Introduction
Glutaric acid, a C5 chain dicarboxylic acid, is used as the building
block for synthesizing nylon and plasticizer
(J. Adkins, J. Jordan, & D. R. Nielsen,
2013; Bermúdez, León, Alemán, &
Muñoz-Guerra, 2000; W. Li et al., 2019;
Zhang, Gao, Guo, Guo, Kang, Xiao, Yan,
Tao, Zhang, & Dong, 2018). Currently, glutaric acid is prepared using
nitric acid oxidation and separated from a mixture of dicarboxylic
acids, but its yield is not high
(Castellan, Bart, & Cavallaro, 1991;
Nishikido, Tamura, & Fukuoka, 1979;
Niu, Draths, & Frost, 2002;
Polen, Spelberg, & Bott, 2013;
Sato, Aoki, & Noyori, 1998). The most
notable natural route for glutarate production was discovered inPseudomonas putida , which could utilize L-lysine degradation to
produce glutarate by the 5-aminovalerate (AMV)
pathway(JC & JR, 1977;
Revelles, Espinosa-Urgel, Fuhrer, Sauer,
& Ramos, 2005; Revelles, Wittich, &
Ramos, 2007; Zhang, Gao, Guo, Guo, Kang,
Xiao, Yan, Tao, Zhang, & Dong, 2018). In order to avoid the supply of
precursor lysine and improve the glutarate production, the lysine
overproducingCorynebacterium glutamicumoverexpressed the AMV pathway from P. putida to produce glutaric
acid in high titers and yields (J. Adkins,
J. Jordan, & D. R. Nielsen, 2013; Kim et
al., 2019; Maria, Gideon, Michael,
Christoph, & Judith, 2016; Rohles et
al., 2018; Shin et al., 2016). At the
same time, in order to better study the AMV pathway, those genes were
also heterologous expressed in Escherichia coli(J. Adkins, J. Jordan, & D. R. Nielsen,
2013; Park et al., 2013). The maximum
titer of 0.82 g/L for glutarate was reached after 48 hours of
fermentation (J. Adkins, J. Jordan, & D.
R. Nielsen, 2013). In order to obtain higher output, lysine and α-KG
were supplemented to the cell catalytic system resulting in 1.7 g/L
glutarate in E. coli WL3110
strain(Park et al., 2013). Recently,
glutarate titer was also greatly improved through whole cell
bio-catalyst or whole-cell immobilized
(Hong et al., 2018;
S.-Y. Yang et al., 2019). And Li et
al used the native lysine catabolic pathway in E. coli to
synthesize glutarate and finally achieved both high titer and high
yield(W. Li et al., 2019).
The above all results are based on AMV pathway or lysine carbolic
pathway. In addition to the AMV pathway, a variety of attempts for the
glutarate production and exploration were made in E. coli .
Recently, the reverse adipic acid degradation pathway (RADP) was
utilized to balance the production of adipate and glutarate and finally
achieved 4.8 g/L glutarate (Zhao, Huang,
et al., 2018; Zhao, Li, & Deng, 2018).
Then the glutarate in RADP was accumulated to 6.3 g/L by malonate
absorption(Sui et al., 2020). The RADP
seems to be a potential pathway to produce glutarate from glucose inE. coli . The RADP contained the following enzymes: Tfu_0875
(β-ketothiolase), Tfu_2399 (3-hydroxyacyl-CoA dehydrogenase), Tfu_0067
(3-hydroxyadipyl-CoA dehydrogenase), Tfu_1647
(5-carboxy-2-pentenoyl-CoA reductase) and Tfu_2576-7 (adipyl-CoA
synthetase) (Sui et al., 2020;
Zhao, Huang, et al., 2018;
Zhao, Li, & Deng, 2018) (Fig. 1). The
RADP could convert acetyl-CoA and malonyl-CoA to glutaric acid and
malonyl-CoA was crucial for the glutarate production. Therefore,
cerulenin was used for increasing the amount of malonyl-CoA available by
inhibiting fatty acid synthesis
pathway(Heath & Rock, 1995;
Rogers & Church, 2016;
Zhao, Li, & Deng, 2018). And matB(malonic acid synthetase) and matC (malonic acid carrier protein)
were overexpressed, absorb the malonate to improve the intracellular
malonyl-CoA(Sui et al., 2020;
Wu, Du, Zhou, & Chen, 2013). However,
only increasing the content of intracellular malonyl-CoA was not
sufficient for glutarate production. Both precursors of glutaric acid
should be considered to increase production.
