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Metabolic Engineering
journal homepage: www.elsevier.com/locate/ymben

Increased lysine production by flux coupling of the tricarboxylic acid cycle and the lysine biosynthetic pathway—Metabolic engineering of the availability of succinyl-CoA in Corynebacterium glutamicum
Stefanie Kind, JudithBecker, Christoph Wittmann n
Technische Universit¨t Braunschweig, Institute of Biochemical Engineering, Gaußstr. 17, D-38106 Braunschweig, Germany a

a r t i c l e i n f o
Article history: Received 3 April 2012 Received in revised form 5 July 2012 Accepted 23 July 2012 Keywords: Succinylase pathway Succinyl-CoA synthetase 13 C metabolic flux Lysine TCA cycle Glyoxylate shunt Systems metabolicengineering Escherichia coli

abstract
In this study, we demonstrate increased lysine production by flux coupling using the industrial work horse bacterium Corynebacterium glutamicum, which was mediated by the targeted interruption of the tricarboxylic acid (TCA) cycle at the level of succinyl-CoA synthetase. The succinylase branch of the lysine production pathway functions as the bridging reaction toconvert succinyl-CoA to succinate in this aerobic bacterium. The mutant C. glutamicum DsucCD showed a 60% increase in the yield of lysine when compared to the advanced lysine producer which was used as parent strain. This mutant was highly vital and exhibited only a slightly reduced specific growth rate. Metabolic flux analysis with 13C isotope studies confirmed that the increase in lysine productionwas mediated by pathway coupling. The novel strain exhibited an exceptional flux profile, which was closer to the optimum performance predicted by in silico pathway analysis than to the large set of lysine-producing strains analyzed thus far. Fluxomics and transcriptomics were applied as further targets for next-level strain engineering to identify the back-up mechanisms that were activated upondeletion of the enzyme in the mutant strain. It seemed likely that the cells partly recruited the glyoxylate shunt as a by-pass route. Additionally, the a-ketoglutarate decarboxylase pathway emerged as the potential compensation mechanism. This novel strategy appears equally promising for Escherichia coli, which is used in the industrial production of lysine, wherein this bacterium synthesizeslysine exclusively by succinyl-CoA activation of pathway intermediates. The channeling of a high flux pathway into a production pathway by pathway coupling is an interesting metabolic engineering strategy that can be explored to optimize bioproduction in the future. & 2012 Elsevier Inc. All rights reserved.

1. Introduction With a continuously increasing market production volume of 1.5 million tonsper year, lysine is an important industrial amino acid. Since only the L-isomer of lysine is bioactive as a feed supplement, lysine production utilizes the fermentative production by the Gram-positive soil bacterium Corynebacterium glutamicum (Becker and Wittmann, 2012a). The high industrial relevance of C. glutamicum has constantly stimulated efforts to analyze and modify the underlying metabolicand regulatory networks for improved lysine yield, titer and productivity in a targeted manner. In recent years, sophisticated experimental and computational tools in systems biology have provided an excellent platform to overcome the limitations of classical strain engineering and have initiated the era of metabolic engineering,

n

Corresponding author. Fax: þ49 531 391 7652. E-mail address:c.wittmann@tu-braunschweig.de (C. Wittmann).

and more recently, systems metabolic engineering (Atsumi et al., 2008; Becker and Wittmann, 2012b; Kind et al., 2010a; Lee et al., 2005; Ohnishi et al., 2002). C. glutamicum possesses a functionally split pathway for the biosynthesis of lysine (Schrumpf et al., 1991), which is closely connected to the carbon core pathways (Fig. 1). Metabolic...