Engineering Enzyme Specificity Using Computational Design of a Defined-Sequence Library
Engineered biosynthetic pathways have the potential to produce high-value molecules from inexpensive feedstocks, but a key limitation is engineering enzymes with high activity and specificity for new reactions. Here, we developed a method for combining structure-based computational protein design wi...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | en_US |
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Elsevier
2015
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| Online Access: | http://hdl.handle.net/1721.1/96044 https://orcid.org/0000-0003-0437-3157 |
| _version_ | 1811088625234870272 |
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| author | Lippow, Shaun M. Moon, Tae Seok Basu, Subhayu Yoon, Sang-Hwal Li, Xiazhen Chapman, Brad A. Robison, Keith Lipovšek, Daša Prather, Kristala L. Jones |
| author2 | Massachusetts Institute of Technology. Department of Chemical Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Chemical Engineering Lippow, Shaun M. Moon, Tae Seok Basu, Subhayu Yoon, Sang-Hwal Li, Xiazhen Chapman, Brad A. Robison, Keith Lipovšek, Daša Prather, Kristala L. Jones |
| author_sort | Lippow, Shaun M. |
| collection | MIT |
| description | Engineered biosynthetic pathways have the potential to produce high-value molecules from inexpensive feedstocks, but a key limitation is engineering enzymes with high activity and specificity for new reactions. Here, we developed a method for combining structure-based computational protein design with library-based enzyme screening, in which inter-residue correlations favored by the design are encoded into a defined-sequence library. We validated this approach by engineering a glucose 6-oxidase enzyme for use in a proposed pathway to convert D-glucose into D-glucaric acid. The most active variant, identified after only one round of diversification and screening of only 10,000 wells, is approximately 400-fold more active on glucose than is the wild-type enzyme. We anticipate that this strategy will be broadly applicable to the discovery of new enzymes for engineered biological pathways. |
| first_indexed | 2024-09-23T14:04:57Z |
| format | Article |
| id | mit-1721.1/96044 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T14:04:57Z |
| publishDate | 2015 |
| publisher | Elsevier |
| record_format | dspace |
| spelling | mit-1721.1/960442022-09-28T18:16:00Z Engineering Enzyme Specificity Using Computational Design of a Defined-Sequence Library Lippow, Shaun M. Moon, Tae Seok Basu, Subhayu Yoon, Sang-Hwal Li, Xiazhen Chapman, Brad A. Robison, Keith Lipovšek, Daša Prather, Kristala L. Jones Massachusetts Institute of Technology. Department of Chemical Engineering Massachusetts Institute of Technology. Synthetic Biology Center Prather, Kristala L. Jones Moon, Tae Seok Yoon, Sang-Hwal Engineered biosynthetic pathways have the potential to produce high-value molecules from inexpensive feedstocks, but a key limitation is engineering enzymes with high activity and specificity for new reactions. Here, we developed a method for combining structure-based computational protein design with library-based enzyme screening, in which inter-residue correlations favored by the design are encoded into a defined-sequence library. We validated this approach by engineering a glucose 6-oxidase enzyme for use in a proposed pathway to convert D-glucose into D-glucaric acid. The most active variant, identified after only one round of diversification and screening of only 10,000 wells, is approximately 400-fold more active on glucose than is the wild-type enzyme. We anticipate that this strategy will be broadly applicable to the discovery of new enzymes for engineered biological pathways. United States. Office of Naval Research. Young Investigator Program (Grant N000140510656) National Science Foundation (U.S.) (Synthetic Biology Engineering Research Center. Grant EEC-0540879) MIT Faculty Start-up Fund Codon Devices, Inc. 2015-03-17T16:10:57Z 2015-03-17T16:10:57Z 2010-12 2010-10 Article http://purl.org/eprint/type/JournalArticle 10745521 http://hdl.handle.net/1721.1/96044 Lippow, Shaun M., Tae Seok Moon, Subhayu Basu, Sang-Hwal Yoon, Xiazhen Li, Brad A. Chapman, Keith Robison, Daša Lipovšek, and Kristala L.J. Prather. “Engineering Enzyme Specificity Using Computational Design of a Defined-Sequence Library.” Chemistry & Biology 17, no. 12 (December 2010): 1306–1315. © 2010 Elsevier Ltd. https://orcid.org/0000-0003-0437-3157 en_US http://dx.doi.org/10.1016/j.chembiol.2010.10.012 Chemistry and Biology Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf Elsevier Elsevier |
| spellingShingle | Lippow, Shaun M. Moon, Tae Seok Basu, Subhayu Yoon, Sang-Hwal Li, Xiazhen Chapman, Brad A. Robison, Keith Lipovšek, Daša Prather, Kristala L. Jones Engineering Enzyme Specificity Using Computational Design of a Defined-Sequence Library |
| title | Engineering Enzyme Specificity Using Computational Design of a Defined-Sequence Library |
| title_full | Engineering Enzyme Specificity Using Computational Design of a Defined-Sequence Library |
| title_fullStr | Engineering Enzyme Specificity Using Computational Design of a Defined-Sequence Library |
| title_full_unstemmed | Engineering Enzyme Specificity Using Computational Design of a Defined-Sequence Library |
| title_short | Engineering Enzyme Specificity Using Computational Design of a Defined-Sequence Library |
| title_sort | engineering enzyme specificity using computational design of a defined sequence library |
| url | http://hdl.handle.net/1721.1/96044 https://orcid.org/0000-0003-0437-3157 |
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