17.2 GLUTAMIC ACID PRODUCTION BY CORYNEBACTERIUM
17.2.2 Metabolic Engineering of Glutamic Acid Production by
To improve bioproduction using cells, both genetic modification of metabolic path- ways and process operation strategies are important. Metabolic engineering can be defined as a methodology for improvement strategies of production of target product(s) and cellular characteristics by genetic engineering. Here, the metabolic engineering approach of glutamic acid production byC. glutamicumis reviewed.
17.2.2.1 Metabolic Flux Redistribution by the Induction of Glutamic Acid Production in Corynebacterium Glutamicum Decrease in ODHC activity occurs during glutamic acid production byC. glutamicumand can be induced by limiting the amount of biotin as well as the addition of Tween 40 and penicillin.
Thus, the metabolic fluxes toward 2-oxoglutarate are redistributed to succinyl- CoA or glutamic acid biosynthesis by triggering treatments for glutamic acid production.
We analyzed the impact of enhanced enzyme activity around the 2-oxoglutarate node (i.e., isocitrate dehydrogenase (ICDH) and GDH; Figure 17.2) and decreased ODHC activity due to the biotin limitation on the glutamic acid production byC. glu- tamicumby metabolic flux redistribution analysis (Shimizu et al., 2003). There was a small impact of changes in ICDH and GDH activities on metabolic flux redistribution toward glutamic acid production, but the change in ODHC activity had a significant impact on glutamic acid production and metabolic flux redistribution, indicating that the ODHC is an important enzyme in the overproduction of glutamic acid inC. glu- tamicum. Moreover, we also analyzed the metabolic flux redistribution during Tween 40-triggered glutamic acid production byC. glutamicumand demonstrated that the metabolic flux is redistributed toward glutamic acid production by the addition of Tween 40 (Shirai et al., 2005).
17.2.2.2 The Importance of Anaplerotic Reactions in Glutamic Acid Pro- duction by Corynebacterium Glutamicum In glutamic acid production, a supply of oxaloacetate is required for the TCA cycle to maintain 2-oxoglutarate biosynthesis. Anaplerotic reactions produce oxaloacetate from intermediate metabo- lites in glycolysis because the intermediate metabolites in the TCA cycle are used for anabolic reactions, including glutamic acid biosynthesis.C. glutamicumcarries two anaplerotic reactions, each of which is catalyzed by phosphoenolpyruvate carboxy- lase (PPC) and pyruvate carboxylase (PC). PPC encoded by theppcgene can produce oxaloacetate from phosphoenolpyruvate and CO2, and PC encoded bypyccan also produce oxaloacetate from pyruvate and CO2(O’Regan et al., 1989; Peters-Wendisch et al., 1998).
Our group analyzed the roles of PPC and PC in inducing glutamic acid production by biotin limitation. PC requires biotin as a cofactor for enzyme activity (Sato et al.,
FIGURE 17.3 The involvement of anaplerotic reactions in Tween 40-triggered glutamic acid production byCorynebacterium glutamicum.13C-metabolic flux analysis ofC. glutamicum during Tween 40-induced glutamic acid overproduction was performed and metabolic flux distributions on anaplerotic reactions and the 2-oxoglutarate branch are shown. The left and right values in each box represent the fluxes in the growth and glutamic acid production phases.
The flux for glucose uptake is normalized to 100.
2008). Therefore, under limiting biotin concentrations, PPC was proposed to supply oxaloacetate in glutamic acid production. Indeed, theppcknockout in theC. glutam- icumstrain was not able to produce glutamic acid at limiting biotin concentrations, whereas the pyc knockout strain produced glutamic acid. 13C tracer experiments showed that the flux of anaplerotic reactions in thepycknockout mutant was lower than that in the wild-typeC. glutamicumat limiting biotin concentrations, suggesting that PPC is important for glutamic acid production under biotin limitation conditions.
