Increased Hydrogen Production by Genetic Engineering of Escherichia coli - Genetic Engineering: Recent Developments in Applications - page 7

Table 2. Hydrogen yields for strain W3110 with or without defined chromosomal gene disruptions.
The specific hydrogen yield of strain ZF2 (W3110∆hybC) was 22% higher
than W3110 (Table 2). Of the three E. coli uptake hydrogenases, hydrogenase
2 is believed to be the more active one based on in vitro kinetic studies [8]. The
double mutant ZF5 (W3110∆focA∆hybC) did not produce more hydrogen than
the single mutants ZF1 (W3110∆focA) and ZF2 (W3110∆hybC) for reasons
that remain unclear. Inactivation of the metabolic enzyme PEPC resulted in ap-
proximately 14% increase in specific hydrogen yields.
If all of the pyruvate is directed towards the PFL reaction the theoretical maxi-
mum yield of hydrogen from a hexose sugar is 2 mols of H2/mol glucose [7].
Strain ZF3 (W3110∆narL) also displayed the best molar yield of hydrogen (mols
of H2/mol of glucose) of approximately 0.96 compared to 0.54 for W3110 (Ta-
ble 2). The utility of eliminating transcriptional repression of multiple operons
in anaerobic metabolism is highlighted by the effects of the narL gene deletion.
This deletion confers a 1.5-fold increase in specific hydrogen yield and a 2-fold
increase in molar hydrogen yield, while retaining the growth characteristics of the
wild-type strain W3110.
To examine the partitioning of glucose in the two high hydrogen yielding
strains the distribution of fermentation products of strains ZF1 (W3110∆focA)
and ZF3 (W3110∆narL) were compared to that of W3110 (Table 3). These two
strains carried out a balanced fermentation, with over 90% of the added glucose
converted to products. No remaining glucose was detected in the medium by
HPLC and spectrophotometric assays. Medium from strain ZF1 (W3110∆focA)
contained 40% less formate compared to strains W3110 and ZF3 (W3110∆narL),
consistent with less formate being secreted from the cell due to the disruption
of the channel for formate. Lactate formation was increased by about 37% in
strain ZF1 (W3110∆focA) compared to W3110, perhaps from the efficient in-
duction of ldhA due to lower intra-cellular pH from formate accumulation, or by
the inhibition of PFL activity and/or biosynthesis by formate. The formation of
ethanol was lowered by 17% and 33% in strains ZF1 (W3110∆focA) and ZF3
(W3110∆narL), respectively, compared to that of W3110. These two strains ac-
cumulated succinate and acetate to levels similar to those of strain W3110.