Pantothenic acid biosynthesis in zymomonas
View Patent ↗Zymomonas is unable to synthesize pantothenic acid and requires this essential vitamin in growth medium. Zymomonas strains transformed with an operon for expression of 2-dehydropantoate reductase and aspartate 1-decarboxylase were able to grow in medium lacking pantothenic acid. These strains may be used for ethanol production without pantothenic acid supplementation in seed culture and fermentation media.
1. A recombinant bacterial strain of the genus Zymomonas comprising a heterologous nucleic acid molecule encoding a polypeptide having 2-dehydropantoate reductase activity and a heterologous nucleic acid molecule encoding a polypeptide having aspartate 1-decarboxylase activity wherein said recombinant bacterial strain can produce ethanol in the absence of externally supplied pantothenic acid.
2. A recombinant bacterial strain of claim 1 wherein the polypeptide having 2-dehydropantoate reductase activity is an enzyme belonging to the EC 1.1.1.169 class.
3. A recombinant bacterial strain of claim 2 wherein the polypeptide having 2-dehydropantoate reductase activity has at least ten conserved amino acid positions selected from the group consisting of G at position 7, G at position 9, G at position 12, K at position 72, N at position 98, G at position 99, K at position 176, N at position 180, N at position 184, E at position 210, S at position 244, D or S at position 248, and E at position 256, as compared to the E. coli 2-dehydropantoate reductase of SEQ ID NO:4.
4. A recombinant bacterial strain of claim 1 wherein the polypeptide having aspartate 1-decarboxylase activity is an enzyme belonging to the EC 4.1.1.11 class.
5. A recombinant bacterial strain of claim 4 wherein the polypeptide having aspartate 1-decarboxylase activity has at least eight conserved amino acid positions selected from the group consisting of K at position 9, H at position 11, Y at position 22, G at position 24, S at position 25, G at position 52, R at position 54, T at position 57, Y at position 58, N at position 72, G at position 73, and I at position 86, as compared to the E. coli aspartate 1-decarboxylase of SEQ ID NO:7.
6. A recombinant bacterial strain of claim 4 wherein the polypeptide having aspartate 1-decarboxylase activity has the five conserved amino acid positions K at position 9, Y at position 22, G at position 24, T at position 57, and Y at position 58, as compared to the E. coli aspartate 1-decarboxylase of SEQ ID NO:7.
7. A recombinant bacterial strain of claim 1 wherein the strain grows in media that lacks pantothenic acid.
8. The recombinant bacterial strain of claim 1 wherein the strain comprises genetic modifications which enhance the production of ethanol.
9. A process for producing a Zymomonas strain that synthesizes pantothenic acid comprising:
a) providing a bacterial strain of the genus Zymomonas;
b) introducing a heterologous nucleic acid molecule encoding a polypeptide having 2-dehydropantoate reductase activity; and
c) introducing a heterologous nucleic acid molecule encoding a polypeptide having aspartate 1-decarboxylase activity;
wherein steps b) and c) may be in either order or simultaneous, and wherein 2-dehydropantoate reductase activity and aspartate 1-decarboxylase activity are both expressed in the strain produced by steps (a), (b), and (c).
10. A method for the producing ethanol comprising:
a) providing a recombinant bacterial strain of the genus Zymomonas comprising a heterologous nucleic acid molecule encoding a polypeptide having 2-dehydropantoate reductase activity and a heterologous nucleic acid molecule encoding a polypeptide having aspartate 1-decarboxylase activity; and
b) contacting the strain of (a) with fermentation medium under conditions whereby the strain produces ethanol.
11. A method according to claim 10 wherein the polypeptide having 2-dehydropantoate reductase activity is an enzyme belonging to the EC 1.1.1.169 class.
12. A method according to claim 11 wherein the polypeptide having 2-dehydropantoate reductase activity has at least ten conserved amino acid positions selected from the group consisting of G at position 7, G at position 9, G at position 12, K at position 72, N at position 98, G at position 99, K at position 176, N at position 180, N at position 184, E at position 210, S at position 244, D or S at position 248, and E at position 256, as compared to the E. coli 2-dehydropantoate reductase of SEQ ID NO:4.
13. A method according to claim 10 wherein the polypeptide having aspartate 1-decarboxylase activity is an enzyme belonging to the EC 4.1.1.11 class.
14. A method according to claim 13 wherein the polypeptide having aspartate 1-decarboxylase activity has at least eight conserved amino acid positions selected from the group consisting of K at position 9, H at position 11, Y at position 22, G at position 24, S at position 25, G at position 52, R at position 54, T at position 57, Y at position 58, N at position 72, G at position 73, and I at position 86, as compared to the E. coli aspartate 1-decarboxylase of SEQ ID NO:7.
15. A method according to claim 13 wherein the polypeptide having aspartate 1-decarboxylase activity has the five conserved amino acid positions K at position 9, Y at position 22, G at position 24, T at position 57, and Y at position 58, as compared to the E. coli aspartate 1-decarboxylase of SEQ ID NO:7.
16. A method according to claim 10 wherein prior to step (b) the bacterial strain of (a) is contacted with medium that lacks or has a sub-optimal amount of pantothenic acid wherein a seed culture is produced to inoculate the fermentation medium of (b).
17. A method according to claim 10 or 16 wherein the fermentation medium lacks or has a sub-optimal amount of pantothenic acid.