Microorganisms and methods for producing vanillin
View Patent ↗The present invention concerns a recombinant strain belonging to the order of Actinomycetales, wherein at least one gene encoding an enzyme having vanillin reductase activity is non-functional. The present invention is also related to a process for producing vanillin or a precursor thereof, comprising the culture of a recombinant strain in an appropriate medium comprising a substrate, and recovery of the produced vanillin or precursor thereof.
1. A recombinant strain belonging to the order of Actinomycetales, wherein at least one gene encoding an enzyme having vanillin reductase activity is non-functional, wherein
said gene encoding an enzyme having vanillin reductase activity comprises a sequence having at least 90% of nucleic acid identity with a sequence selected in a group comprising the sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9; and/or
said enzyme having vanillin reductase activity has an amino acid sequence comprising at least 90% of amino acid identity with a sequence selected in a group comprising the sequences: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10; and/or
at least one gene chosen among the genes comprising the sequences as shown in SEQ ID NO: 1 (vr1), SEQ ID NO: 3 (vr2), SEQ ID NO: 5 (vr3), SEQ ID NO: 7 (vr4) and SEQ ID NO: 9 (vr5) is non-functional.
2. The recombinant strain of claim 1 , wherein said strain is from the genus Amycolatopsis or Streptomyces.
3. The recombinant strain of claim 2 , wherein said strain is the strain Amycolatopsis sp. accessible under number ATCC 39116.
4. The recombinant strain of claim 1 , wherein at least one endogenous gene encoding an enzyme having vanillin reductase activity has been deleted.
5. The recombinant strain of claim 1 , wherein at least one endogenous gene encoding an enzyme having vanillin reductase activity has been replaced, totally or partially, with a DNA cassette.
6. The recombinant strain of claim 1 , wherein at least one endogenous gene encoding an enzyme having vanillin reductase activity has been inactivated by introduction of a DNA cassette.
7. The recombinant strain of claim 5 , wherein the DNA cassette has been removed from the genome to obtain a marker-less recombinant strain.
8. The recombinant strain of claim 5 , wherein the DNA cassette comprises two sequences (1) and (2), each one having a length of about 2 kb.
9. The recombinant strain of claim 1 , wherein said gene encoding an enzyme having vanillin reductase activity comprises a sequence having at least 95% of nucleic acid identity with a sequence selected in a group comprising the sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9.
10. The recombinant strain of claim 1 , wherein said enzyme having vanillin reductase activity has an amino acid sequence comprising at least 95% of amino acid identity with a sequence selected in a group comprising the sequences: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10.
11. The recombinant strain of claim 1 , wherein at least one gene chosen among the genes comprising the sequences as shown in SEQ ID NO: 1 (vr1), SEQ ID NO: 3 (vr2), SEQ ID NO: 5 (vr3), SEQ ID NO: 7 (vr4) and SEQ ID NO: 9 (vr5) is non-functional.
12. The recombinant strain of claim 1 , wherein at least two genes encoding an enzyme having vanillin reductase activity are non-functional among the genes presenting the sequences as shown in SEQ ID NO: 1 (vr1), SEQ ID NO: 3 (vr2), SEQ ID NO: 5 (vr3), SEQ ID NO: 7 (vr4) and SEQ ID NO: 9 (vr5).
13. The recombinant strain of claim 1 , wherein the five genes comprising the sequences as shown in SEQ ID NO: 1 (vr1), SEQ ID NO: 3 (vr2), SEQ ID NO: 5 (vr3), SEQ ID NO: 7 (vr4) and SEQ ID NO: 9 (vr5) are non-functional.
14. The recombinant strain of claim 1 , wherein at least one gene encoding an enzyme having vanillin dehydrogenase activity is non-functional among the genes presenting the sequences as shown in SEQ ID NO: 1 (vr1), SEQ ID NO: 3 (vr2), SEQ ID NO: 5 (vr3), SEQ ID NO: 7 (vr4) and SEQ ID NO: 9 (vr5).
15. A process for producing vanillin, comprising culturing the recombinant strain of claim 1 in an appropriate medium comprising a substrate, and recovering the produced vanillin.
16. The recombinant strain of claim 6 , wherein the DNA cassette has been removed from the genome to obtain a marker-less recombinant strain.
17. The recombinant strain of claim 6 , wherein the DNA cassette comprises two sequences (1) and (2), each one having a length of about 2 kb.
18. A process for producing vanillin, comprising culturing the recombinant strain of claim 4 in an appropriate medium comprising a substrate, and recovering the produced vanillin.
19. A process for producing vanillin, comprising culturing the recombinant strain of claim 5 in an appropriate medium comprising a substrate, and recovering the produced vanillin.
20. A process for producing vanillin, comprising culturing the recombinant strain of claim 6 in an appropriate medium comprising a substrate, and recovering the produced vanillin.
21. A process for producing vanillin, comprising culturing the recombinant strain of claim 11 in an appropriate medium comprising a substrate, and recovering the produced vanillin.
22. A process for producing vanillin, comprising culturing the recombinant strain of claim 13 in an appropriate medium comprising a substrate, and recovering the produced vanillin.