IP Library › Granted Patent US 11,447,754

Granted Patent B2

US 11,447,754 · App. 17/038,971 · Granted Sep 20, 2022

In vitro methods of chemical conversion using non-stereospecific glutathione lyases

Inventors: Timothy James Donohue (Middleton, WI); Daniel Leo Gall (Madison, WI); Wayne S. Kontur (Madison, WI); Hoon Kim (Madison, WI); John Ralph (Madison, WI); Daniel R. Noguera (Madison, WI); Brian Fox (Madison, WI); Craig Bingman (Fitchburg, WI)

Assignee: Wisconsin Alumni Research Foundation

C12N9/1088 C12P7/22 C12P7/26 C12N9/88 C12Y114/16005 C12Y205/01018

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Quick Facts

Patent No.: US 11,447,754
App. No.: 17/038,971
Granted: Sep 20, 2022
Kind: B2

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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.

Claims (211)

1. A method of chemical conversion, comprising contacting a first compound in vitro with a non-stereospecific glutathione lyase to yield a second compound, wherein:

the non-stereospecific glutathione lyase comprises:

an amino acid sequence at least 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);

SEQ ID NO: 30 (ecYfcG);

SEQ ID NO: 32 (ssYghU);

and

SEQ ID NO: 36 (PcUre2pB 1);

and

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 );

the first compound has a structure of Formula I or a salt thereof:

wherein:

R 1 , R 2 , and R 3 are each independently —H, —OH, —O-alkyl, —O-lignin, or -lignin;

R 4 is —H, —OH, —SH, —COOH, —SO 3 H, or —O-lignin; and

SG is glutathione bound in an S or R configuration; and

the second compound has a structure of Formula II or a salt thereof:

wherein:

R 1 , R 2 , and R 3 are each independently —H, —OH, —O-alkyl, —O-lignin, or -lignin; and

R 4 is —H, —OH, —SH, —COOH, —SO 3 H, or —O-lignin.

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);

SEQ ID NO: 30 (ecYfcG);

SEQ ID NO: 32 (ssYghU);

and

SEQ ID NO: 36 (PcUre2pB1).

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);

residues 21-313 of SEQ ID NO: 28 (recombinant

ecYghU);

SEQ ID NO: 30 (ecYfcG);

SEQ ID NO: 32 (ssYghU);

and

SEQ ID NO: 36 (PcUre2pB1).

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);

residues 21-313 of SEQ ID NO: 28 (recombinant

ecYghU);

SEQ ID NO: 30 (ecYfcG);

SEQ ID NO: 32 (ssYghU);

and

SEQ ID NO: 36 (PcUre2pB1).

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 non-stereospecific glutathione lyase comprises an amino acid sequence at least 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).

9. The method of claim 8 , 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).

10. The method of claim 8 , 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);

residues 21-313 of SEQ ID NO: 28 (recombinant

ecYghU);

and

SEQ ID NO: 32 (ssYghU).

11. The method of claim 8 , 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);

residues 21-313 of SEQ ID NO: 28 (recombinant

ecYghU);

and

SEQ ID NO: 32 (ssYghU).

12. The method of claim 8 , wherein the non-stereospecific glutathione lyase comprises at least seven of: