Increased Hydrogen Production by Genetic Engineering of Escherichia coli - Genetic Engineering: Recent Developments in Applications - page 4

the biosynthesis of structural polypeptides without placing an unnecessary meta-
bolic burden on the cells [25]. Here, we describe increases in specific and molar
yields of hydrogen achieved by the modification of the focA, ppc, narL, and fnr
genes involved in precursor transport and metabolism, and the global regulation
of fermentative hydrogen production by E. coli.
results and discussion
Strategy for Genetic Modification
The fermentation pathways initiate by the non-oxidative cleavage of pyruvate to
acetyl-CoA and formate by PFL; formate is subsequently metabolized to hydro-
gen and carbon dioxide by FHL. Our strategy for improving fermentative hydro-
gen production involved the modification of precursor transport and metabolism,
and of regulatory elements, to direct the flow of the two key precursor metabolites
pyruvate and formate towards hydrogen, and achieve increased expression of both
structural and auxiliary components of the FHL enzyme system. The biochemi-
cal reactions and regulatory elements that were modified by the inactivation of
specific chromosomal genes, or by plasmid-directed gene over-expression are illus-
trated schematically in Figure 1; the functions of the corresponding gene products
are summarized in Table 1.
The inactivation of the four selected genes in the chromosome of E. coli strain
W3110 was hypothesized to increase the cellular concentrations of pyruvate and
formate, reduce the re-oxidation of evolved hydrogen, and relieve the repression
of genes required for PFL and FHL biosynthesis. The ppc gene encoding phos-
phoenolpyruvate carboxylase (PEPC) was disrupted to increase cellular pyruvate
concentrations available for formate biosynthesis [6], [7]. The gene encoding the
formate transporter, focA, was inactivated to elevate intra-cellular formate levels
for FHL biosynthesis and conversion to hydrogen [10]. E. coli synthesizes two
uptake hydrogenases, hydrogenase1 and 2, which oxidize hydrogen to protons
with the release of electrons. In this study the hybC gene, which encodes the large
catalytic subunit of hydrogenase 2, was inactivated to reduce the oxidation of
hydrogen [26]. The global transcriptional regulator NarL represses transcription
of the structural genes of FHL (fdhF), PFL (pflB), and of the genes of the nik
operon, which are required for the transport and metabolism of nickel for Hydro-
genase cofactor biosynthesis [16]. To eliminate the repression described, the narL
gene was inactivated.
The global transcriptional regulators Fnr, ArcAB and IhfAB activate multiple
genes of anaerobic pyruvate and formate metabolism. Targets for these global
regulators include fhlA (the specific transcriptional activator of the fhl regulon),