17.2 GLUTAMIC ACID PRODUCTION BY CORYNEBACTERIUM
17.2.1 Glutamic Acid Production by Corynebacterium Glutamicum
A fermentation process has been developed for the direct production of glutamic acid from sugar and ammonia inC. glutamicum. At present,C. glutamicumis also a widely used host microorganism for other amino acids, such as lysine, arginine, threonine, and valine.C. glutamicumis a high G+C Gram-positive, rod-shaped, and facultative anaerobic bacterium. In addition, the genome DNA of this microorganism has been sequenced (Ikeda and Nakagawa, 2003; Kalinowski et al., 2003; Yukawa et al., 2007).
C. glutamicumis regarded as a useful host species for producing chemicals, such as organic acids (Okino et al., 2005; Wendisch et al., 2006), alcohols (Blombach et al., 2011), secreted proteins (Kikuchi et al., 2003), and amino acids other than glutamic acid (Nakayama et al., 1961; Sano and Shiio, 1970; Shiio and Nakamori, 1970;
Tsuchida et al., 1975; Ohnishi et al., 2002; Blombach et al., 2008; Becker et al., 2011; Hasegawa et al., 2012, 2013). This section reviews the molecular mechanism of glutamic acid production byC. glutamicum.
17.2.1.1 Triggers for Glutamic Acid Overproduction in Corynebacterium Glutamicum Glutamic acid is derived from 2-oxogluratrate, a metabolite in the TCA cycle, and produced in a one-step reaction catalyzed by glutamate dehydro- genase (GDH) in the presence of high concentrations of ammonia (Figure 17.2).
C. glutamicum is a biotin (vitamin B7)-auxotrophic microorganism. A wild-type strains ofC. glutamicumcannot secrete glutamic acid when excess biotin is in the culture medium. However, at limiting biotin concentrations,C. glutamicumproduces
FIGURE 17.2 Metabolic reactions around 2-oxoglutarate node in the TCA cycle. Regulation of 2-oxoglutarate dehydrogenase complex (ODHC) activity via phosphorylation status of OdhI protein is also shown. ICDH, isocitrate dehydrogenase; GDH, glutamate dehydrogenase.
significant amounts of glutamic acid (Shiio et al., 1962). Because significant amounts of biotin are found in the raw material required for glutamic acid production (e.g., molasses), other strategies were required to induce glutamic acid overproduction in the presence of excess biotin. Treatments with fatty acid ester surfactants (e.g., Tween 40) andβ-lactam antibiotics (e.g., penicillin) have been reported to induce glutamic acid production byC. glutamicum(Nara et al., 1964; Takinami et al., 1965). These treatments are thought to affect the cell surface structure ofC. glutamicum. Biotin is a cofactor of the enzyme required for the biosynthesis of fatty acids, which are com- ponents of cytoplasmic membrane. Tween 40 and penicillin affect the synthesis of fatty acids and peptidoglycans, which are components of the cytoplasmic membrane and cell wall, respectively. Therefore, the glutamic acid produced byC. glutamicum has been proposed to passively leak through the cell surface. However, based on dif- ferences between intracellular and extracellular glutamic acid levels, this leak model of glutamic acid cannot explain the significant amounts (more than 60–80 g/L) of glutamic acid produced.
17.2.1.2 Metabolic Change and Its Molecular Mechanism During Glu- tamic Acid Production by Corynebacterium Glutamicum In 1971, a change in the enzyme activity of the 2-oxoglutarate dehydrogenase complex (ODHC)
was observed during fermentation of glutamic acid byC. glutamicum(Shigu and Terui, 1971). The ODHC is located at branch points between the TCA cycle and glutamic acid biosynthesis catalyzed by GDH. Kawahara et al., later observed that ODHC activity decreased during glutamic acid overproduction induced by biotin lim- itation as well as the addition of Tween 40 and penicillin (Kawahara et al., 1997). The decrease in ODHC activity induced the accumulation of 2-oxoglutarate, the substrate for GDH. As a result, the metabolic flux toward glutamic acid production increased.
In 2006, Niebisch et al. (2006) discovered the protein OdhI, which is involved in decreasing ODHC activity (Figure 17.2). The OdhI protein directly interacted with a catalytic subunit of ODHC, OdhA (E1o), inhibited ODHC activity, and regulated interactions between OdhI and OdhA via phosphorylation. Furthermore, the non- phosphorylated OdhI interacted with OdhA, whereas phosphorylated OdhI could not (Krawczyk et al., 2010). Kinases (PknA, PknB, and PknG) and a phosphatase (Ppp) for phosphorylating and dephosphorylating OdhI were also identified (Niebisch et al., 2006; Schultz et al., 2007, 2009). Intracellular levels of non-phosphorylated OdhI were also enhanced by induction treatments (e.g., biotin limitation as well as the addi- tion of Tween 40 and penicillin) for glutamic acid overproduction byC. glutamicum (Boulahya et al., 2010; Kim et al., 2011).
Recently, proteins consisting of AceE, AceF, Lpd, and OdhA form a supercomplex inC. glutamicum. The AceE, AceF, and Lpd proteins are the E1p, E2p, and E3p subunits of pyruvate dehydrogenase complex (PDHC), respectively (Hoffelder et al., 2010). In most organisms, ODHC consists of E1o, E2o, and E3o, and E3o is Lpd.
However, in the case of C. glutamicumand other related species Mycobacterium tuberculosis, the gene encoding the E2o protein in the ODHC is not found in the genome. For ODHC activity, the E2 protein in ODHC may potentially be shared with the AceF protein in the supercomplex. During glutamic acid production, PDHC activity decreased as well as ODHC activity (Hasegawa et al., 2008). Additional studies on the relationship between the PDHC and ODHC supercomplexes, OdhI, and ODHC activity in glutamic acid production need to be performed.
Glutamic acid overproduction by C. glutamicum is triggered by biotin limita- tion as well as the addition of Tween 40 and penicillin. These treatments affect the integrity of theC. glutamicumcell surface. Therefore, some reports have proposed a model of the passive leakage of glutamic acid through the membrane and investigated the relationship between changes in the cell surface and glutamic acid production (Hoischen and Kr¨amer, 1990; Hirasawa et al., 2000, 2001; Hashimoto et al., 2006).
Recently, a mechanosensitive channel protein NCgl1221 was reported to contribute to glutamic acid production in C. glutamicum. The NCgl1221 gene was identified in the analysis of theC. glutamicum odhAdisruptant (Nakamura et al., 2007). The NCgl1221 disruptant could not secrete glutamic acid outside of cells. Mechanosensi- tive channel proteins can sense changes in membrane tension. The NCgl1221 protein senses changes in membrane tension by its conformational change. Conformational changes in the NCgl1221 protein appear to induce glutamic acid overproduction by C. glutamicum, which is induced by biotin limitation as well as the addition of Tween 40 and penicillin, and the NCgl1221 protein functions as an exporter of glutamic acid.
Hashimoto et al., reported that the NCgl1221 protein functions as a mechanosensitive
channel through which glutamic acid can leak across the cell membrane by passive diffusion (Hashimoto et al., 2010, 2012).