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.