Production of Alkenes by Combined Enzymatic Conversion of 3-Hydroxyalkanoic Acids

Abstract

The present invention relates to a method for generating alkenes through a biological process. More specifically, the invention relates to a method for producing alkenes (for example propylene, ethylene, 1-butylene, isobutylene or isoamylene) from molecules of the 3-hydroxyalkanoate type.

1 - 19 . (canceled) 
     
     
         20 . A method for producing an alkene comprising the conversion of a 3-hydroxyalkanoate into said alkene by:
 (i) a first enzyme having an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate; and   (ii) a second enzyme being different from the first enzyme and having an activity of converting said 3-phosphonoxyalkanoate into said alkene.   
     
     
         21 . The method of  claim 20 , wherein the first enzyme is a mevalonate diphosphate (MDP) decarboxylase and the second enzyme is a different mevalonate diphosphate (MDP) decarboxylase. 
     
     
         22 . The method of  claim 20  wherein:
 (i) the first enzyme is selected from the group consisting of:
 (A) a protein comprising the amino acid sequence as shown in SEQ ID NO: 1 or a protein comprising an amino acid sequence which is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 1 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 1; 
 (B) a protein comprising the amino acid sequence as shown in SEQ ID NO: 2 or a protein comprising an amino acid sequence which is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 2 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 2; 
 (C) a protein comprising the amino acid sequence as shown in SEQ ID NO: 3 or a protein comprising an amino acid sequence which is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 3 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 3; and 
 (D) a protein comprising the amino acid sequence as shown in SEQ ID NO: 4 or a protein comprising an amino acid sequence which is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 4 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 4; and 
 
 (ii) the second enzyme is selected from the group consisting of:
 (a) a protein comprising the amino acid sequence as shown in SEQ ID NO: 5 or a protein comprising an amino acid sequence which is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 5 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 5; 
 (b) a protein comprising the amino acid sequence as shown in SEQ ID NO: 6 or a protein comprising an amino acid sequence which is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 6 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 6; 
 (c) a protein comprising the amino acid sequence as shown in SEQ ID NO: 7 or a protein comprising an amino acid sequence which is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 7 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 7; 
 (d) a protein comprising the amino acid sequence as shown in SEQ ID NO: 8 or a protein comprising an amino acid sequence which is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 8 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 8; 
 (e) a protein comprising the amino acid sequence as shown in SEQ ID NO: 9 or a protein comprising an amino acid sequence which is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 9 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 9 
 (f) a protein comprising the amino acid sequence as shown in SEQ ID NO: 10 or a protein comprising an amino acid sequence which is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 10 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 10; 
 (g) a protein comprising the amino acid sequence as shown in SEQ ID NO: 11 or a protein comprising an amino acid sequence which is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 11 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 11; 
 (h) a protein comprising the amino acid sequence as shown in SEQ ID NO: 12 or a protein comprising an amino acid sequence which is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 12 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 12; 
 (i) a protein comprising the amino acid sequence as shown in SEQ ID NO: 13 or a protein comprising an amino acid sequence which is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 13 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 13; 
 (j) a protein comprising the amino acid sequence as shown in SEQ ID NO: 14 or a protein comprising an amino acid sequence which is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 14 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 14; and 
 (k) a protein comprising the amino acid sequence as shown in SEQ ID NO: 15 or a protein comprising an amino acid sequence which is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 15 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 15. 
 
 
     
     
         23 . The method of  claim 22 , wherein:
 (a) the first enzyme is a protein comprising the amino acid sequence as shown in SEQ ID NO: 2 or a protein comprising an amino acid sequence which is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 2 and showing an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 2; and   (b) the second enzyme is a protein comprising the amino acid sequence as shown in SEQ ID NO: 10 or a protein comprising an amino acid sequence which is at least 75% identical to the amino acid sequence shown in SEQ ID NO: 10 and showing an activity of converting said 3-phosphonoxyalkanoate into said alkene which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 10.   
     
     
         24 . The method of  claim 20 , wherein the 3-hydroxyalkanoate is 3-hydroxypropionate and the alkene is ethylene. 
     
     
         25 . The method of  claim 20 , wherein the 3-hydroxyalkanoate is 3-hydroxybutyrate and the alkene is propylene. 
     
     
         26 . The method of  claim 20 , wherein the 3-hydroxyalkanoate is 3-hydroxyvalerate and the alkene is 1-butylene. 
     
     
         27 . The method of  claim 20 , wherein the hydroxyalkanoate is 3-hydroxy-3-methylbutyrate and the alkene is isobutylene. 
     
     
         28 . The method of  claim 20 , wherein the 3-hydroxyalkanoate is 3-hydroxy-3-methylvalerate and the alkene is isoamylene. 
     
     
         29 . The method of  claim 20 , characterized in that the conversion step is carried out in vitro, in a cell-free system. 
     
     
         30 . The method of  claim 20  characterized in that the method is carried out in the presence of a microorganism producing said enzymes as defined in (i) and (ii) of  claim 20 . 
     
     
         31 . The method of  claim 20 , characterized by the use of a multicellular organism producing said enzymes as defined in (i) and (ii) of  claim 20 . 
     
     
         32 . The method of  claim 20 , comprising a step of collecting gaseous alkenes degassing out of the reaction. 
     
     
         33 . A multicellular organism or a microorganism which produces at least two enzymes, wherein one enzyme is selected from (i) and the other enzyme is selected from (ii), wherein (i) and (ii) are as follows:
 (i) a first enzyme having an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate; and   (ii) a second enzyme being different from the first enzyme and having an activity of converting said 3-phosphonoxyalkanoate into said alkene.   
     
     
         34 . A method of using the multicellular organism or microorganism of  claim 33  to produce alkenes, comprising the steps of:
 (i) culturing the multicellular organism or microorganism of  claim 33  with 3-hydroxyalkanoate for a sufficient period of time to allow for the conversion of a 3-hydroxyalkanoate to an alkene; and 
 (ii) recovering said alkene. 
 
     
     
         35 . A composition comprising the microorganism of  claim 33 , a suitable culture medium and a 3-hydroxyalkanoate compound or a carbon source that can be converted by the microorganism to a 3-hydroxyalkanoate compound. 
     
     
         36 . A method of using a combination of at least two enzymes to convert a 3-hydroxyalkanoate to an alkene, wherein the method comprises:
 (i) contacting a 3-hydroxyalkanoate with:
 (a) a first enzyme having an activity of converting the 3-hydroxyalkanoate into the corresponding 3-phosphonoxyalkanoate; and 
 (b) a second enzyme being different from the first enzyme and having an activity of converting said 3-phosphonoxyalkanoate into said alkene; 
   (ii) converting said 3-hydroxyalkanoate to an alkene.   
     
     
         37 . A method for producing an alkene comprising the step of enzymatically converting a 3-phosphonoxyalkanoate into the corresponding alkene by use of an enzyme which can catalyze the conversion via decarboxylation and dephosphorylation. 
     
     
         38 . The method of  claim 20 , wherein the method is carried out with ATP, dATP, ADP, AMP, an NTP other than ATP, a dNTP or pyrophosphate as co-substrate.