US2013267696A1PendingUtilityA1

Producing alpha-olefins using polyketide synthases

47
Individually held — no corporate assignee on recordPriority: Sep 28, 2010Filed: Sep 28, 2011Published: Oct 10, 2013
Est. expirySep 28, 2030(~4.2 yrs left)· nominal 20-yr term from priority
Inventors:Jeffrey L. FortmanLeonard KatzEric J. SteenJay D. Keasling
C12P 5/007C12P 5/005C12P 5/02C12N 9/00C12P 5/026C12N 15/52C12Y 203/01C12N 9/1029
47
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Claims

Abstract

The present invention provides for a polyketide synthase (PKS) capable of synthesizing an α-olefin, such as 1-hexene or butadiene. The present invention also provides for a host cell comprising the PKS and when cultured produces the α-olefin.

Claims

exact text as granted — not AI-modified
1 . A non-naturally occurring polyketide synthase (PKS), or functional variant thereof, capable of synthesizing an α-olefin. 
     
     
         2 . The PKS of  claim 1 , wherein the α-olefin is not a compound synthesized by a naturally occurring PKS. 
     
     
         3 . The PKS of  claim 1 , wherein the PKS is a hybrid PKS comprising modules, domains, and/or portions thereof, or functional variant thereof, from two or more PKSs. 
     
     
         4 . The PKS of  claim 3 , wherein the PKS comprises a terminal module comprising ST, and TE, or functional variant thereof, of  Lyngbya majuscula  CurM. 
     
     
         5 . The PKS of  claim 4 , wherein the PKS comprises a terminal module comprising KR, ACP, ST, and TE, or functional variant thereof, of  Lyngbya majuscula  CurM. 
     
     
         6 . The PKS of  claim 5 , wherein the PKS comprises a terminal module comprising AT, KR, ACP, ST, and TE, or functional variant thereof, of  Lyngbya majuscula  CurM. 
     
     
         7 . The PKS of  claim 3 , wherein the PKS comprises at least one terminal module comprising a ST and a TE, or functional variant thereof, described in Tables 2-4. 
     
     
         8 . The PKS of  claim 3 , wherein the PKS comprises a loading module which incorporates acrylyl-CoA. 
     
     
         9 . The PKS of  claim 3 , wherein the PKS comprises the loading module is a DEBS proprionyl-CoA specific loading domain modified to accept acrylyl-CoA. 
     
     
         10 . The PKS of  claim 9 , wherein the PKS comprises a module in which a terminal carbon is incorporated as a methyl group which is later oxidized. 
     
     
         11 . The PKS of  claim 1 , wherein the α-olefin has the following chemical structure: 
       
         
           
           
               
               
           
         
         wherein each R 1  is independently —H or —CH 3 , each R 2  is independently —H or —OH, n is an integer from 1 to 10, αβ is (1) a single bond or (2) a double bond, with the proviso that when an αβ is a double bond then the corresponding R 2  is H. 
       
     
     
         12 . The PKS of  claim 11 , wherein the α-olefin is 1-hexene, 1-decene, or (E)-deca-1,5-diene. 
     
     
         13 . The PKS of  claim 1 , wherein the α-olefin is an aromatic α-olefin having the following chemical structure: 
       
         
           
           
               
               
           
         
         wherein R 3  is —H, —OH, —NH 3 , or —NO 2 . 
       
     
     
         14 . The PKS of  claim 1 , wherein the α-olefin has the following chemical structure: 
       
         
           
           
               
               
           
         
         wherein R is one of the following structures 
       
       
         
           
           
               
               
           
         
       
     
     
         15 . A recombinant nucleic acid encoding the polyketide synthase (PKS) of  claim 1 . 
     
     
         16 . A replicon comprising the recombinant nucleic acid 14, wherein the replicon is capable of stable maintenance in a host cell. 
     
