| Summary: | We demonstrate that hydrogen production can be increased by random mutagenesis
using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and that hydrogen production can be
further increased in the chemically-mutagenized strain by targeted gene deletion and
overexpression of genes related to formate metabolism. Chemical mutagenesis of Escherichia
coli BW25113 hyaB hybC hycE::kan/pBS(Kan)-HycE to form strain 3/86 resulted in 109 +/- 0.5-
fold more hydrogen; 3/86 lacks functional hydrogen uptake hydrogenases 1 and 2, has hydrogenproducing
hydrogenase 3 inactivated from the chromosome, and has constitutively active
hydrogenase 3 based on expression of the large subunit of hydrogenase 3 from a high copy
number plasmid. Deleting fdoG, which encodes formate dehydrogenase O, (that diverts formate
from hydrogen), from chemical mutagen 3/86 increased hydrogen production 188 +/- 0.50-fold
(relative to the unmutagenized strain), and deletion of hycA, which encodes the repressor of
formate hydrogen lyase (FHL), increased hydrogen production 232 +/- 0.50-fold. Deleting both
fdoG and hycA increased hydrogen production 257 +/- 0.50-fold, and overexpressing fhlA along
with the fdoG hycA mutations increased hydrogen 308 +/- 0.52-fold. Whole-transcriptome
analysis of chemical mutagen 3/86 revealed 89 genes were induced and 31 genes were repressed.
In an effort to identify chromosomal mutations in chemical mutagen 3/86, we performed
comparative genome sequencing and identified two chromosomal loci with mutations in coding regions of ftnA and yebJ; however, neither gene was related to the increased hydrogen
production as determined by the close vial (short) hydrogen assay.
In addition, transposon mutagenesis, which is one of the most efficient strategies for
creating random mutations in the genomic DNA, was performed in two different strains: E. coli
BW25113 hyaB hybC hycA fdoG::kan/pCA24N-FhlA and E. coli MG1655 to identify beneficial
mutations for hydrogen production. As a result of screening 461 E. coli BW25113 hyaB hybC
hycA fdoG::kan/pCA24N-FhlA transformants and 1000 E. coli MG1655 transformants, three
interesting mutations have been discovered in E. coli BW25113 hyaB hybC hycA
fdoG::kan/pCA24N-FhlA transformants (gpsA, dipZ, glgP) and 1 beneficial mutation in E. coli
MG1655 transformants (malT). When any of these genes gpsA, dipZ, or glgP is disrupted by Tn5
insertion, hydrogen production decreases 17, 3 and 8-fold, respectively. Additionally, when malT
gene is disrupted by Tn5 insertion, hydrogen increases 3.4-fold.
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