Biobutanol - Biotechnology in China: Biofuels and Bioenergy - page 91

half those of its control strain 2018p (EA2018 bearing pIMP1-Pptb vector) in P2
medium. In addition, the authors found that regulating the culture pH by adding
calcium carbonate can increase the butanol titer of strain 2018adc. Regulating the
in-vivo electron flow in this strain by adding methyl viologen could also increase
the yield of butanol from 57 to 70.8%. This work is believed to the first example of
a TargeTron-based knockout mutant of C. acetobutylicum defective in central
fermentative metabolism [ 28 ].
2.3.2 Engineering the Robustness of C. acetobutylicum
To improve the aero- and solvent tolerance of C. acetobutylicum, Zhu et al. cloned
and over-expressed the gshAB genes from E. coli into C. acetobutylicum
DSM1731 to biosynthesize glutathione (GSH), and thus increased the robustness
of C. acetobutylicum and achieved better solvent production [ 22 ]. The gshA
gene encodes c-glutamylcysteine synthetase (c-GCS) and the gshB gene encodes
glutathione synthetase (GS) in E. coli, via which glutathione is synthesized.
Zhu et al. constructed plasmids pITA, pITB, and pITAB on the base of parent plasmid
pITF [ 10 ] so as to introduce gshA, gshB, and gshAB into C. acetobutylicum
DSM1731, respectively. Biosynthesis of GSH in C. acetobutylicum DSM1731
increased the aerotolerance and butanol-producing capability, and decreased the
growth inhibition of butanol. Strain DSM1731(pITAB) produced 19.7 g/L total
solvents, including 14.8 g/L butanol, 3.7 g/L acetone, and 1.2 g/L ethanol in 64 h of
fermentation. Production of butanol by DSM1731_pITAB increased by 66 and 37%
when compared to the control strain DSM1731_pITB and wild-type strain
(DSM1731), respectively. It was postulated that GSH might be involved in scav-
enging reactive oxygen species (ROS) and maintenance of redox balance in cells
with concomitant improvement in aerotolerance. Furthermore, scavenging ROS
helps cells resist solvent, acid and osmotic pressure, thus increase the butanol
tolerance, which resulted in increased butanol production. This study showed for the
first time that GSH biosynthesis in C. acetobutylicum could increase the production
of butanol and improve the butanol tolerance. The results demonstrated that intro-
ducing this metabolic redundancy could improve the robustness of the host to achieve
better solvent production, which is one of the most important physiological func-
tionalities for efficient production of fuels and chemicals [ 55 ]. Therefore, strategies
of exploiting and engineering functional metabolic redundancy may also help
microbes to better adapt themselves to the industrial environments.
2.3.3 Improvement of Xylose Utilization
For converting lignocellulosic hydrolysate into biofuels, it is important for
C. acetobutylicum to utilize a variety of carbohydrates, including pentose and
hexose. Lignocellulose is the most abundant renewable resource, and D-xylose
is a significant component of the hydrolysate [ 20 , 58 ]. Making good use of