COMPOSITIONS COMPRISING AND METHODS FOR PRODUCING BETA-HYDROXY FATTY ACID ESTERS

1 . A recombinant microorganism comprising

a heterologous polynucleotide sequence encoding a polypeptide having an ester synthase (EC 2.3.1.75) activity, wherein the recombinant microorganism produces an ester composition comprising a beta-hydroxy fatty ester in the presence of a carbon source.

2 . The recombinant microorganism of claim 1 , further comprising a heterologous polynucleotide sequence encoding a polypeptide having a thioesterase (EC 3.1.2.14 or EC 3.1.1.5) and an acyl-CoA synthase (EC 2.3.1.86) activity.

3 . A method of producing a fatty ester composition comprising beta-hydroxy fatty esters, the method comprising culturing the recombinant microorganism of claim 2 in the presence of a carbon source under conditions effective to produce a fatty ester composition comprising beta-hydroxy fatty esters in a culture.

4 . The method of claim 3 , wherein the beta-hydroxy fatty esters include beta-hydroxy methyl esters or beta-hydroxy ethyl esters.

5 . The method of claim 4 , wherein the conditions include the presence of methanol.

6 . The method of claim 5 , wherein the methanol is included in or added to the culture.

7 . The method of claim 5 , wherein the methanol is produced by the recombinant microorganism.

8 . The method of claim 4 , wherein the composition comprises beta-hydroxy ethyl esters.

9 . The method of claim 8 , wherein the conditions include the presence of ethanol.

10 . The method of claim 9 , wherein the ethanol is included in or added to the culture.

11 . The method of claim 9 , wherein the ethanol is produced by the recombinant microorganism.

12 . The method of claim 3 , wherein the microorganism is a bacterium.

13 . The method of claim 3 , wherein the microorganism is a yeast.

14 . The method of claim 12 , wherein the bacterium is of the species Escherichia coli.

15 . The method of claim 3 , wherein the thioesterase has at least 90% amino acid sequence identity to a thioesterase encoded by tes A or ′tesA from E. coli.

16 . The method of claim 3 , wherein the acyl-CoA synthase has at least 90% amino acid sequence identity to an acyl-CoA synthase encoded by fadD from E. coli.

17 . The method of claim 3 , wherein the recombinant microorganism is engineered to have reduced expression of a fatty acid degradation enzyme or an outer membrane protein receptor.

18 . The method of claim 17 , wherein the microorganism is of the species Escherichia coli and has reduced expression of fhuA.

19 . The method of claim 17 , wherein the microorganism is of the species Escherichia coli and has reduced expression of fadE.

20 . The method of claim 3 , wherein the thioesterase is a tesA engineered to have enhanced ability to use acyl-ACP as a substrate, relative to a corresponding wild-type tesA.

21 . The method of claim 3 , wherein the polynucleotide sequence encoding the polypeptide having the ester synthase (EC 2.3.1.75) activity is located on a plasmid.

22 . The method of claim 3 , wherein the polynucleotide encoding a polypeptide having the ester synthase (EC 2.3.1.75) activity is integrated into a chromosome of the microorganism.

23 . The method of claim 3 , wherein the composition has a fatty acid methyl ester or fatty acid ethyl ester titer of at least about 5 g/L.

24 . The method of claim 23 , wherein the composition has a fatty acid methyl ester titer of at least about 45 g/L.

25 . The method of claim 23 , wherein beta-hydroxy methyl esters or beta-hydroxy ethyl esters comprise at least 5% of the total methyl or ethyl esters.

26 . The method of claim 4 , further comprising separating fatty acid methyl esters or fatty acid ethyl esters from the culture to form an enriched fatty acid methyl ester or fatty acid ethyl ester fraction.

27 . The method of claims 26 , further comprising polishing the enriched fatty acid methyl ester or fatty acid ethyl ester fraction.

28 . The method of claim 3 , wherein the composition has a percentage of total fatty acid methyl esters as follows:

Methyl octanoate (C8:0)

 0-5%

Methyl decanoate (C10:0)

 0-2%;

Methyl dodecanoate (C12:0):

 0-5%;

Methyl dodecenoate (C12:1):

 0-10%;

Methyl tetradecanoate (C14:0):

30-50%;

Methyl 7-tetradecenoate (C14:1):

 0-10%;

Methyl hexadecanoate (C16:0):

 0-15%;

Methyl 9-hexadecenoate (C16:1):

10-40%; and

Methyl 11-octadecenoate (C18:1):

 0-15%[[;]].

29 . The method of claim 28 , wherein the fatty acid methyl esters comprise at least 5% of beta-hydroxyl esters.

30 . (canceled)

31 . The method of claim 3 , further comprising purifying the beta-hydroxy fatty esters to form a beta-hydroxy fatty ester enriched fraction.

32 . The method of claim 3 , wherein the carbon source is biomass.

33 . A composition produced by the method of claim 3 .

34 . A biodiesel composition comprising a fatty acid methyl ester composition wherein a percentage of total fatty acid methyl esters is as follows:

Methyl octanoate (C8:0)

 0-5%;

Methyl decanoate (C10:0)

 0-2%;

Methyl dodecanoate (C12:0):

 0-5%;

Methyl dodecenoate (C12:1):

 0-10%;

Methyl tetradecanoate (C14:0):

30-50%;

Methyl 7-tetradecenoate (C14:1):

 0-10%;

Methyl hexadecanoate (C16:0):

 0-15%;

Methyl 9-hexadecenoate (C16:1):

10-40%; and

Methyl 11-octadecenoate (C18:1):

 0-15%

with at least 5% of the fatty acid methyl esters comprising beta-hydroxy methyl esters.

35 . The method of claim 4 , wherein the composition comprises beta-hydroxy methyl esters.