Functional enhancement of microorganisms to minimize production of acrylamide
The present disclosure provides yeast transformed with a nucleic acid molecule (GAT1) to reduce nitrogen catabolite repression of asparagine transport/degradation and/or overexpress genes (ASP1 or ASP3) encoding cell-wall or extracellular proteins involved in asparagine degradation and/or genes (AGP1 or GNP1 or GAP1) encoding proteins involved in asparagine transport under food preparation/processing conditions. The genetically modified yeast has enhanced ability to reduce acnlamide concentration in foods prepared by heating. Also provided are methods and uses of the transgenic yeast for reducing acnlamide in a food product and food products having reduced acrylamide content prepared using the transgenic yeast.
1. A microorganism transformed with at least two nucleic acid molecules, wherein the two nucleic acid molecules are from at least two of the following:
(a) a nucleic acid molecule to reduce nitrogen catabolite repression;
(b) a nucleic acid molecule to overexpress a gene encoding an extracellular protein involved in asparagine degradation; and
(c) a nucleic acid molecule to overexpress a gene encoding a protein involved in asparagine transport.
2. The microorganism of claim 1 , wherein the nucleic acid molecule of (b) encodes a cell-wall asparaginase.
3. The microorganism of claim 2 , wherein the asparaginase is encoded by ASP3 or wherein the asparaginase is Asp3p.
4. The microorganism of claim 1 , wherein the nucleic acid molecule of (c) encodes an amino acid transporter.
5. The microorganism of claim 4 , wherein the amino acid transporter is encoded by GAP1, AGP1, GNP1, DIP5, AGP2 or AGP3 or is Gap1p, Agp1p, Gnp1p, Dip5p Agp2p or Agp3p.
6. The microorganism of claim 1 , wherein the nucleic acid molecule of (a) modifies the activity of a regulatory factor of nitrogen catabolite repression.
7. The microorganism of claim 6 , wherein the regulatory factor is encoded by URE2, GAT1, TOR1, TOR2, DAL80, GLN3 or GZF3 or is Ure2p, Gat1p, Tor1p, Tor2p, Dal80p, Gln3p or Gzf3p.
8. The microorganism of claim 6 , wherein the nucleic acid molecule of (a) comprises a URE2 deletion cassette or encodes.
9. The microorganism of claim 1 , wherein the microorganism is yeast.
10. The microorganism of claim 1 , wherein at least one of the nucleic acid molecules is operatively linked to a constitutively active promoter.
11. The microorganism of claim 1 transformed with a first and a second nucleic acid molecule, wherein the first nucleic acid molecule encodes Asp3p and the second nucleic acid molecule encodes Gap1p or Gat1p.
12. A method for reducing asparagine during food preparation or processing or for reducing acrylamide in a food product comprising
a) transforming a microorganism with at least two nucleic acid molecules, wherein the two nucleic acid molecules are from at least two of the following:
(i) a nucleic add molecule to reduce nitrogen catabolite repression;
(ii) a nucleic acid molecule to overexpress a gene encoding an extracellular protein involved in asparagine degradation; and
(iii) a nucleic acid molecule to overexpress a gene encoding a protein involved in asparagine transport;
b) adding the microorganism to food under the preparation or processing conditions;
wherein the microorganism reduces nitrogen catabolite repression and/or overexpresses the gene encoding the extracellular protein involved in asparagine degradation and/or the gene encoding the protein involved in asparagine transport thereby reducing asparagine during the food preparation or processing or reducing acrylamide in the food product.
13. The method of claim 12 , wherein the nucleic acid molecule of a) (ii) encodes a cell-wall asparaginase.
14. The method of claim 13 , wherein the asparaginase is encoded by ASP3 or wherein the asparaginase is Asp3p.
15. The method of claim 12 , wherein the nucleic acid molecule of a) (iii) encodes an amino acid transporter.
16. The method of claim 15 , wherein the amino acid transporter is encoded by GAP1, AGP1, GNP1, DIP5, AGP2 or AGP3 or is Gap1p, Agp1p, Gnp1p, Dip5p Agp2p or Agp3p.
17. The method of claim 12 , wherein the nucleic acid of a) (i) encodes a protein that modifies the activity of a regulatory factor of nitrogen catabolite repression in the microorganism.
18. The method of claim 17 , wherein the regulatory factor is encoded by URE2, GAT1, TOR1, TOR2, DAL80, GLN3 or GZF3 or is Ure2p, Gat1p, Tor1p, Tor2p, Dal80p, Gln3p or Gzf3p.
19. The method of claim 17 , wherein the nucleic acid of a) (i) comprises a URE2 deletion cassette.
20. The method of claim 12 , wherein the microorganism is yeast.
21. The method of claim 12 , wherein at least one of the nucleic acid molecules is operatively linked to a constitutively active promoter.
22. The method of claim 12 , wherein the food product is a vegetable-based food product, a beverage, a bakery product, a grain product, a fruit, legume, dairy or meat product.