Methods and compositions for production of aromatic and other compounds in yeast
The present disclosure includes methods and components for production of valuable industrial compounds in yeast. In an embodiment, the present invention provides a nucleic acid construct with increased stability for gene expression or gene editing comprising: a nucleic acid sequence encoding one or more of SEQ ID NO: 1-8 (CENs 1-8); and one or more regulatory elements functional in a yeast cell. In an embodiment of the present invention the nucleic acid constructs are vectors, preferably episomal vectors. High expression promoters, as well as methods for increasing production of compounds such as aromatics are disclosed.
1. A modified recombinant yeast cell for producing an aromatic compound, comprising
a) an expression system that comprises at least one nucleotide sequence that encodes a transketolase, TKL1, capable of being expressed;
b) an expression system that comprises at least one nucleotide sequence that encodes a pentafunctional protein for aromatic amino acid biosynthesis (ARO1), wherein the nucleotide sequence has the mutation aro1 D920A , capable of being expressed; wherein the expression of a protein involved in regulating the efficient localization of Golgi network proteins (RIC1), a pentafunctional protein for aromatic amino acid biosynthesis (ARO1) and a 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (ARO4) are downregulated or absent, and wherein said modified yeast cell produces higher titers of aromatic compounds compared to wild-type yeast cell of the same strain.
2. The modified yeast cell of claim 1 , wherein said cell further comprises a third expression system that comprises at least one nucleotide sequence that encodes ribose-5-phosphate ketol-isomerase (RKI1).
3. The modified yeast cell of claim 1 , wherein said cell is of Saccharomyces spp.
4. The modified yeast cell of claim 1 , wherein said mutant pentafunctional protein for aromatic amino acid biosynthesis (ARO1) is aro1 D1409A-D920A .
5. The modified yeast cell of claim 3 , wherein said cell is Saccharomyces cerevisiae.
6. The modified yeast cell of claim 1 , wherein said cell is of Scheffersomyces spp.
7. The modified yeast cell of claim 6 , wherein said cell is Scheffersomyces stipitis.
8. The modified yeast cell of claim 1 , wherein said cell is Saccharomyces uvae, Saccharomyces kluyveri, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Pichia methanolica, Pichia kluyveri, Yarrowia lipolytica, Candida sp., Candida utilis, Candida cacaoi, Geotrichum sp., and/or Geotrichum fermentans.
9. The modified yeast cell of claim 1 , wherein said modified yeast cell produces higher titers of shikimic acid compared to wild-type yeast cell of the same strain.
10. The modified yeast cell of claim 1 , wherein said modified yeast cell produces higher titers of muconic acid compared to wild-type yeast cell of the same strain.
11. The modified yeast cell of claim 1 , wherein said cell further comprises a third expression system that comprises at least one nucleotide sequence that encodes aro4 K220L .
12. A cell, tissue, or organ comprising the modified yeast cell of claim 1 .