Host cell modified to produce 2-pyrrolidone
View Patent ↗The present invention provides for a genetically modified host cell comprising a 2-pyrrolidone synthase, or an enzymatically active fragment thereof, heterologous to the host cell.
1. A genetically modified host cell comprising a 2-pyrrolidone synthase comprising an amino acid sequence having at least 80% identity with SEQ ID NO:1, wherein the amino acid sequence comprises one or more of the following conserved amino acid motifs or sites: acyl-activating enzyme (AAE) consensus motif (residues 137, 140-145, and 147-148), acyl-activating enzyme (AAE) consensus motif (residues 140, 257-258, 279-284, 357, 369, 372, 382, and 458), AMP binding site (residues 140, 180-181, 227, 229-230, 233, 257-258, 279-284, 357, 369, 372, 379-382, and 439), and CoA binding site (residues 180, 229-230, 233, 257, 379-381, 433, and 439) heterologous to the host cell, wherein the 2-pyrrolidone synthase catalyzes the following reaction:
2. The genetically modified host cell of claim 1 , wherein host cell is capable of synthesizing Compound 1 or uptaking Compound 1 from the environment or culture.
3. The genetically modified host cell of claim 2 , wherein host cell further comprises one or more enzymes of a pathway for synthesizing Compound 1 from a carbon source.
4. The genetically modified host cell of claim 3 , wherein pathway for synthesizing Compound 1 from a carbon source that is native to the host cell.
5. The genetically modified host cell of claim 3 , wherein pathway for synthesizing Compound 1 from a carbon source is heterologous to the host cell.
6. The genetically modified host cell of claim 1 , wherein host cell lacks betaine-CoA ligase.
7. The genetically modified host cell of claim 1 , wherein n is an integer from 1 to 20.
8. The genetically modified host cell of claim 7 , wherein n is an integer from 1 to 10.
9. The genetically modified host cell of claim 8 , wherein n is an integer from 1 to 7.
10. The genetically modified host cell of claim 9 , wherein n is an integer from 1 to 3.
11. A method of producing a Compound 2 in a genetically modified host cell, comprising:
(a) providing the genetically modified host cell of claim 1 ,
(b) culturing the genetically modified host cell in a medium under a suitable condition such that the culturing results in the genetically modified host cell producing a Compound 2.
12. The method of claim 11 , further comprising introducing one or more nucleic acid(s) into the host cell encoding the enzyme operably linked to a suitable promoter capable of transcription in the host cell, and optionally encoding the one or more enzyme(s) of a pathway for synthesizing Compound 1 from a carbon source; wherein the introducing step is prior to the culturing step.
13. The method of claim 11 , further comprising separating Compound 2 from the host cell and/or the medium, wherein the separating step is subsequent, concurrent or partially concurrent with the culturing step.
14. The genetically modified host cell of claim 1 , wherein the 2-pyrrolidone synthase comprising an amino acid sequence comprising the following conserved amino acid motifs or sites: acyl-activating enzyme (AAE) consensus motif (residues 137, 140-145, and 147-148), acyl-activating enzyme (AAE) consensus motif (residues 140, 257-258, 279-284, 357, 369, 372, 382, and 458), AMP binding site (residues 140, 180-181, 227, 229-230, 233, 257-258, 279-284, 357, 369, 372, 379-382, and 439), and CoA binding site (residues 180, 229-230, 233, 257, 379-381, 433, and 439).
15. The genetically modified host cell of claim 1 , wherein the 2-pyrrolidone synthase comprises an amino acid sequence having at least 90% identity with SEQ ID NO:1.
16. The genetically modified host cell of claim 14 , wherein the 2-pyrrolidone synthase comprises an amino acid sequence having at least 95% identity with SEQ ID NO:1.
17. The genetically modified host cell of claim 15 , wherein the 2-pyrrolidone synthase comprises an amino acid sequence having at least 99% identity with SEQ ID NO:1.