Conversion of S-lignin compounds to useful intermediates
The present disclosure relates to a genetically modified microbial cell that includes a first genetic modification resulting in the expression of an exogenous vanillate demethylase, such that the microbial cell is capable of metabolizing an S-lignin decomposition product and producing 2-pyrone-4,6-dicarboxylate (PDC).
1. A genetically modified Pseudomonas sp. comprising:
a first genetic modification of a first gene encoding an exogenous vanillate demethylase, wherein the first gene has a sequence that is at least 80% identical to at least one of SEQ ID NO: 11 or SEQ ID NO: 13, and wherein the expressed exogenous vanillate demethylase has a sequence that is at least 80% identical to at least one of SEQ ID NO: 12 or SEQ ID NO: 14; and
a second genetic modification of a second gene encoding an exogenous protocatechuate dioxygenase, wherein the second gene has a sequence that is at least 80% identical to at least one of SEQ ID NO: 5 or SEQ ID NO: 7, and wherein the exogenous protocatechuate dioxygenase has a sequence that is at least 80% identical to at least one of SEQ ID NO: 6 or SEQ ID NO: 8; wherein:
the Pseudomonas sp. metabolizes a S-lignin decomposition product, and
the Pseudomonas sp. produces 2-pyrone-4,6-dicarboxylate (PDC).
2. The genetically modified Pseudomonas sp. of claim 1 , wherein the Pseudomonas sp. is selected from the group consisting of P. putida, P. fluorescens , or P. stutzeri.
3. The genetically modified Pseudomonas sp. of claim 1 , wherein the exogenous dioxygenase is derived from a bacterium.
4. The genetically modified Pseudomonas sp. of claim 1 , wherein the exogenous dioxygenase comprises a LigAB.
5. The genetically modified Pseudomonas sp. of claim 1 , wherein the Pseudomonas sp. metabolizes at least one of a G-lignin decomposition product or an H-lignin decomposition product.
6. The genetically modified Pseudomonas sp. of claim 1 , wherein the exogenous vanillate demethylase demethylates vanillate.
7. The genetically modified Pseudomonas sp. of claim 1 , wherein the exogenous vanillate demethylase cannot demethylate 3-O-methylgallate.
8. The genetically modified Pseudomonas sp. of claim 1 , wherein the S-lignin decomposition molecule comprises at least one of syringaldehyde, syringate, or 3-0 methylgallate.
9. The genetically modified Pseudomonas sp. of claim 5 , wherein the G-lignin decomposition molecule comprises ferulate.
10. A method for lignin valorization, the method comprising:
converting an S-lignin decomposition product to 2-pyrone-4,6-dicarboxylate (PDC) utilizing a genetically modified Pseudomonas sp., wherein:
the genetically modified Pseudomonas sp. comprises:
a first genetic modification of a first gene encoding an exogenous vanillate demethylase, wherein the first gene has a sequence that is at least 80% identical to at least one of SEQ ID NO: 11 or SEQ ID NO: 13 and wherein the expressed exogenous vanillate demethylase has a sequence that is at least 80% identical to at least one of SEQ ID NO: 12 or SEQ ID NO: 14; and
a second genetic modification of a second gene encoding an exogenous protocatechuate dioxygenase, wherein the second gene has a sequence that is at least 80% identical to at least one of SEQ ID NO: 5 or SEQ ID NO: 7 and wherein the exogenous protocatechuate dioxygenase has a sequence that is at least 80% identical to at least one of SEQ ID NO: 6 or SEQ ID NO: 8.
11. The genetically modified Pseudomonas sp. of claim 1 wherein the first genetic modification consists of replacing a native vanAB gene with the first genetic modification.
12. The genetically modified Pseudomonas sp. of claim 11 wherein the first genetic modification consists of replacing a native chromosomal vanAB gene with the first genetic modification.
13. The genetically modified Pseudomonas sp. of claim 1 wherein the second genetic modification consists of replacing a native pcaHG gene with the second genetic modification.
14. The genetically modified Pseudomonas sp. of claim 1 wherein the second genetic modification consists of replacing a native chromosomal pcaHG gene with the second genetic modification.
15. The genetically modified Pseudomonas sp. of claim 1 wherein the first genetic modification consists of replacing a native chromosomal vanAB gene with the first genetic modification and wherein the second genetic modification consists of replacing a native chromosomal pcaHG gene with the second genetic modification.
16. The genetically modified Pseudomonas sp. of claim 15 wherein 2-pyrone-4,6-dicarboxylate (PDC) is produced by the cell at a concentration of up to 3.65 mM.