PRODUCING ALPHA-OLEFINS USING POLYKETIDE SYNTHASES
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.
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.