COMPOSITIONS COMPRISING MILK FAT TRIGLYCERIDES PRODUCED BY MICROBIAL FERMENTATION
The present disclosure relates to microbial hosts and methods for biosynthesis of compositions comprising milk fat triglycerides.
1 . A microbial cell for producing milk fat triglycerides, wherein the cell expresses a biosynthetic pathway comprising at least one heterologous enzyme, the biosynthetic pathway producing triglycerides having short chain fatty acids esterified at sn-3, and wherein the cell produces triglycerides having at least about 5% C4 and C6 fatty acids esterified at sn-3 on a molar basis.
2 . The microbial cell of claim 1 , wherein the cell produces triglycerides having at least about 10% C4 and C6 fatty acids esterified at sn-3 on a molar basis.
3 . (canceled)
4 . The microbial cell of claim 1 , wherein;
the triglycerides further comprise C12 to C18 fatty acids esterified at sn-3;
the triglycerides comprise C4:0, C6:0, C8:0, and C10:0 fatty acids esterified at sn-3; and/or
the triglycerides have predominately C12 to C18 fatty acids esterified at sn-1 and sn-2 on a molar basis.
5 .- 9 . (canceled)
10 . The microbial cell of claim 1 , wherein the biosynthetic pathway comprises one or more heterologous fatty acyl-CoA synthetase (ACS) having specificity for short chain fatty acid substrates.
11 . The microbial cell of claim 10 , wherein at least one heterologous ACS comprises an amino acid sequence that is at least 70% identical to an amino acid sequence selected from SEQ ID NOs: 1-7.
12 . (canceled)
13 . The microbial cell of claim 11 , wherein the microbial cell expresses a heterologous ACS comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.
14 . (canceled)
15 . The microbial cell of claim 1 , wherein the biosynthetic pathway comprises a heterologous diacylglycerol O-acyltransferase (DGA) having specificity for short chain fatty acid CoA substrate.
16 . The microbial cell of claim 15 , wherein the DGA comprises an amino acid sequence that is at least 70% identical to an amino acid sequence selected from SEQ ID NOs: 8-17.
17 .- 18 . (canceled)
19 . The microbial cell of claim 1 , wherein the microbial cell expresses a conjugated linoleic acid (CLA) isomerase.
20 . The microbial cell of claim 19 , wherein the CLA isomerase comprises an amino acid sequence that is at least about 75% identical to an amino acid sequence selected from SEQ ID NO: 19.
21 . The microbial cell of claim 1 , wherein the microbial cell comprises a modification that results in decreased amount or activity of one or more endogenous diglyceride acyltransferases.
22 . (canceled)
23 . The microbial cell of claim 1 , wherein the cell has one or more genetic modifications that reduce C16:1 fatty acid production and/or increase C18:1 fatty acid production.
24 .- 25 . (canceled)
26 . The microbial cell of claim 23 , wherein the microbial cell expresses at least one heterologous OLE1 enzyme converting C18:0 to C18:1, and which is optionally selected from an enzyme that comprises an amino acid sequence having at least 80% sequence identity to one of SEQ ID NOS: 22 to 30.
27 . The microbial cell of claim 23 , wherein the microbial cell has a deletion, inactivation, or reduced expression or activity of one or more fatty acid elongase enzymes.
28 . The microbial cell of claim 1 , wherein the microbial cell expresses one or more heterologous fatty acid synthases (FAS), optionally where the heterologous FAS enzymes produce one or more of C10:0, C12:0, and C14:0 fatty acids.
29 . The microbial cell of claim 28 , at least one heterologous fatty acid synthase comprises a subunit comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 20 (FAS1), and a subunit comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 21 (FAS2).
30 . The microbial cell of claim 1 , further comprising a biosynthetic pathway producing one or more lactones.
31 .- 38 . (canceled)
39 . The microbial cell of claim 1 , wherein the cell produces β-carotene through a heterologous biosynthetic pathway.
40 .- 43 . (canceled)
44 . The microbial cell of any one of claims 1 to 43 , wherein the microbial cell is a yeast or fungal cell or a bacterial cell.
45 .- 46 . (canceled)
47 . The microbial cell of claim 44 , wherein the yeast or fungal cell is Yarrowia lipolytica or Yarrowia phangngensis.
48 .- 52 . (canceled)
53 . A method for making a composition comprising milk fat triglycerides, comprising: culturing a microbial cell of claim 1 in the presence of C4:0, C6:0, C8:0, C10:0, C12:0, and C14:0 fatty acid substrates, and recovering the composition from the culture.
54 .- 60 . (canceled)
61 . A composition comprising milk fat triglycerides produced according to the method of claim 1 .
62 .- 63 . (canceled)