In vitro methods for processing lignin and other aromatic compounds

Patent metadata

Field	Value
Publication number	US-10829745-B2
Application number	US-201816103275-A
Country	US
Kind code	B2
Filing date	Aug 14, 2018
Priority date	Aug 14, 2017
Publication date	Nov 10, 2020
Grant date	Nov 10, 2020

Abstract

Enzymes for depolymerizing lignin. The enzymes include dehydrogenases, β-etherases, and glutathione lyases. The dehydrogenases can comprise one or more or LigD, LigO, LigN, and LigL. The β-etherases can comprise one or more of LigE, LigF, LigP, and BaeA. The glutathione lyases can comprise any one or more of LigG and a number of non-stereospecific, optionally recombinant glutathione lyases derived from Sphingobium sp. SYK-6, Novosphingobium aromaticivorans, Escherichia coli, Streptococcus sanguinis, Phanerochaete chrysosporium, and other microorganisms. The enzymes can be combined in compositions and/or used in methods of processing lignin or other aromatic compounds in vitro.

First claim

We claim:

1. A method of processing lignin, comprising contacting lignin comprising β-O-4 ether linkages in vitro with:
a dehydrogenase comprising at least one of LigD, LigO, LigN, and LigL;
a β-etherase comprising at least one of LigE, LigF, LigP, and an enzyme comprising a first polypeptide having an amino acid sequence of SEQ ID NO:40 or an amino acid sequence at least about 95% identical thereto and a second polypeptide having an amino acid sequence of SEQ ID NO:42 or an amino acid sequence at least about 95% identical thereto; and
a non-stereospecific glutathione lyase comprising an amino acid sequence at least about 80% identical to any of:
SEQ ID NO:18 (NaGST Nu );
residues 21-313 of SEQ ID NO:20 (recombinant NaGST Nu );
SEQ ID NO:22 (SYK6GST Nu );
residues 21-324 of SEQ ID NO:24 (recombinant SYK6GST Nu );
SEQ ID NO:26 (ecYghU);
residues 21-313 of SEQ ID NO:28 (recombinant ecYghU); and
SEQ ID NO:32 (ssYghU),

wherein the non-stereospecific glutathione lyase comprises at least four of:
threonine or a conservative variant of threonine at a position corresponding to position 51 of SEQ ID NO:18 (NaGST Nu );
asparagine or a conservative variant of asparagine at a position corresponding to position 53 of SEQ ID NO:18 (NaGST Nu );
glutamine or a conservative variant of glutamine at a position corresponding to position 86 of SEQ ID NO:18 (NaGST Nu );
lysine, a conservative variant of lysine, arginine, or a conservative variant of arginine at a position corresponding to position 99 of SEQ ID NO:18 (NaGST Nu );
isoleucine or a conservative variant of isoleucine at a position corresponding to position 100 of SEQ ID NO:18 (NaGST Nu );
glutamate or a conservative variant of glutamate at a position corresponding to position 116 of SEQ ID NO:18 (NaGST Nu );
serine, threonine, a conservative variant of serine, or a conservative variant of threonine at a position corresponding to position 117 of SEQ ID NO:18 (NaGST Nu ); and
arginine or a conservative variant of arginine at a position corresponding to position 177 of SEQ ID NO:18 (NaGST Nu ).

2. The method of claim 1 , wherein the non-stereospecific glutathione lyase comprises an amino acid sequence at least about 85% identical to any of:
SEQ ID NO:18 (NaGST Nu );
residues 21-313 of SEQ ID NO:20 (recombinant NaGST Nu );
SEQ ID NO:22 (SYK6GST Nu );
residues 21-324 of SEQ ID NO:24 (recombinant SYK6GST Nu );
SEQ ID NO:26 (ecYghU);
residues 21-313 of SEQ ID NO:28 (recombinant ecYghU); and
SEQ ID NO:32 (ssYghU).

3. The method of claim 1 , wherein the non-stereospecific glutathione lyase comprises an amino acid sequence at least about 90% identical to any of:
SEQ ID NO:18 (NaGST Nu );
residues 21-313 of SEQ ID NO:20 (recombinant NaGST Nu );
SEQ ID NO:22 (SYK6GST Nu );
residues 21-324 of SEQ ID NO:24 (recombinant SYK6GST Nu );
SEQ ID NO:26 (ecYghU); and
residues 21-313 of SEQ ID NO:28 (recombinant ecYghU).

4. The method of claim 1 , wherein the non-stereospecific glutathione lyase comprises an amino acid sequence at least about 95% identical to any of:
SEQ ID NO:18 (NaGST Nu );
residues 21-313 of SEQ ID NO:20 (recombinant NaGST Nu );
SEQ ID NO:22 (SYK6GST Nu );
residues 21-324 of SEQ ID NO:24 (recombinant SYK6GST Nu );
SEQ ID NO:26 (ecYghU); and
residues 21-313 of SEQ ID NO:28 (recombinant ecYghU).

