encoding a linkage sequence between the secretion signal polypeptide and the protein or polypeptide of interest. 
     
     
         139 . The method of  claim 128 , wherein the protein or polypeptide of interest is native to the organism from which the host cell is derived, and wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, of the protein or polypeptide of interest comprises a native amino terminus. 
     
     
         140 . The method of  claim 128 , wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, of the recombinant protein is active. 
     
     
         141 . The method of  claim 128 , wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the recombinant protein is expressed in a periplasmic compartment. 
     
     
         142 . The method of  claim 128 , wherein the host cell is a Pseudomonad cell. 
     
     
         143 . The method of  claim 142 , wherein the cell is a  P. fluorescens  cell. 
     
     
         144 . The method of  claim 128 , wherein the cell is an  E. coli  cell.