     
         17 . The replicon of  claim 16 , wherein the replicon is a plasmid or vector. 
     
     
         18 . The replicon of  claim 17 , wherein the vector is an expression vector. 
     
     
         19 . A host cell comprising the recombinant nucleic acid of  claim 15 . 
     
     
         20 . A host cell comprising the replicon of  claim 16 . 
     
     
         21 . The host cell of  claim 19 , wherein the host cell when cultured produces the α-olefin. 
     
     
         22 . The host cell of  claim 21 , wherein the host cell comprises a PKS which produces butadiene comprising a loading module comprising an acrylyl-ACP starter, and further comprises one or more nucleic acids encoding propionyl-CoA ligase (synthetase) (EC 6.2.1.17) and acyl-CoA dehydrogenase (mammalian) (EC 1.3.99.3), or functional variants thereof, wherein when cultured and optionally provided with propionate the PKS is capable of synthesizing acrylyl-CoA and producing butadiene. 
     
     
         23 . The host cell of  claim 21 , wherein the host cell comprises a PKS which produces butadiene comprising a loading module comprising an acrylyl-ACP starter, and further comprises one or more nucleic acids encoding lactate dehydrogenase (EC 6.2.1.17), lactate CoA transferase (EC 2.8.3.1), propionyl-CoA ligase (synthetase) (EC 6.2.1.17), and lactoyl-CoA dehydratase (EC 4.2.164), or functional variants thereof, wherein when cultured and optionally provided with propionate and glucose the PKS is capable of synthesizing acrylyl-CoA and producing butadiene. 
     
     
         24 . The host cell of  claim 21 , wherein the host cell comprises a PKS which produces butadiene comprising a loading module comprising an acrylyl-ACP starter, and further comprises one or more nucleic acids encoding acetyl-CoA carboxylase (EC 6.4.1.2), malonyl-CoA reductase (EC 1.2.1.-), 3-hydroxypropionate:CoA ligase, and 3-hydroxypropionyl-CoA hydratase (EC 4.2.1.17), or functional variants thereof, wherein when cultured the PKS is capable of synthesizing acrylyl-CoA and producing butadiene 
     
     
         25 . The host cell of  claim 21 , wherein the host cell comprises a PKS which produces butadiene comprising a loading module comprising an acrylyl-ACP starter, and further comprises one or more nucleic acids encoding propionyl-CoA ligase (synthetase) (EC 6.2.1.17), the AccA/PccB gene products and lactoyl-CoA dehydratase (EC 4.2.164), and lactate dehydrogenase (EC 6.2.1.17), lactate CoA transferase (EC 2.8.3.1), propionyl-CoA ligase (synthetase) (EC 6.2.1.17), and lactoyl-CoA dehydratase (EC 4.2.164), or acyl-CoA dehydrogenase, or functional variants thereof, wherein when cultured and optionally provided with propionate the PKS is capable of synthesizing acrylyl-CoA and producing butadiene. 
     
     
         26 . A method of producing an α-olefin, comprising: providing a host cell of  claim 21 , and culturing said host cell in a suitable culture medium such that the α-olefin is produced. 
     
     
         27 . The method of  claim 26 , further comprising isolating the isolating the α-olefin. 
     
     
         28 . The method of  claim 27 , further comprising polymerizing the α-olefin to produce a polymer. 
     
     
         29 . The method of  claim 26 , wherein the α-olefin is a polyolefin. 
     
     
         30 . The method of  claim 29 , wherein the polyolefin is butadiene, isoprene, or (E)-deca-1,5-diene. 
     
     
         31 . A composition comprising an α-olefin isolated from the host cell of  claim 21 , and trace residues and/or contaminants of the host cell. 
     
     
         32 . The composition of  claim 31 , wherein the α-olefin is polyolefin. 
     
     
         33 . The composition of  claim 32 , wherein the polyolefin is butadiene, isoprene, or (E)-deca-1,5-diene.

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