Enzymatic biosynthesis of lactones
The disclosure relates to methods for the production of natural lactones by bacteria under physiological conditions. The methods employ ybgC proteins having lactonizing activity.
1 . A method for enzymatically producing a lactone under physiological conditions, the method comprising culturing a recombinant bacterium in the presence of a hydroxy acyl-CoA substrate, thereby producing said lactone, wherein said recombinant bacterium expresses a heterologous acyl-CoA thioesterase and a heterologous acyl-CoA synthetase protein, wherein:
the acyl-CoA thioesterase comprises a YbgC protein, wherein the YbgC protein has at least 85% amino acid sequence identity to any one of the amino acid sequence of SEQ ID NOs: 1-6, and wherein the YbgC protein converts a hydroxy acyl-CoA substrate to a lactone;
the hydroxy acyl-CoA substrate is a member selected from a 4-hydroxy acyl-CoA, a 5-hydroxy acyl-CoA, and a 6-hydroxy acyl-CoA; and
the YbgC protein converts the 4-hydroxy acyl-CoA to a γ-lactone, the YbgC protein converts the 5-hydroxy acyl-CoA to a δ-lactone, and the YbgC protein converts the 6-hydroxy acyl-CoA to a ε-lactone.
2 . The method of claim 1 , wherein the YbgC protein is overexpressed in said recombinant bacterium.
3 . The method of claim 1 , further comprising exogenously adding a fatty acid derivative molecule to the culture medium to produce said hydroxy acyl-CoA substrate from the recombinant bacterium.
4 . The method of claim 3 , wherein the fatty acid derivative molecule is a 4-hydroxy fatty acid derivative, a 5-hydroxy fatty acid derivative or a 6-hydroxy fatty acid derivative.
5 . The method of claim 4 , wherein the fatty acid derivative molecule is the 4-hydroxy fatty acid derivative and the recombinant bacterium produces 4-hydroxy acyl-CoA.
6 . The method of claim 5 , wherein the 4-hydroxy fatty acid derivative is 4-hydroxy decanoic acid, 4-hydroxy octanoic acid, 4-hydroxy dodecanoic acid, or 4-hydroxy tetranoic acid.
7 . The method of claim 4 , wherein the fatty acid derivative molecule is the 5-hydroxy fatty acid derivative and the recombinant bacterium produces 5-hydroxy acyl-CoA.
8 . The method of claim 7 , wherein the 5-hydroxy fatty acid derivative is 5-hydroxy decanoic acid.
9 . The method of claim 4 , wherein the fatty acid derivative molecule is the 6-hydroxy fatty acid derivative and the recombinant bacterium produces 6-hydroxy acyl-CoA.
10 . The method of claim 9 , wherein the 6-hydroxy fatty acid derivative is 6-hydroxy hexanoic acid or 6-hydroxy decanoic acid.
11 . The method of claim 1 , wherein the YbgC protein has at least 85% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1.
12 . The method of claim 1 , wherein the recombinant bacterium further expresses a second heterologous thioesterase and a heterologous hydroxylating enzyme.
13 . The method of claim 12 , wherein:
the second thioesterase is a member selected from the group consisting of the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24; and
the hydroxylating enzyme is a member selected from the group consisting of the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO; 29, SEQ ID NO: 30, and SEQ ID NO: 31.
14 . The method of claim 1 , wherein the recombinant bacterium produces the lactone from a simple carbon source.
15 . The method of claim 1 , wherein the recombinant bacterium comprises an endogenous acyl-CoA dehydrogenase that is attenuated or deleted or a 3-ketoacyl-CoA thiolase or dual 3-hydroxyacyl-CoA-dehydrogenase/dehydratase that is attenuated or deleted.
16 . The method of claim 1 , wherein the acyl-CoA synthetase is overexpressed.
17 . The method of claim 1 , further comprising recovering the lactone from the culture medium or isolating the lactone from the recombinant bacterium, or both.
18 . The method of claim 1 , wherein the lactone is produced without a chemical conversion step or acidification of the culture medium.