PMID:19581479

Citation	Sanchez-Torres, V, Maeda, T and Wood, TK (2009) Protein engineering of the transcriptional activator FhlA To enhance hydrogen production in Escherichia coli. Appl. Environ. Microbiol. 75:5639-46
Abstract	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.
Links	PubMed PMC2737898 Online version:10.1128/AEM.00638-09
Keywords	Artificial Gene Fusion; DNA Mutational Analysis; Escherichia coli/metabolism; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Formates/metabolism; Gene Expression Profiling; Gene Expression Regulation, Developmental; Gene Order; Genes, Reporter; Hydrogen/metabolism; Mutant Proteins/genetics; Mutant Proteins/metabolism; Mutation, Missense; Polymerase Chain Reaction/methods; Protein Engineering; Trans-Activators/genetics; Trans-Activators/metabolism; Up-Regulation; beta-Galactosidase/genetics; beta-Galactosidase/metabolism
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Growth medium for transcription profiling experiments: Cultures were grown in modified complex medium (Maeda et al. 2008) supplemented with 20 mM formate and Cm (30 ug/ml). I tracked the description of the medium from Maeda et al. 2008 to Rachman et al. 1997 (doi:10.1016/S0922-338X(97)80142-0).

The modified complex-formate medium contained (per liter):

• 100 mM formate
• 5.0 g yeast extract
• 5.0 g tryptone
• 7.0 g K₂HPO₄
• 5.5 g KH₂PO₄
• 0.5 g L-cysteine-HCl-H₂O
• 1.0 g (NH₄)₂SO₄
• 0.25 g MgSO₄^.7H₂O
• 0.012 g CaCl₂^.2H₂O
• 0.029 g Co(NO₃)₂^.6H₂O
• 0.039 g Fe(NH₄)₂SO₄^.6H₂O
• 2.0 mg nicotinic acid
• 0.172 mg Na₂SeO₃
• 0.02 mg NiCl₂
• 10 ml trace element solution (0.5 g MnCl₂^.4H₂O, 0.1 g H₃BO₃, 0.01 g AlK(SO₄)₂^.H₂O, 0.001 g CuCl₂^.2H₂O, and 0.5 g Na₂EDTA per liter)

Note: Rachman et al.'s original complex medium contained 20 g glucose per liter.

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