Methods and Compositions for Producing Alkenes of Various Chain Length
The NonA alkene synthase in Synechococcus sp. displays selective synthesis of 1-nonadecene. Heterologous recombination of a domain, i.e. the acyl binding domain, with other acyl binding proteins, affects acyl substrate chain-length binding selectivity and therefore the chain-length of the synthesized 1-alkenes. Compositions and methods are provided to selectively synthesize 1-alkenes of various chain lengths.
1 . An isolated or recombinant chimeric NonA alkene synthase comprising a heterologous acyl binding pocket.
2 . The alkene synthase of claim 1 , wherein said heterologous acyl binding pocket comprises a polypeptide sequence selected from the group consisting of:
a. SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16; and
b. a polypeptide sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 8, SEQ ID NO: 12, or SEQ ID NO: 16.
3 . An isolated or recombinant polynucleotide encoding the heterologous acyl binding pocket of claim 1 , comprising or consisting of:
a. SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 34;
b. a nucleotide sequence that is a degenerate variant of SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 34;
c. a nucleic acid sequence at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.9% identical to SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 34;
d. a nucleic acid sequence that encodes a polypeptide at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 8, SEQ ID NO: 12, or SEQ ID NO: 16; or
e. a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 34.
4 . An isolated or recombinant polypeptide comprising or consisting of:
a. SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 29; or
b. a polypeptide sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 29.
5 . An isolated or recombinant polynucleotide comprising or consisting of:
a. SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 26;
b. a nucleotide sequence that is a degenerate variant of SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 26;
c. an nucleotide sequence at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.9% identical to SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 26;
d. a nucleic acid sequence that encodes a polypeptide having the amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 29;
e. a nucleic acid sequence that encodes a polypeptide at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 29; or
f. a nucleic acid sequence that hybridizes under stringent conditions to a nucleic acid sequence that encodes a polypeptide having the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 26.
6 . The isolated or recombinant polynucleotide of claim 3 or claim 5 , wherein the nucleic acid sequence is operably linked to one or more expression control sequences.
7 . A vector comprising the isolated or recombinant polynucleotide of claim 3 or claim 5 .
8 . A fusion protein comprising the polypeptide of claim 2 or claim 4 fused to a heterologous amino acid sequence.
9 . A host cell comprising the isolated or recombinant polynucleotide of claim 3 or claim 5 .
10 . The host cell of claim 9 , wherein the host cell is selected from the group consisting of prokaryotes, eukaryotes, yeasts, filamentous fungi, protozoa, algae and synthetic cells.
11 . The host cell of claim 9 , wherein the host cell is a photoautotroph.
12 . The host cell of claim 9 , wherein the host cell is E. coli.
13 . The host cell of claims 9 wherein the host cell produces carbon-based products of interest.
14 . An isolated antibody or antigen-binding fragment or derivative thereof which binds selectively to the polypeptide of claim 3 or claim 5 .
15 . A method for producing a carbon-based product of interest comprising:
a. culturing a host cell to produce the carbon-based product of interest, wherein said host cell comprises an engineered chimeric alkene synthase comprising a heterologous acyl binding pocket; and
b. isolating the carbon-based product of interest.
16 . The method of claim 15 , wherein the chimeric alkene synthase is derived from NonA.
17 . The method of claim 16 , wherein said NonA comprises SEQ ID NO: 2.
18 . The method of claim 16 , wherein said NonA comprises SEQ ID NO: 24.
19 . The method of claim 15 , wherein the heterologous acyl binding pocket comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 16.
20 . The method of claim 15 , wherein said chimeric alkene synthase selectively synthesizes one or more alkenes with specific chain lengths.