Identification and optimization of a novel thermo- and solvent stable ketol-acid reductoisomerase for cell free isobutanol biosynthesis

Steven Reiße; Daniel Garbe; Thomas Brück

doi:10.1016/j.biochi.2014.10.024

Identification and optimization of a novel thermo- and solvent stable ketol-acid reductoisomerase for cell free isobutanol biosynthesis

Steven Reiße, Daniel Garbe, Thomas Brück

Werner Siemens-Chair of Synthetic Biotechnology

Technical University of Munich

Research output: Contribution to journal › Article › peer-review

7 Scopus citations

Abstract

Due to its enhanced energy content and hydrophobicity, isobutanol is flagged as a next generation biofuel and chemical building block. For cellular and cell-free isobutanol production, NADH dependent (over NADPH dependent) enzyme systems are desired. To improve cell-free isobutanol processes, we characterized and catalytically optimized a NADH dependent, thermo- and solvent stable ketol-acid reductoisomerase (KARI) derived from the bacterium Meiothermus ruber (Mr). The wild type Mr-KARI has the most temperature tolerant KARI specific activity reported to date. The KARI screening procedure developed in this study allows accelerated molecular optimization. Thus, a KARI variant with a 350% improved activity and enhanced NADH cofactor specificity was identified. Other KARI variants gave insights into Mr-KARI structure-function relationships.

Original language	English
Pages (from-to)	76-84
Number of pages	9
Journal	Biochimie
Volume	108
DOIs	https://doi.org/10.1016/j.biochi.2014.10.024
State	Published - Jan 2015

Keywords

Biocatalysis
Cell-free
Isobutanol
Ketol-acid reductoisomerase
Meiothermus ruber
Thermophilic enzymes

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10.1016/j.biochi.2014.10.024

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title = "Identification and optimization of a novel thermo- and solvent stable ketol-acid reductoisomerase for cell free isobutanol biosynthesis",

abstract = "Due to its enhanced energy content and hydrophobicity, isobutanol is flagged as a next generation biofuel and chemical building block. For cellular and cell-free isobutanol production, NADH dependent (over NADPH dependent) enzyme systems are desired. To improve cell-free isobutanol processes, we characterized and catalytically optimized a NADH dependent, thermo- and solvent stable ketol-acid reductoisomerase (KARI) derived from the bacterium Meiothermus ruber (Mr). The wild type Mr-KARI has the most temperature tolerant KARI specific activity reported to date. The KARI screening procedure developed in this study allows accelerated molecular optimization. Thus, a KARI variant with a 350% improved activity and enhanced NADH cofactor specificity was identified. Other KARI variants gave insights into Mr-KARI structure-function relationships.",

keywords = "Biocatalysis, Cell-free, Isobutanol, Ketol-acid reductoisomerase, Meiothermus ruber, Thermophilic enzymes",

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T1 - Identification and optimization of a novel thermo- and solvent stable ketol-acid reductoisomerase for cell free isobutanol biosynthesis

AU - Reiße, Steven

AU - Garbe, Daniel

AU - Brück, Thomas

PY - 2015/1

Y1 - 2015/1

N2 - Due to its enhanced energy content and hydrophobicity, isobutanol is flagged as a next generation biofuel and chemical building block. For cellular and cell-free isobutanol production, NADH dependent (over NADPH dependent) enzyme systems are desired. To improve cell-free isobutanol processes, we characterized and catalytically optimized a NADH dependent, thermo- and solvent stable ketol-acid reductoisomerase (KARI) derived from the bacterium Meiothermus ruber (Mr). The wild type Mr-KARI has the most temperature tolerant KARI specific activity reported to date. The KARI screening procedure developed in this study allows accelerated molecular optimization. Thus, a KARI variant with a 350% improved activity and enhanced NADH cofactor specificity was identified. Other KARI variants gave insights into Mr-KARI structure-function relationships.

AB - Due to its enhanced energy content and hydrophobicity, isobutanol is flagged as a next generation biofuel and chemical building block. For cellular and cell-free isobutanol production, NADH dependent (over NADPH dependent) enzyme systems are desired. To improve cell-free isobutanol processes, we characterized and catalytically optimized a NADH dependent, thermo- and solvent stable ketol-acid reductoisomerase (KARI) derived from the bacterium Meiothermus ruber (Mr). The wild type Mr-KARI has the most temperature tolerant KARI specific activity reported to date. The KARI screening procedure developed in this study allows accelerated molecular optimization. Thus, a KARI variant with a 350% improved activity and enhanced NADH cofactor specificity was identified. Other KARI variants gave insights into Mr-KARI structure-function relationships.

KW - Biocatalysis

KW - Cell-free

KW - Isobutanol

KW - Ketol-acid reductoisomerase

KW - Meiothermus ruber

KW - Thermophilic enzymes

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JO - Biochimie

JF - Biochimie

ER -

Identification and optimization of a novel thermo- and solvent stable ketol-acid reductoisomerase for cell free isobutanol biosynthesis

Abstract

Keywords

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