We also analyzed the contribution of anaplerotic reactions to the overproduction of glutamic acid triggered by Tween 40 using precise metabolic flux analysis based on
13C metabolic flux analysis, in which the metabolic flux distribution is estimated from the13C enrichment in proteinogenic amino acids (Shirai et al., 2007) (Figure 17.3).
PPC was observed to be active during cell growth, whereas PC became active by the addition of Tween 40, inducing glutamic acid production.
17.2.2.3 Metabolic Engineering Approaches for the Improvement of Glutamic Acid Production by Corynebacterium Glutamicum The enhancement of glutamic acid production by C. glutamicum can be achieved by metabolic engineering approaches. The following are engineering targets for enhancing glutamic acid production: ODHC, regulation of ODHC activity, and anaplerotic reactions. For example,odhAdisruption without triggers or decreasing OdhA levels by expressing odhA antisense RNA with triggering treatments can increase glutamic acid production by C. glutamicum (Asakura et al., 2007; Kim et al., 2009). Our research group showed that the overexpression of theodhI gene encoding the protein that inhibits ODHC activity increases glutamic acid production byC. glutamicumwithout triggering treatments (Kim et al., 2010). As for anaplerotic reactions, glutamic acid production triggered by Tween 60, which has the same effect as Tween 40, was enhanced by the overexpression ofpycand decreased by the pycdisruptant (Peters-Wendisch et al., 2001).
17.2.2.4 Glutamic Acid Production by Corynebacterium Glutamicum from Renewable Resources Recent studies on glutamic acid production by C. glutamicumusing renewable resources demonstrated that metabolic engineering could be applied toC. glutamicumto utilize biomass resources and waste by-products produced during industrial chemical production.
C. glutamicumcannot consume pentose sugars, such as xylose and arabinose, which are obtained by hydrolyzing lignocellulosic biomass. Therefore, the construc- tion of recombinant strains ofC. glutamicumto convert pentose sugars to other chemi- cals was attempted. AC. glutamicumrecombinant strain expressingEscherichia coli xylAand/orxylBgenes encoding xylose isomerase and xylulokinase, respectively, was able to grow on xylose (Kawaguchi et al., 2006). Moreover, aC. glutamicum strain expressing theE. coli araBAD genes was able to grow on arabinose as the sole carbon source (Kawaguchi et al., 2008). Schneider et al. (2011) successfully produced glutamic acid from arabinose in a strain harboringE. coli araBADgenes by adding ethambutol, which is an inhibitor of cell wall biosynthesis inMycobacteria and can induce glutamic acid production byC. glutamicum(Radmacher et al., 2005).
Gopinath et al. (2011) constructed a strain expressingE. coli xylAandaraBADgenes, which could grow on both xylose and arabinose. Moreover, this strain produced glu- tamic acid upon the addition of ethambutol from a mixture of xylose, arabinose, rice straw, and wheat bran hydrolysates.
Glycerol is one of the major by-products in biodiesel production, and utilizing glycerol is an important objective in metabolic engineering for chemicals production.
BecauseC. glutamicumdoes not grow well on glycerol as the sole carbon source, a C. glutamicumrecombinant strain that could consume glycerol as a carbon source needed to be constructed. Notably,E. colipossesses the glycerol assimilation pathway, and the introduction of theE. coligenes (e.g.,glpP,glpK, andglpD) involved in this pathway intoC. glutamicumled to the production of glutamic acid from glycerol (Rittmann et al., 2008).
The other way to utilize biomass resources for chemical production via fer- mentation is to express and secrete the carbohydrate-degrading enzymes. Recently,
recombinantC. glutamicumstrains secreting endoglucanases derived from various microorganisms were constructed and produced glutamic acid fromβ-glucan upon the addition of Tween 40 (Tsuchidate et al., 2011). In addition, a recombinantC. glu- tamicum strain expressingα-amylase fromStreptococcus bovison the cell surface was constructed and successfully produced glutamic acid from starch (Yao et al., 2009).