Oxygen tolerant hydrogenase by mutating electron supply pathway

Patent metadata

Field	Value
Publication number	US-10221401-B2
Application number	US-201615255833-A
Country	US
Kind code	B2
Filing date	Sep 2, 2016
Priority date	Sep 8, 2015
Publication date	Mar 5, 2019
Grant date	Mar 5, 2019

Abstract

Compositions and methods are provided for an O2 tolerant Fe—Fe hydrogenase. The hydrogenases of the invention comprise specific amino acid substitutions relative to the native, or wild-type enzymes.

First claim

What is claimed is:

1. A modified Fe—Fe hydrogenase comprising at least one amino acid substitution relative to the wild-type sequence, said modified Fe—Fe hydrogenase has
[i] at least 80% sequence identity to SEQ ID NO: 1, and at least one amino acid substitution at L192, G194, T356 or S357;
[ii] at least 96% sequence identity to SEQ ID NO: 2, and at least one amino acid substitution at L191, G193, N355 and/or D356;
[iii] at least 80% sequence identity to SEQ ID NO: 3, and at least one amino acid substitution at L192, G194, T356 or A357;
[iv] at least 80% sequence identity to SEQ ID NO: 4, and at least one amino acid substitution at L192, G194, T356 or A357; or
[v] at least 80% sequence identity to SEQ ID NO: 5, and at least one amino acid substitution at L191, G193, N355 or D356;
wherein the modified enzyme retains at least 10% of the initial specific activity following exposure to 0.01 atm. O 2 for 5 minutes.

2. The modified hydrogenase of claim 1 , wherein the Fe—Fe hydrogenase is derived from a Clostridium species.

3. The modified hydrogenase of claim 1 , wherein the amino acid substitution is selected from A156C, M166C, G194C, Q195C, I197C, A156C+L191C, G158C+I197C, N160C+T161C, N160C+A165C, N160C+L192C, N160C+Q195C, N160C+I197C, T161C+G194C, T161C+I197C, T163C+N189C, T163C+Q195C, T163C+I197C, Y164C+Q195C, A165C+N189C, A165C+L192C, A165C+Q195C, A165C+I197C, M166C+Q195C, M166C+I197C, F185C+I197C, N189C+G194C, N189C+I197C, L191C+L192C, L191C+I197C, Q195C+I197C, L192F, L192W, L192S, L192D, L192G, P301C, T356C, S357C, P301C+T356C, P301C+A498C, P301C+G502C, G302C+T356C, G302C+S357C, G302C+A498C, W303C+G507C, P354C+G508C, T356C+S357C, S357C+A498C, S357C+N505C, T356V, T356I, T356L, T356P, S357A, S357V, S357I, S357L, S357T, S357P, T356V+S357T, T356V+S357V, T356V+S357P, where numbering is made relative to SEQ ID NO:1.

4. The modified hydrogenase of claim 3 , wherein the amino acid substitutions is one or more of L192G, G194C, T356V, S357T.

5. The modified hydrogenase of claim 4 , wherein the hydrogenase comprises the amino acid sequence set forth in SEQ ID NO:6.

6. The modified hydrogenase of claim 3 , wherein the at least one amino acid substitutions that provides for faster H 2 generation is selected from A156C, G158C, M166C, L192C, Q195C, G185C+I197C, N160C+L192C, T163C+Y164C, Y164C+I197C, A165C+L191C, L192E, L192G, P301C, T356C, M498C, N505C+P301C, N505C, T356C+S357C, S357A, S357V, S357I, S357P, T356V+S357T.

Assignees

• Univ Leland Stanford Junior

Inventors

• Swartz James Robert
• KOO Jamin

Classifications

• C12P3/00

  Preparation of elements or inorganic compounds except carbon dioxide {(recovery of carbon dioxides as by-products C12F3/02)} · CPC title

• C12Y112/02001

  Cytochrome-c3 hydrogenase (1.12.2.1) · CPC title

• C12Y112/07002

  Ferredoxin hydrogenase (1.12.7.2) · CPC title

• C12N9/0067 Primary

  acting on hydrogen as donor (1.12) · CPC title