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Applied and Environmental Microbiology, September 2009, p. 5639-5646, Vol. 75, No. 17
0099-2240/09/$08.00+0 doi:10.1128/AEM.00638-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Protein Engineering of the Transcriptional Activator FhlA To Enhance Hydrogen Production in Escherichia coli
,
Viviana Sanchez-Torres,1
Toshinari Maeda,1,
and
Thomas K. Wood1,2,3*
Artie McFerrin Department of Chemical Engineering,1 Department of Biology,2 Zachry Department of Civil and Environmental Engineering, 220 Jack E. Brown Building, Texas A&M University, College Station, Texas 77843-31223
Received 18 March 2009/ Accepted 25 June 2009
Escherichia coli produces H2 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 CO2 from formate and hydrogenase 3 (encoded by hycGE) for synthesizing H2 from 2H+ and 2e–. FHL protein production is activated by the 
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 H2 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 PfdhF and Phyc is less sensitive to formate regulation, and with FhlA363 (E363G), Phyc transcription increases but Phyp transcription decreases and hydrogen production is less affected by the repressor HycA.
* Corresponding author. Mailing address: Artie McFerrin Department of Chemical Engineering, 220 Jack E. Brown Building, Texas A&M University, College Station, TX 77843-3122. Phone: (979) 862-1588. Fax: (979) 865-6446. E-mail: Thomas.Wood{at}chemail.tamu.edu
Published ahead of print on 6 July 2009.
Supplemental material for this article may be found at http://aem.asm.org/.
Present address: Department of Biological Functions and Engineering, Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-ku, Kitakyushu 808-0196, Japan.
Applied and Environmental Microbiology, September 2009, p. 5639-5646, Vol. 75, No. 17
0099-2240/09/$08.00+0 doi:10.1128/AEM.00638-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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