Introduction Corynebacterium L 1 2 4 L 2 5 6 7 8 9 10 3 11 12 11 Escherichia coli Saccharomyces cerevisiae 12 13 14 Corynebacterium glutamicum 4 + C. glutamicum 15 C. glutamicum 16 17 18 C. glutamicum 17 19 3 3 C. glutamicum Materials and methods Microorganism and fermentation conditions Corynebacterium glutamicum 9114 3 Corynebacterium glutamicum 9114 Analytical methods 3 2 2 2 20 Extraction and assay of the GDH and LDH 1 \documentclass[12pt]{minimal}
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\begin{document}$$\alpha\hbox{-}{\hbox{ketoglutarate}}({\hbox{a-KG}}) + {\hbox{NH}}^{ + }_{4} + {\hbox{NADPH}}{\mathop \Leftrightarrow \limits^{{\text{GDH}}} }{\hbox{Glutamate}}+{\hbox{NADP}},$$\end{document} 2 \documentclass[12pt]{minimal}
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\begin{document}$${\hbox{Pyruvate}} + {\hbox{NADH}}{\mathop \Leftrightarrow \limits^{{\text{LDH}}} }{\hbox{Lactate}} + {\hbox{NAD}}.$$\end{document} g 4 On-line control system 3 3 \documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} & {\hbox{AGT}}(k) = {\hbox{AGT}}(k - 1) + C_{0} {\left[ {{\hbox{RQ}}(k) - {\hbox{RQ}}_{\hbox{set}} } \right]} + C_{1} {\left[ {{\hbox{RQ}}(k - 1) - {\hbox{RQ}}_{\hbox{set}} } \right],} \\ & \hbox{where}\quad C_{0} = K_{C} {\left({1 + \frac{1}{{\tau _{\rm I} }}} \right)}\quad C_{1} = - K_{C} \\ \end{aligned} $$\end{document} 3 k set K C I C I Results and discussion The changing patterns of glutamate and lactate production, as well as the GDH and LDH activities at different DO control levels 1 Fig. 1 a filled circle filled triangle open circle open triangle b filled circle open circle filled triangle open triangle c filled circle filled triangle d filled circle filled triangle  1 1 The changing patterns of glutamate and lactate production, as well as the GDH and LDH activities at anaerobic fermentation condition 2 2 Fig. 2 a filled circle open triangle solid line b filled circle open triangle  The glucose consumption rates (glycolysis rates) at different DO control levels 3 Fig. 3 Open circle open triangle  The mechanism analysis of lactate overflow and the control strategy of BMC C. glutamicum 3 4 r 5 2 r 6 1 4 r 4 5 Fig. 4 Broken line solid line bold solid line a b c  Fig. 5 Comparison of RQ and calculated TCA metabolic flux rate at different DO control levels  1 3 r 1 r 6 r 5 r 4 2 1 4 5 r 1 r 6 r 1 r 4 r 5 r 6 r 4 r 1 4 Possibility of using and realizing the BMC strategy r 6 r 4 r 1 r 4 5 r 4 2 2 2 2 5 The performance and experimental result of the BMC strategy 6 K C I 6 set set set set Fig. 6 Filled circle open triangle open circle filled square  2 r 4 7 r 4 Fig. 7 The calculated TCA metabolic fluxes of the BMC strategy and the DO constant controls  1 2 2 r 4 2 2 4 r 4 Table 1 The materials and carbon balances of the balanced metabolic control (BMC) and dissolved oxygen (DO) constant controls Batch no. Glucose consumed (g) Glutamate produced (g) Lactate produced (g) a 2 b 2 DO = 10% 839.2 384.0 102.0 90.8 352.9   C-content 335.6 156.7 40.8 46.9 96.2 101.5 28.6 DO = 50% 530.2 265.2 3.2 77.1 262.9   C-content 212.1 108.2 1.3 39.8 71.7 104.2 33.9 RQ-BMC 716.7 406.4 0.4 60.7 335.9   C-content 286.7 165.9 0.18 31.4 91.6 100.8 31.9 a C. glutamicum 4.71 8.02 1.92 b 2 2 Table 2 The summarized results of the BMC and DO constant control Batch no. Conversion rate (%) Glutamate concentration (g/L) Lactate concentration (g/L) Productivity (g/L/h) Constant DO controls  050331 (DO = 10%) 49.38 83.00 27.90 2.86  050526 (DO = 10%) 43.62 91.50 25.50 2.69  DO = 10%, average 46.50 87.25 26.45 2.77  050407 (DO = 50%) 44.51 74.20 1.00 2.56  050512 (DO = 50%) 53.40 72.80 0.80 2.43  DO = 50%, average 48.96 73.50 0.90 2.49  050427 (DO = 30%) 55.55 83.20 18.60 2.19 Balanced metabolic control by RQ (RQ-BMC)  050509 (RQ-BMC) 56.71 101.60 0.11 2.99  050516 (RQ-BMC) 49.80 98.80 1.04 2.60  RQ-BMC, average 53.26 100.20 0.58 2.80 Summary A novel fermentation optimization method—the “balanced metabolic control” (BMC) strategy was proposed and successfully used by feedback controlling RQ to regulate the TCA metabolic flux rate at an appropriate level to achieve the metabolic balance among glycolysis, glutamate synthesis, and TCA metabolic flux for glutamate fermentation. The proposed BMC strategy greatly improved the fermentation performance in the two terms of maximal glutamate concentration and the lactate overflow repression. The maximal glutamate concentration increased by about 15% compared with the best results of the DO constant controls, and furthermore, the lactate overproduction occurred in the DO constant control cases could be completely relieved when using the BMC strategy. As a result, the proposed BMC strategy is expected to be applicable for optimization of other aerobic amino acids fermentations in potential.