Increased Hydrogen Production by Genetic Engineering of Escherichia coli - Genetic Engineering: Recent Developments in Applications - page 2

transcriptional regulator protein FNR at levels above natural abundance had
a synergistic effect on increasing the hydrogen yield in the ∆ focA genetic back-
ground. The modification of global transcriptional regulators to modulate the
expression of multiple operons required for the biosynthesis of formate hydro-
genlyase represents a practical approach to improve hydrogen production.
introduction
The present emphasis on the diversification of global energy sources, while reduc-
ing the consumption of fossil fuels, has placed renewed interest on hydrogen as
an alternative fuel and its synthesis through microbe-mediated bioconversions.
Microbes have evolved unique mechanisms for hydrogen generation, and some of
these mechanisms are being explored for biotechnological applications including
nitrogenase-mediated and fermentative hydrogen production [1]. Efforts to im-
prove hydrogen production have been focused on pathway redirection, identifica-
tion and engineering of oxygen-tolerant hydrogenases, improvement in hydrogen
molar yields and development of efficient hydrogen separation techniques from
bioreactor headspace [1]-[5].
The production of hydrogen by facultative anaerobic organisms such as E. coli
is a characteristic of mixed acid fermentation. Under anaerobic conditions, the
fermentation products comprise a mixture of ethanol, succinate, lactate, acetate,
and formate (Fig. 1). Succinate is produced via the succinate pathway. A key reac-
tion in this pathway is carboxylation of phosphoenolpyruvate (PEP) to the four-
carbon compound oxaloacetate by phosphoenolpyruvate carboxylase (PEPC) [6].
PEP is also converted to pyruvate which is subsequently converted to acetyl-CoA
and formate by pyruvate formate lyase (PFL), and to lactate by lactate dehydro-
genase (LDH). Formate hydrogenlyase (FHL) catalyzes the conversion of for-
mate to carbon dioxide and hydrogen [7], [8]. The intracellular level of formate
is determined by the rates of biosynthesis and metabolism of formate, and also
by formate transporter FocA which is a membrane protein involved in formate
transport into and out of the cell [9]. Knockout of the focA gene results in intra-
cellular accumulation of formate [10]. The FHL enzyme complex is comprised of
formate dehydrogenase H (FDHH), and a hydrogen-evolving hydrogenase (hy-
drogenase 3) [11], [12]. The biosynthesis of PFL and FHL are up-regulated by the
action of multiple transcriptional regulators including the global transcriptional
factors Fnr, ArcAB and integration host factor (IHF) [13]-[15], and repressed by
the dual transcriptional regulator NarL (Table 1)[16]. The transcription of the fhl
regulon is regulated by the primary and secondary transcriptional activator FHLA
and ModE [17], [18]. The expression of fhlA is activated by Fnr in response to
the cellular redox state [19], [20]. The biosynthesis of FDHH also requires the