3.2 Improving glutarate production by increasing precursors
Due to the mutual cooperation of metabolism and regulatory network in
the cell, the biosynthesis of essential metabolites (such as CoA
derivative biosynthesis) was generally retained at a relatively stable
level and the derivative was rarely overexpressed
(Pitera, Paddon, Newman, & Keasling,
2007). Therefore, the metabolic fluxes resulting in the production of
the CoA precursors in RADP were considered as the optimization
objectives by metabolic engineering. Specifically, four strategies were
adopted in order to increase the usability of acetyl-CoA and malonyl-CoA
for glutarate production. These methods included 1) overexpression of
acetyl-CoA carboxylase (ACC) for the conversion of acetyl-CoA and
malonyl-CoA; 2) overexpression ofacs (acetyl-CoA synthase)
for acetate uptake pathway; 3) Blocking of the acetate competition
pathway by deleting the ackA (acetate kinase) gene; 4) Blocking
of the acetate synthesis pathway by deleting the poxB (pyruvate
dehydrogenase) gene.
It was worth noting that the dissolved oxygen levels had a crucial
effect on the glutarate production(J.-L.
Yu et al., 2017; Zhao, Li, & Deng,
2018). Subsequently, the different recombinant strains were fermented
under different dissolved oxygen conditions (aerobic condition,
microaerobic condition, anaerobic condition) for glutarate production
(Fig. 3). Overexpression of ACC (acetyl-CoA carboxylase, accABCD )
gene from E. coli by Bgl41461 changed the glutarate production
concentration by 0.93, 0.82, 0.99-fold than the control strain Bgl4146
under same aerobic condition, microaerobic condition and anaerobic
condition, respectively. And the maximum glutarate titer by Bgl41461 was
0.34 g/L under microaerobic conditions. Overexpression of ACC (two
subunits of acetyl-CoA carboxylase, accBC and dtsR1 ) fromC. glutamicum (Gande et al., 2007;
Miyahisa et al., 2005) alone Bgl41462
changed the glutarate production concentration by 1.27, 1.06, 0.78-fold
than the control Bgl4146 under same aerobic condition, microaerobic
condition and anaerobic condition, respectively. And the maximum
glutarate titer by Bgl41462 was 0.44 g/L under microaerobic conditions.
The glutarate titer was better improved on Bgl41462 by expressingaccBC and dtsR1 and microaerobic condition was useful for
glutarate fermentation. But we found the production of acetic acid was
affected by dissolved oxygen during the fermentation process and the
by-product acetate titer was relatively high in microaerobic condition
compare to other conditions in Bgl4146(Fig.
S1)(Akesson, Hagander, & Axelsson, 2001).
At the same time, the over-accumulation of the metabolic by-product
acetate inhibited cell growth(Luli &
Strohl, 1990; Nakano, Rischke, Sato, &
Märkl, 1997) and production of recombinant
proteins(Glazyrina et al., 2010;
Lee, 1996), which was one of the barriers
to achieving high yield and production of target compound.
In order to balance glutarate and acetate production, overexpressing the
single acs (acetyl-CoA synthase) gene in E. coli was
proven to be the best natural approach for acetate digestion
(Lin, Castro, Bennett, & San, 2006;
Zha et al., 2009). Therefore, we
introduced the acs from E. coli to degrade acetate to
increase the available acetyl-CoA in Bgl41463. As shown in Fig. 3,
overexpression of acs from E. coli only to digest acetic
acid into acetyl-CoA by Bgl41463 increasing the glutarate production by
0.94, 0.89, 1.05-fold than the control Bgl4146 under same aerobic
condition, microaerobic condition and anaerobic condition, respectively.
And the maximum glutarate titer by Bgl41463 was 0.37 g/L under
microaerobic conditions. However, those results were not ideal for only
increasing the content of intracellular acetyl-CoA or malonyl-CoA. Both
precursors of glutaric acid should be considered together to increase
glutarate production. Therefore, the well overexpressing accBCand dtsR1 was tried to co-express with acs to enhance the
glutarate production. Overexpression of acs , accBC anddtsR1 together by Bgl414164 increased the glutarate production
concentration by 1.38, 1.19, 1.54-fold than the control Bgl4146 under
same aerobic, microaerobic and anaerobic condition, respectively. And
the glutarate production had been greatly improved, reaching 0.49 g/L by
Bgl41464 under microaerobic condition. This may be due to the increase
in the content of acetyl-CoA and malonyl-CoA
(Zhu et al., 2014). The better production
strain Bgl41464 was applied for the further study under the microaerobic
condition.