| Protein engineering of the transcriptional activator FhlA To enhance hydrogen production in Escherichia coli. | |
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MedLine Citation:
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PMID: 19581479 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Escherichia coli produces H(2) from formate via the formate hydrogenlyase (FHL) complex during mixed acid fermentation; the FHL complex consists of formate dehydrogenase H (encoded by fdhF) for forming 2H(+), 2e(-), and CO(2) from formate and hydrogenase 3 (encoded by hycGE) for synthesizing H(2) from 2H(+) and 2e(-). FHL protein production is activated by the sigma(54) transcriptional activator FhlA, which activates transcription of fdhF and the hyc, hyp, and hydN-hypF operons. Here, through random mutagenesis using error-prone PCR over the whole gene, as well as over the fhlA region encoding the first 388 amino acids of the 692-amino-acid protein, we evolved FhlA to increase H(2) production. The amino acid replacements in FhlA133 (Q11H, L14V, Y177F, K245R, M288K, and I342F) increased hydrogen production ninefold, and the replacements in FhlA1157 (M6T, S35T, L113P, S146C, and E363K) increased hydrogen production fourfold. Saturation mutagenesis at the codons corresponding to the amino acid replacements in FhlA133 and at position E363 identified the importance of position L14 and of E363 for the increased activity; FhlA with replacements L14G and E363G increased hydrogen production (fourfold and sixfold, respectively) compared to FhlA. Whole-transcriptome and promoter reporter constructs revealed that the mechanism by which the FhlA133 changes increase hydrogen production is by increasing transcription of all of the genes activated by FhlA (the FHL complex). With FhlA133, transcription of P(fdhF) and P(hyc) is less sensitive to formate regulation, and with FhlA363 (E363G), P(hyc) transcription increases but P(hyp) transcription decreases and hydrogen production is less affected by the repressor HycA. |
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Authors:
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Viviana Sanchez-Torres; Toshinari Maeda; Thomas K Wood |
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Publication Detail:
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Type: Journal Article; Research Support, U.S. Gov't, Non-P.H.S. Date: 2009-07-06 |
Journal Detail:
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Title: Applied and environmental microbiology Volume: 75 ISSN: 1098-5336 ISO Abbreviation: Appl. Environ. Microbiol. Publication Date: 2009 Sep |
Date Detail:
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Created Date: 2009-08-28 Completed Date: 2009-10-22 Revised Date: 2010-09-27 |
Medline Journal Info:
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Nlm Unique ID: 7605801 Medline TA: Appl Environ Microbiol Country: United States |
Other Details:
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Languages: eng Pagination: 5639-46 Citation Subset: IM |
Affiliation:
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Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, TX 77843-3122, USA. |
| Data Bank Information | |
Bank Name/Acc. No.:
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GEO/GSE13902 |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Artificial Gene Fusion DNA Mutational Analysis Escherichia coli / metabolism* Escherichia coli Proteins / genetics*, metabolism* Formic Acids / metabolism Gene Expression Profiling Gene Expression Regulation, Developmental Gene Order Genes, Reporter Hydrogen / metabolism* Mutant Proteins / genetics, metabolism Mutation, Missense Polymerase Chain Reaction / methods Protein Engineering* Trans-Activators / genetics*, metabolism* Up-Regulation beta-Galactosidase / genetics, metabolism |
| Chemical | |
Reg. No./Substance:
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0/Escherichia coli Proteins; 0/Formic Acids; 0/Mutant Proteins; 0/Trans-Activators; 131689-36-6/fhlA protein, E coli; 1333-74-0/Hydrogen; 64-18-6/formic acid; EC 3.2.1.23/beta-Galactosidase |
| Comments/Corrections | |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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