3.3 Improving glutarate by acetate uptake pathway
The strain Bgl41464 tried to uptake acetate directly from the medium for
furthermore enhancing glutarate titer. Before that, the natural acetate
degradation way was explored individually. We firstly tested the growth
of Bgl4146 under different acetate concentrations (Fig. S2). The acetate
growth and degradation were measured in M9 medium (pH 7.0) supplemented
with acetate as the only carbon source. But 3 g/L and 6 g/L acetate
could be consumed completely about 48h and 96h, respectively. In order
to grow better and accelerate the consumption of acetate, we cultivated
Bgl41464 in the fermentation medium with and without glucose (Fig. 4A
and 4B). In order to improve the content of acetyl-CoA, the competition
route of acetic acid was destroyed by eliminating the ackA gene
and the acetate synthesis pathway was destroyed by knockout of thepoxB gene for obtaining more acetyl-CoA. Because Zhu et alfound that a combination of the three manipulations (deletion ofackA , overexpression of acs , accBC anddtsR1 ) caused a 16.3-fold increasing in the intracellular
malonyl-CoA level than that in wild-type E.
coli (Zhu et al., 2014). Therefore, we
explored the impact of the deletions of acetate synthesis pathway
(ackA or poxB ) for glutarate production in E. coli(Fig. 4A and 4B). We found that the glutarate production with glucose
fermentation was about three times than without glucose fermentation.
And the glutarate production reached a maximum of 0.70 g/L under 6 g/L
acetate with strain Bgl51464 (BL21(DE3) ΔarcA ΔldhAΔatoB ΔpflB ΔackA carrying pAD1, pAD4, pAD6 and
pACC4).