Affiliation:
1. Artie McFerrin Department of Chemical Engineering
2. Department of Biology
3. Zachry Department of Civil and Environmental Engineering, 220 Jack E. Brown Building, Texas A&M University, College Station, Texas 77843-3122
Abstract
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 σ
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.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
39 articles.
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