Engineered hosts with exogenous ligninase and uses thereof
View Patent ↗The present invention relates to methods and engineered microbial hosts useful for treating lignin or a derivative thereof. In some embodiments, the host has one or more exogenous nucleic acid sequences that encode a ligninase (e.g., a laccase and/or a peroxidase).
1. An engineered Bacillus host comprising one or more exogenous nucleic acid sequences, wherein at least one exogenous nucleic acid sequence encodes a fungal laccase and a fungal peroxidase;
wherein the laccase comprises an amino acid sequence having at least 90% sequence identity to one of SEQ ID NOs: 1-4, 10, and 11;
wherein the peroxidase comprises an amino acid sequence having at least 80% sequence identity to one of SEQ ID NOs: 20-25; and
wherein the at least one exogenous nucleic acid sequence encodes the peroxidase in a sequence that is codon-optimized for the host.
2. The host of claim 1 , wherein the peroxidase is a versatile peroxidase.
3. The host of claim 1 , wherein the at least one exogenous nucleic acid sequence encodes the laccase and the peroxidase in a sequence that is codon-optimized for the host.
4. The host of claim 1 , wherein the laccase comprises an amino acid sequence having at least 97% sequence identity to one of SEQ ID NOs: 1-4, 10, and 11.
5. The host of claim 1 , wherein the peroxidase comprises an amino acid sequence having at least 97% sequence identity to one of SEQ ID NOs:20-25.
6. A method of treating lignin or a derivative thereof, the method comprising:
providing an engineered microbial host comprising one or more exogenous nucleic acid sequences, wherein at least one exogenous nucleic acid sequence encodes a laccase and a peroxidase; and
introducing the engineered microbial host to a source comprising lignin or a derivative thereof at a pH of from about 4 to 5;
wherein the laccase comprises an amino acid sequence having at least 90% sequence identity to one of SEQ ID NOs: 1-4, 10, and 11;
wherein the peroxidase comprises an amino acid sequence having at least 80% sequence identity to one of SEQ ID NOs: 20-25;
wherein the host is a member of the genus Bacillus ; and
wherein the at least one exogenous nucleic acid sequence encodes the peroxidase in a sequence that is codon-optimized for the host.
7. The method of claim 6 , wherein the laccase comprises an amino acid sequence having at least 97% sequence identity to one of SEQ ID NOs: 1-4, 10, and 11.
8. The method of claim 6 , wherein the peroxidase comprises an amino acid sequence having at least 97% sequence identity to one of SEQ ID NOs:20-25.
9. The method of claim 6 , wherein the host is a Bacillus subtilis bacterium.
10. The method of claim 6 , wherein the one or more exogenous nucleic acid sequences is provided as an expression vector.
11. The method of claim 6 , wherein the pH is 4 to 5.
12. The method of claim 6 , wherein the source comprises a media comprising a citric acid buffer.
13. The method of claim 6 , wherein the source comprises lignin.
14. The method of claim 13 , wherein the source comprises one or more˜-O-4 linkages.
15. The method of claim 6 , wherein the peroxidase comprises an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NOs: 20-25.
16. The method of claim 6 , wherein the laccase comprises an amino acid sequence having at least 99% sequence identity to one of SEQ ID NOs: 1-4, 10, and 11.
17. A method of treating lignin, the method comprising:
providing an engineered microbial host comprising one or more exogenous nucleic acid sequences, wherein at least one exogenous nucleic acid sequence encodes a laccase and/or a peroxidase; and
introducing the engineered microbial host to a source comprising lignin at a pH of from about 4 to about 6;
wherein the source comprises a media comprising a citric acid buffer;
wherein the host is a member of the genus Bacillus;
wherein the at least one exogenous nucleic acid sequence encodes at least the peroxidase, the peroxidase comprising an amino acid sequence having at least 99% sequence identity to one of SEQ ID NOs: 20-25; and
wherein the at least one exogenous nucleic acid sequence encodes the peroxidase in a sequence that is codon-optimized for the host.
18. The method of claim 17 , wherein the source comprises corn liquefied corn meal, corn steep liquor, corn stover, or corn fiber.
19. The method of claim 17 , wherein the peroxidase is one of SEQ ID NOs: 20-25.
20. The method of claim 17 , wherein the pH is about 4 to 5.