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.