5. The method of claim 1 , wherein the non-stereospecific glutathione lyase comprises at least seven of:
asparagine or a conservative variant of asparagine at a position corresponding to position 25 of SEQ ID NO:18 (NaGST Nu );
threonine or a conservative variant of threonine at a position corresponding to position 51 of SEQ ID NO:18 (NaGST Nu );
asparagine or a conservative variant of asparagine at a position corresponding to position 53 of SEQ ID NO:18 (NaGST Nu );
glutamine or a conservative variant of glutamine at a position corresponding to position 86 of SEQ ID NO:18 (NaGST Nu );
lysine, a conservative variant of lysine, arginine, or a conservative variant of arginine at a position corresponding to position 99 of SEQ ID NO:18 (NaGST Nu );
isoleucine or a conservative variant of isoleucine at a position corresponding to position 100 of SEQ ID NO:18 (NaGST Nu );
glutamate or a conservative variant of glutamate at a position corresponding to position 116 of SEQ ID NO:18 (NaGST Nu );
serine, threonine, a conservative variant of serine, or a conservative variant of threonine at a position corresponding to position 117 of SEQ ID NO:18 (NaGST Nu );
tyrosine or a conservative variant of tyrosine at a position corresponding to position 166 of SEQ ID NO:18 (NaGST Nu );
arginine or a conservative variant of arginine at a position corresponding to position 177 of SEQ ID NO:18 (NaGST Nu ); and
tyrosine or a conservative variant of tyrosine at a position corresponding to position 224 of SEQ ID NO:18 (NaGST Nu ).

6. The method of claim 1 , wherein the contacting occurs in the presence of a glutathione (GSH) reductase that catalyzes reduction of glutathione disulfide (GSSG).

7. The method of claim 6 , wherein the GSH reductase comprises an amino acid sequence at least about 95% identical to SEQ ID NO:38 (AvGR).

8. The method of claim 1 , wherein the contacting releases at least one of a monomeric phenylpropanoid unit and a monomeric flavone.

9. The method of claim 1 , wherein the contacting releases at least one of a monomeric guaiacyl phenylpropanoid unit, a monomeric syringyl phenylpropanoid unit, a monomeric p-hydroxyphenyl phenylpropanoid unit, and a monomeric tricin unit.

10. The method of claim 1 , wherein the lignin comprises an average molecular weight (MW) of from about 600 to about 20,000.

11. The method of claim 1 , wherein the dehydrogenase comprises at least one of LigD and LigO and at least one of LigL and LigN.

12. The method of claim 1 , wherein the dehydrogenase comprises LigD and LigN.

13. The method of claim 1 , wherein the β-etherase comprises LigF and at least one of LigE, LigP, and the enzyme comprising the first polypeptide having the amino acid sequence of SEQ ID NO:40 or the amino acid sequence at least about 95% identical thereto and the second polypeptide having the amino acid sequence of SEQ ID NO:42 or the amino acid sequence at least about 95% identical thereto.

14. The method of claim 1 , wherein the β-etherase comprises LigF and LigE.

15. The method of claim 1 , wherein the non-stereospecific glutathione lyase comprises an amino acid sequence at least about 95% identical to SEQ ID NO:18 (NaGST Nu ).

16. The method of claim 1 , wherein:
the dehydrogenase comprises at least one of LigD and LigO and at least one of LigL and LigN;
the β-etherase comprises LigF and at least one of LigE, LigP, and the enzyme comprising the first polypeptide having the amino acid sequence of SEQ ID NO:40 or the amino acid sequence at least about 95% identical thereto and the second polypeptide having the amino acid sequence of SEQ ID NO:42 or the amino acid sequence at least about 95% identical thereto;
the non-stereospecific glutathione lyase comprises an amino acid sequence at least about 95% identical to SEQ ID NO:18 (NaGST Nu ); and
the contacting occurs in the presence of a glutathione (GSH) reductase that catalyzes reduction of glutathione disulfide (GSSG) and comprises an amino acid sequence at least about 95% identical to SEQ ID NO:38 (AvGR).

17. The method of claim 1 , wherein:
the dehydrogenase comprises LigD and LigN;
the β-etherase comprises LigF and LigE;
the non-stereospecific glutathione lyase comprises an amino acid sequence at least about 95% ide

Assignees

• Wisconsin Alumni Res Found

Inventors

• Donohue Timothy James
• Gall Daniel Leo
• Kontur Wayne S
• Kim Hoon
• Ralph John
• Noguera Daniel R
• Fox Brian
• Bingman Craig

Classifications

• C12Y114/16005

  Alkylglycerol monooxygenase (1.14.16.5) · CPC title

• C12N9/1088 Primary

  Glutathione transferase (2.5.1.18) · CPC title

• C12P7/26

  Ketones · CPC title

• C12N9/88

  Lyases (4.) · CPC title

• C12Y205/01018

  Glutathione transferase (2.5.1.18) · CPC title