Microorganisms for the production of insect pheromones and related compounds

• US 10,308,962 B1
• Filed: 05/18/2018
• Issued: 06/04/2019
• Est. Priority Date: 11/18/2015
• Status: Active Grant

• Current Assignee: Provivi, Inc.
• Original Assignee: Provivi, Inc.
• Inventors: Leonard, Effendi, Meinhold, Peter, Wampler, Keith, Coelho, Pedro, Sheppard, Micah, Heel, Thomas
• Primary Examiner(s): Raghu, Ganapathirama
• Application Number:
  US15/983,706
• Time in Patent Office: 382 Days
• CPC Class Codes: A01N 31/02 Acyclic compounds
• CPC Class Codes: A01N 63/14 Insects
• CPC Class Codes: A01N 63/30 Microbial fungi; Substances produced thereby or obtained therefrom
• CPC Class Codes: A01N 63/50 Isolated enzymes; Isolated proteins peptides A01N37/46
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12P 7/04 acyclic
• CPC Class Codes: C12P 7/62 Carboxylic acid esters
• CPC Class Codes: C12P 7/6436 Fatty acid esters
• CPC Class Codes: C12P 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12R 2001/645 Fungi ; Processes using fungi
• CPC Class Codes: C12Y 102/0105 Long-chain-fatty-acyl-CoA reductase (1.2.1.50)
• CPC Class Codes: C12Y 114/19001 Stearoyl-CoA 9-desaturase (1.14.19.1), i.e. DELTA9-desaturase
• CPC Class Codes: C12Y 114/19005 DELTA11-fatty-acid desaturase (1.14.19.5)

Abstract

The present application relates to recombinant microorganisms useful in the biosynthesis of unsaturated C₆-C₂₄ fatty alcohols, aldehydes, and acetates which may be useful as insect pheromones, fragrances, flavors, and polymer intermediates. The C₆-C₂₄ fatty alcohols, aldehydes, and acetates described herein may be used as substrates for metathesis reactions to expand the repertoire of target compounds and pheromones. The application further relates to recombinant microorganisms co-expressing a pheromone pathway and a pathway for the production of a toxic protein, peptide, oligonucleotide, or small molecule suitable for use in an attract-and-kill pest control approach. Also provided are methods of producing unsaturated C₆-C₂₄ fatty alcohols, aldehydes, and acetates using the recombinant microorganisms, as well as compositions comprising the recombinant microorganisms and/or optionally one or more of the product alcohols, aldehydes, or acetates.

Claims

1. A recombinant Yarrowia lipolytica microorganism capable of producing a mono- or poly-unsaturated C₆-C₂₄ fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄ fatty acid, comprising:

a heterologous nucleic acid molecule encoding and expressing a Helicoverpa zea fatty acyl desaturase that catalyzes the conversion of a saturated C₆-C₂₄ fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄ fatty acyl-CoA, wherein the mono- or poly-unsaturated C₆-C₂₄ fatty acyl-CoA is converted to the corresponding mono- or poly-unsaturated C₆-C₂₄ fatty alcohol.

2. The recombinant Yarrowia lipolytica microorganism of claim 1, wherein the Helicoverpa zea fatty acyl desaturase is a Z11 desaturase.

3. The recombinant Yarrowia lipolytica microorganism of claim 1, wherein the heterologous nucleic acid molecule encoding a Helicoverpa zea fatty acyl desaturase comprises an amino acid sequence encoded by SEQ ID NO:

27 or a functionally active and expressed variant thereof.

4. The recombinant Yarrowia lipolytica microorganism of claim 1, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion of the fao1 gene.

5. The recombinant Yarrowia lipolytica microorganism of claim 1, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion of pox1, pox2, pox3, pox4, pox5, and pox6 genes.

6. The recombinant Yarrowia lipolytica microorganism of claim 1, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion of fao1, pox1, pox2, pox3, pox4, pox5, and pox6 genes.

7. The recombinant Yarrowia lipolytica microorganism of claim 1, wherein the mono- or poly-unsaturated C₆-C₂₄ fatty alcohol is Z11-16:

OH.

8. A method of producing a mono- or poly-unsaturated C₆-C₂₄ fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄ fatty acid, comprising:

cultivating the recombinant Yarrowia lipolytica microorganism of claim 1, in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated C₆-C₂₄ fatty alcohol.

9. The method of claim 8, wherein said unsaturated C₆-C₂₄ fatty alcohol is an insect fatty alcohol.

10. The method of claim 8, wherein said unsaturated C₆-C₂₄ fatty alcohol is an insect pheromone.

11. The recombinant Yarrowia lipolytica microorganism of claim 1, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion of adh1, adh2, adh3, adh4, adh5, adh6, and adh7 genes.

12. The recombinant Yarrowia lipolytica microorganism of claim 1, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion of fao1, adh1, adh2, adh3, adh4, adh5, adh6, and adh7 genes.

13. The recombinant Yarrowia lipolytica microorganism of claim 1, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion of fao1, adh1, adh2, adh3, adh4, adh5, adh6, adh7, pox1, pox2, pox3, pox4, pox5, and pox6 genes.