The acetyl-CoA could be transformed to malonyl-CoA by catalyzing
acetyl-CoA carboxylase (ACC) (S. J. Li &
Cronan, 1992; Lussier, Colatriano,
Wiltshire, Page, & Martin, 2012; Xu, Li,
Zhang, Stephanopoulos, & Koffas, 2014;
Zhu, Wu, Du, Zhou, & Chen, 2014). But
acetyl-CoA mainly flew to the tricarboxylic acid (TCA) cycle to maintain
basic metabolism and growth, whereas only a minor proportion was used by
ACC to synthesize malonyl-CoA(Tokuyama et
al., 2019), and a diminutive proportion was engaged in fatty acid
synthesis(Zha, Rubin-Pitel, Shao, & Zhao,
2009) in E. coli. Therefore, it was important to balance the
supply and demand of acetyl-CoA and malonyl-CoA to flow to the target
product. Here, our research is based on the previous RADP pathway to
produce glutaric acid (Fig. 1). Firstly, we constructed a transformation
system of acetyl-CoA to malonyl-CoA for enhancing the available
intracellular malonyl-CoA in E. coli . Furthermore, the acetic
acid uptake pathway was introduced into the metabolic pathway and the
degradation pathway was knocked out to increase and balance
intracellular acetyl-CoA and malonyl-CoA. Meanwhile, the dissolved
oxygen was effectively controlled so that more carbon source flew to
CoA. Finally, the optimal strains, Bgl51464 was cultured in a 5‐L
fermenter.
- Materials and methods
- Strains and plasmids
The strains, plasmids, and
primers
are shown in Supplementary
Tables
S1 and S2. In order to promote the transformation of acetyl-CoA to
malonyl-CoA in vivo , theaccABCD (Xu et al., 2014) fromE. coli MG1655 was firstly linearized by PCR using accA-1 F and
accA-1 R, accB-1 F and accB-1 R, accC-1 F and accC-1 R and accD-1 F and
accD-1 R, respectively. Then the pACYCDuet-1 was digested usingBam H I and Avr II and all products were ligase by Gibson
assembly (Gibson et al., 2009) and
transformed into competent cells, forming strain pACC1. The accBCand dtsR1 were amplified from C.
glutamicum (Zhu et al., 2014) using
accBC-2 F and accBC-2 R and dtsR1-2 F and dtsR1-2 R primers,
respectively. The pACYCDuet-1 was linearized using kpn I andAvr II. The all products were assembled like pACC1, resulting in
strain pACC2. To increase the CoA in vivo , the acs fromE. coli MG1655 was amplified using acs-3 F and acs-3 R. Both the
pACYCDuet-1 and the acs fragment were digested using Bam H
I and Not I and ligated by T4 DNA ligase, naming pACC3. TheaccBC and dtsR1 were amplified from pACC2 using accBC-2 F
and dtsR1-2 R. The pACC3 plasmid was digested by kpn I andAvr II and all products were assembled, forming strain pACC4. All
above constructs were screened by colony PCR and Sanger sequencing using
veri-pACYC F and veri-pACYC R. All subsequent plasmid constructions
(packA, and ppoxB) also used the Gibson assembly method as described
above. The sgRNA target gene and knock gene template are shown in
Supplementary Table S3.