Biobutanol - Biotechnology in China: Biofuels and Bioenergy - page 90

C. acetobutylicum SMB009 [ 10 ], using the ClosTron system based on group II
intron insertion. The resulting strain SMB009 lost the type II restriction endonu-
clease activity, and can be transformed with unmethylated DNA as efficiently as
with methylated DNA.
In previous studies, the methylation step is inevitable for transforming foreign
DNA into C. acetobutylicum because of the presence of a type II restriction
endonuclease Cac824I [ 35 ]. The strategy developed by Dong et al. [ 10 ] makes it
easier to genetically modify the clostridial species using unmethylated DNA, and
improves the manipulation efficiency significantly. This will help to advance the
understanding of the clostridial physiology from the molecular level, and to
expand the application of Clostridium strains for medical and biotechnological
purposes.
2.2.4 Electrotransformation of C. acetobutylicum in Air
Clostridia are typically strict anaerobes. The manipulation of C. acetobutylicum is
usually performed in an anaerobic chamber, and is thus laborious and time-
consuming. Dong et al. reported work to demonstrate that electrotransformation of
C. acetobutylicum in air is feasible [ 9 ]. The CAC2634 gene encoding PerR is a
known peroxide regulon repressor. They disrupted CAC2634 in their previously
constructed RM system-deficient C. acetobutylicum mutant SMB009 using the
group II method. The resulted mutant SMB012 was proved to be electrotrans-
formable in air with an efficiency of 1.2-3.1 9 10 3 transformants/lg DNA. The
electrotransformation process of C. acetobutylicum could be significantly simpli-
fied, especially when operating multiple electrotransformations.
2.3 Strain Improvement
2.3.1 Metabolic Engineering
Although some microorganisms such as Escherichia coli, yeast, Lactobacillus
brevis, and Cyanobacteria have been engineered to produce biobutanol, clostridial
strains are still the main butanol-forming strains, due to their excellent fermen-
tation characteristics. Butanol constitutes 60-70% (w/w) of the total ABE solvents
[ 26 , 53 , 54 ], and eliminating the production of acetone or ethanol was expected to
increase butanol ratio. Jiang et al. reported their work on weakening the acetone
pathway to increase the butanol ratio [ 24 ]. Acetoacetate decarboxylase (AADC)
encoded by the adc gene is one of the key enzymes to form acetone. They dis-
rupted the adc gene using TargeTron technology in C. acetobutylicum EA2018
[ 45 ]. The adc-disrupted mutant strain 2018adc showed an increased butanol ratio
from 70 to 80.05%, with an decreased acetone titer of 0.21 g/L. However, the
growth of 2018adc is inhibited and the titers of ethanol and butanol are reduced to