3.4 Glutarate production in 5-L bioreactor
The best strain Bgl51464 was fed-batch fermentation to improve glutarate
production using the glucose, acetic acid and glycerol as a mixture
carbon source (Fig. 5). We finally obtained a 7.97 g/L maximal glutarate
at 63 h, representing a 16.27-fold higher than that of strain Bgl41464
with microaerobic condition in shake flask (Fig. 5). And the
OD600 reached a maximum of 24.3 at 113.5 h. After 30 h
of fermentation, the acetate synthesis and consumption were kept in a
dynamic balance (Fig. 5).
Discussion
Glutaric acid, an important C5 compound, is used in industrial
production. Our team has previously used the reversed adipic acid
degradation pathway (RADP) to produce adipate and glutarate in E.
coli (Sui et al., 2020;
Zhao, Huang, et al., 2018;
Zhao, Li, & Deng, 2018). In this work we
used the previous RADP pathway to enhance glutarate production based onE. coli Bgl4146(Zhao, Li, & Deng,
2018). The precursors of glutaric acid were acetyl-CoA and malonyl-CoA.
In order to increase the precursors of glutarate production, these
methods applied to enhance the precursors were described: 1)
overexpression and optimization of ACC (acetyl-CoA carboxylase) from
different strains to enhance the transformation of acetyl-CoA to
malonyl-CoA; 2) overexpression of acs (acetyl-CoA synthase) to
promote the decomposition of acetate into acetyl-CoA; 3) Combination of
ACC and acs simultaneously to increase 2 CoA precursors for
glutarate output; 4) Knockout of ackA or poxB to decrease
acetate titer. The best strain Bgl51464 produced 7.97 g/L glutarate by
fed-batch fermentation. This can be used as an effective method for the
production of CoA-derived other high value-added chemicals.
The glutarate production was closely related to the dissolved
oxygen(J. L. Yu, X. X. Xia, J. J. Zhong,
& Z. G. Qian, 2017; Zhao, Li, & Deng,
2018). The glutarate titers varied under different dissolved oxygen
levels and microaerobic condition could be suitable to produce glutarate
(Fig. 3). The synthetic precursors of glutaric acid were malonyl-CoA and
acetyl-CoA. Partially impairing the fatty acid pathway and tricarboxylic
acid cycle by microaerobic condition could well drive acetyl-CoA and
malonyl-CoA toward glutarate production while sustaining certain cell
growth and metabolism requirements(Brose,
Golovko, & Golovko, 2016; Zhao, Li, &
Deng, 2018). At the same time, the production of acetic acid was also
affected by dissolved oxygen during the fermentation process and
relatively high in microaerobic condition (Fig.
S1)(Akesson et al., 2001). And the
over-accumulation of the metabolic by-product acetate inhibited cell
growth(Luli & Strohl, 1990;
Nakano et al., 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 compounds
(Mazumdar, Clomburg, & Gonzalez, 2010;
Wolfe, 2005). On the premise of glutarate
production as the first principle, we need to balance the acetate
production in the fermentation. Then, acetyl-CoA synthase (encoded byacs ) was introduced to consume acetate. We finally found that
Bgl41464 was better than other strains and glutarate production reached
0.49 g/L under microaerobic condition (Fig. 3).
The balance between acetyl-CoA and malonyl-CoA was crucial for glutarate
production. Firstly, the acetyl-CoA was transformed to malonyl-CoA by
overexpressing ACC (acetyl-CoA carboxylase). As shown in Fig. 3,
overexpression of ACC (acetyl-CoA carboxylase,accABCD )(My et al., 2013;
Xu et al., 2014) gene from E. coliby Bgl41461 had a certain negative effect on the glutarate production.
And the maximum glutarate titer by Bgl41461 was 0.34 g/L under
microaerobic conditions, which was 0.82-fold that of the control Bgl4146
(Fig. 3). However overexpression of the ACC gene from C.
glutamicum consisting of two subunits, accBC anddtsR1 (Gande et al., 2007;
Miyahisa et al., 2005) alone by Bgl41462
had a great influence on the glutarate production. And the maximum
glutarate titer by Bgl41462 was 0.44 g/L under microaerobic conditions,
which was 1.06-fold that of the control Bgl4146 (Fig. 3). Though the
glutarate titer was improved by overexpression of accBC anddtsR1 in shake flask, it seemed that only increasing the content
of malonyl-CoA was not sufficient for the synthesis of glutaric acid.
Therefore, the acetyl-CoA synthase (encoded by acs ) was
introduced to decompose the metabolic by-product acetic acid to produce
acetyl-CoA. Overexpression of acs , accBC and dtsR1by Bgl41464 increases the maximum titer of glutaric acid to 0.49 g/L
under microaerobic conditions, which was 1.18-fold higher than the
control Bgl4146 (Fig. 3). This combination balanced intracellular CoA
and increased glutarate production.
Acetate can be used as a potential substrate for production of
CoA-derived valuable compounds. However, the ability to digest acetate
was restricted in E. coli due to slow growth and low recombinant
protein expression(Wolfe, 2005). Theacs pathway (overexpression of acetyl-CoA synthetase) and the
reversible pta -ackA pathway (overexpression of
phosphotransacetylase/acetate kinase) were two natural roads for
digesting acetate in E. coli(Wolfe, 2005), the acs pathway was
the best strategy for acetate
decomposition(Lin et al., 2006;
Zha et al., 2009). Therefore, theacs was overexpressed by Bgl41464, resulting in 0.49 g/L
glutarate (Fig. 3). In order to improve the titer, we optimized the
engineering strains and added acetate to the medium. The Bgl51464
finally was fermented and obtained 0.70 g/L glutarate with 6 g/L acetate
in this study while the highest glutarate titer was 0.56 g/L when 10.4
g/L malonic acid was supplemented (Sui et
al., 2020; Thuronyi, Privalsky, & Chang,
2017; Walker et al., 2013;
Wu et al., 2013). Compared with the
previous glutarate fermentation by the uptake of malonate, the uptake of
acetate was cheaper, more efficient and obtained a higher titer in the
shake flask. The acetate had many benefits as a potential fermentation
substrate: 1) Acetate had a cheap source and a variety of
routes(Xiao et al., 2013;
J. Yang & Nie, 2016); 2) Acetate was
easily soluble in water and easily transfers mass during fermentation;
3) Acetate was decomposed into acetyl-CoA, which can be used as an
important precursor for many expensive chemicals. While malonic acid was
a very valuable chemical for the synthesis of many flavors, fragrances,
and pharmaceuticals and it mainly was produced from cyanoacetic acid or
diethyl malonate(Pollak & Romeder,
2000). Importantly, the acetate uptake increased the all precursors of
glutarate (acetyl-CoA and malonyl-CoA) while malonate uptake could only
increase the glutarate precursor, malonyl-CoA. This may be the key
reason for the difference in glutarate titer. Therefore, regardless of
the availability of the added carbon source or the final effect on the
glutarate production, acetate would be a good carbon source for the
fermentation of chemicals.
Acknowledgements
This work was supported by the
National Key R&D Program of China (2019YFA0905502), the
National Natural Science Foundation
of China
(21877053),
the Top-Notch Academic Programs Project of Jiangsu Higher Education
Institutions (TAPP), the National First-class Discipline Program of
Light Industry Technology and Engineering (LITE2018-24), the Fundamental
Research Funds for the Central Universities (JUSRP51705A).
Competing financial interests
The authors declare no competing financial interests.