Variant sucrose transporter polypeptides

Abstract

Variant sucrose transporter polypeptides that enable bacterial growth over a wide range of gene expression levels and sucrose concentrations are described. Additionally, recombinant bacteria comprising these variant sucrose transporter polypeptides, and methods of utilizing the bacteria to produce products such as glycerol and glycerol-derived products are provided

1 - 12 . (canceled) 
     
     
         13 . A recombinant bacterium comprising in its genome or on at least one recombinant construct:
 (a) a nucleotide sequence encoding a variant sucrose transporter polypeptide having an amino acid sequence that has at least 95% identity based on a Clustal W method of alignment to an amino acid sequence selected from the group consisting of SEQ ID NOs:26, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, and 98,and an amino acid at an equivalent position when compared with a reference amino acid sequence of SEQ ID NO:26 selected from the group consisting of:
 (i) alanine at a position equivalent to position 300; and 
 (ii) leucine at a position equivalent to position 300; and 
   (b) a nucleotide sequence encoding a polypeptide having sucrose hydrolase activity;   
       wherein (a) and (b) are each operably linked to the same or a different promoter, further wherein said recombinant bacterium is capable of metabolizing sucrose. 
     
     
         14 . The recombinant bacterium of  claim 13 , wherein the variant sucrose transporter polypeptide further comprises:
 at least one of the following amino acids at an equivalent position when compared with the reference amino acid sequence of SEQ ID NO:26:
 (i) proline at a position equivalent to position 61; 
 (ii) tryptophan at a position equivalent to position 61; 
 (iii) histidine at a position equivalent to position 61; 
 (iv) phenylalanine at a position equivalent to position 61; 
 (v) tyrosine at a position equivalent to position 61. 
   
     
     
         15 . The recombinant bacterium of  claim 14 , wherein the variant sucrose transporter polypeptide comprises an amino acid change from glutamine to histidine at position 353 and an amino acid change from leucine to proline at position 61. 
     
     
         16 . The recombinant bacterium of  claim 13 , wherein the polypeptide having sucrose hydrolase activity is classified as EC 3.2.1.26 or EC 2.4.1.7. 
     
     
         17 . The recombinant bacterium of  claim 13  further comprising in its genome or on at least one recombinant construct, a nucleotide sequence encoding a polypeptide having fructokinase activity. 
     
     
         18 . The recombinant bacterium of  claim 17 , wherein the polypeptide having fructokinase activity is classified as EC 2.7.1.4, EC 2.7.1.3, or EC 2.7.1.1. 
     
     
         19 . The recombinant bacterium of  claim 13 , wherein said bacterium is selected from the group consisting of the genera:  Escherichia, Klebsiella, Citrobacter , and  Aerobacter.    
     
     
         20 . The recombinant bacterium of  claim 19 , wherein said bacterium is  Escherichia coli.    
     
     
         21 . The recombinant bacterium of  claim 13 , wherein the recombinant bacterium produces 1,3-propanediol, glycerol, and/or 3-hydroxypropionic acid. 
     
     
         22 . A process for making glycerol, 1,3-propanediol and/or 3-hydroxypropionic acid from sucrose comprising:
 (a) culturing the recombinant bacterium of  claim 21  in the presence of sucrose; and   (b) optionally, recovering the glycerol, 1,3-propanediol and/or 3-hydroxypropionic acid produced.