Overcoming DAPA aminotransferase bottlenecks in biotin vitamers biosynthesis

Abstract

A method is disclosed for the increased production of biotin and the biotin precursor dethiobiotin using a bacterium that produces a lysine-utilizing DAPA aminotransferase. The method involves the use of a bacterium that is either grown in the presence of lysine or deregulated for lysine biosynthesis.

1 . A method of producing a biotin vitamer by: 
 (a) culturing a bacterium comprising a lysine-utilizing diaminopelargonic acid (DAPA) aminotransferase, said culturing taking place in an environment enriched for lysine, a lysine analog, or a lysine precursor; and    (b) recovering said biotin vitamer.    
   
   
       2 . A method of producing a biotin vitamer by: 
 (a) culturing a bacterium comprising a lysine-utilizing DAPA aminotransferase, wherein said bacterium is deregulated with respect to lysine production; and    (b) recovering said biotin vitamer.    
   
   
       3 . The method of  claim 1  in which the bacterium is engineered to overproduce a lysine-utilizing DAPA aminotransferase.  
   
   
       4 . The method of  claim 2  in which the bacterium is engineered to overproduce a lysine-utilizing DAPA aminotransferase.  
   
   
       5 . The method of  claim 2 , wherein lysine, a lysine analog, or a lysine precursor is exogenously added to the culture.  
   
   
       6 . The method of  claim 1  or  claim 2 , in which lysine, a lysine analog, or a lysine precursor is exogenously added to the culture and totals at least 10 mmoles per liter of culture.  
   
   
       7 . The method of  claim 1  or  claim 2 , in which the biotin vitamer is biotin, dethiobiotin, or diaminopelargonic acid (DAPA).  
   
   
       8 . The method of  claim 1  or  claim 2 , in which the biotin vitamer is dethiobiotin, and, after recovering the dethiobiotin, the method further comprises converting the recovered dethiobiotin to biotin by a separate fermentation, biochemical reaction, or chemical reaction, and recovering biotin.  
   
   
       9 . The method of  claim 1  or  claim 2 , in which the bacterium is resistant to a lysine analog.  
   
   
       10 . The method of  claim 9 , wherein said analog is S-2-aminoethyl-L-cysteine (AEC).  
   
   
       11 . The method of  claim 1  or  claim 2 , in which the bacterium is deregulated with respect to at least one biotin synthetic pathway step in addition to bioA expression.  
   
   
       12 . The method of  claim 1   claim 2 , in which the biotin vitamer is biotin, and the method comprises recovering and purifying the biotin.  
   
   
       13 . The method of  claim 1  or  claim 2 , wherein said bacterium is further engineered to produce a S-adenosylmethionine (SAM)-utilizing DAPA aminotransferase.  
   
   
       14 . The method of  claim 13  in which methionine, S-adenosylmethionine (SAM), or an analog of SAM is added to the culture.  
   
   
       15 . The method of  claim 13  wherein lysine, a lysine analog, or a lysine precursor is added to the culture.  
   
   
       16 . The method of  claim 14 , wherein lysine, a lysine analog, or a lysine precursor is added to the culture.  
   
   
       17 . The method of  claim 15  in which lysine or a lysine analog exogenously added to the culture totals at least 10 mmoles per liter of culture.  
   
   
       18 . The method of  claim 16  in which lysine or a lysine analog exogenously added to the culture totals at least 10 mmoles per liter of culture.  
   
   
       19 . The method of  claim 13  in which the biotin vitamer is biotin, dethiobiotin, or diaminopelargonic acid (DAPA).  
   
   
       20 . The method of  claim 13  in which the biotin vitamer is dethiobiotin, and, after recovering the dethiobiotin, the method further comprises converting the recovered dethiobiotin to biotin by a separate fermentation, biochemical reaction, or chemical reaction, and recovering biotin.  
   
   
       21 . The method of  claim 13  in which the bacterium is deregulated with respect to at least one biotin synthetic pathway step other than bioA expression.  
   
   
       22 . The method of  claim 13  in which the biotin vitamer is biotin, and the method comprises recovering and purifying the biotin.  
   
   
       23 . A bacterium engineered to overproduce a lysine-utilizing DAPA aminotransferase and a SAM-utilizing DAPA aminotransferase.  
   
   
       24 . The bacterial strains BI90 (ATCC 55999) and BI96 (ATCC 202000).  
   
   
       25 . The bacterium of  claim 23 , wherein the strain is further engineered to overproduce the biotin vitamer by engineered deregulation of at least one biotin synthetic step, in addition to bioA expression.  
   
   
       26 . The bacterial strain BI603 (ATCC 202003).  
   
   
       27 . A bacterium engineered to overproduce a lysine-utilizing DAPA aminotransferase, wherein the bacterium is further engineered to overproduce lysine.  
   
   
       28 . The bacterial strain BI641 (ATCC 202002) or BI642 (ATCC 202001).  
   
   
       29 . A biotin vitamer manufactured by the method of  claim 1  or  claim 2 .  
   
   
       30 . A biotin vitamer manufactured by the method by  claim 13 .  
   
   
       31 . A biotin vitamer manufactured by the method of  claim 14.