Protein Engineering of the Transcriptional Activator FhlA To Enhance Hydrogen Production in Escherichia coli

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 σ⁵⁴ 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.