Escherichia coli strains that over-produce l-threonine and processes for their production

Abstract

The invention relates to novel bacterial strains and constructs as well as methods for production of L-amino acids, including but not limited to L-threonine. Such novel bacterial strains may be characterized by, for instance, Escherichia coli strains in which an aspartate semialdehyde dehydrogenase (asd) gene is operably associated with at least one non-native promoter, non-native ribosome binding site, or both.

1 . A strain of  Escherichia coli  comprising the following characteristics:
 (a) a chromosome of said strain contains at least one aspartate semialdehyde dehydrogenase (asd) gene operably associated with at least one constitutive Ptac-like promoter comprising the nucleotide sequence of SEQ ID NO: 16; and   (b) said strain over-produces at least one amino acid when compared to a parent strain identical to said strain except that said asd gene is operably associated with its native promoter.   
     
     
         2 . The strain of claim  0 , wherein said amino acid is selected from the group consisting of L-lysine, L-threonine, L-methionine, L-homoserine, and L-isoleucine. 
     
     
         3 . The strain of claim  0 , further comprising a non-native ribosome binding site operably associated with said asd gene and said constitutive Ptac-like promoter. 
     
     
         4 . The strain of claim  0 , wherein said non-native ribosome binding site is selected from the group consisting of a lac ribosome binding site, a thrA ribosome binding site, a folA ribosome binding site, an araC ribosome binding site, an araB ribosome binding site, a galE ribosome binding site, an ompA ribosome binding site, a trpE ribosome binding site, a lamB ribosome binding site, a MS2 coat ribosome binding site, and a Qβ coat ribosome binding site. 
     
     
         5 . (canceled) 
     
     
         6 . The strain of claim  0 , wherein the expression of asd in said strain is less sensitive to regulation by lysine than said parent strain. 
     
     
         7 . The strain of claim  0 , wherein said constitutive Ptac-like promoter is introduced by at least one of mutagenesis of a native asd promoter and recombination. 
     
     
         8 . A DNA construct comprising an aspartate semialdehyde dehydrogenase gene operably associated with at least one Ptac-like promoter comprising the nucleotide sequence of SEQ ID NO: 16. 
     
     
         9 . A DNA construct according to  claim 8 , wherein said DNA construct further comprises at least one ribosome binding site operably associated with said asd gene and said promoter, wherein said at least one ribosome binding site is not an  E. coli  asd ribosome binding site found in nature. 
     
     
         10 . A DNA construct according to  claim 8 , wherein the construct is a vector. 
     
     
         11 . A DNA construct according to  claim 8 , wherein the construct is the plasmid p4202-133-1. 
     
     
         12 . (canceled) 
     
     
         13 . An  Escherichia coli  host cell of  claim 8  selected from the group consisting of the strains with deposit nos. NRRLB-30824, NRRLB-30825, NRRLB-30826, NRRLB-30827 and TH25.79, wherein said TH25.79 comprises the plasmid p4202-133-1. 
     
     
         14 . A  Escherichia coli  host cell comprising a DNA construct according to  claim 8 , wherein the construct is the plasmid p4202-133-1. 
     
     
         15 . The host cell of  claim 14 , wherein said host cell produces at least one of L-threonine, L-methionine, L-homoserine, L-lysine, and L-isoleucine. 
     
     
         16 . A process for producing at least one amino acid selected from the group consisting of L-threonine, L-methionine, L-isoleucine, L-homoserine, and L-lysine, comprising culturing a strain of claim  0  in a culture medium. 
     
     
         17 . The process of  claim 16 , wherein said strain produces between about 40 and about 200 g/L of L-threonine in about 35 to about 60 hours of growth in culture. 
     
     
         18 . A strain of  claim 1 , wherein said strain has an asd specific activity between about 200 nmol/min/mg and about 30,000 nmol aspartate semialdehyde/min/mg. 
     
     
         19 . A method for making an  Escherichia coli  cell B that over-produces an amino acid X, wherein said cell B is a member of a strain of  E. coli  of  claim 1 , comprising:
 (a) selecting an  Escherichia coli  cell A that produces amino acid X in yield M;   (b) replacing a native asd promoter in said  Escherichia coli  cell A with a constitutive Ptac-like promoter comprising the nucleotide sequence of SEQ ID NO: 16 to produce an  Escherichia coli  cell B that produces said amino acid X in yield N, wherein said yield N is greater than said yield M.   
     
     
         20 . A promoter comprising the nucleotide sequence of SEQ ID NO: 16.