I

TCA cycle

Figure 5.1 Metabolic engineering of central metabolic pathways to increase the synthesis of histidine and aromatic amino acids. Solid and dashed lines represent single and multiple steps, respectively. Solid bars over the arrows represent blocked enzymes, while thick arrows represent amplified enzymes. The following strategies are illustrated. Increasing the production of histidine by increasing the availability of Ribose-5-P, 1- Transketolase-deficient strains. Increasing the production of AA by increasing the supply of erythrose-4-P, 1- Overexpression of transketolases in AA producer. Increasing the production of AA by increasing the availability of PEP, 2- Inactivation of PEP-dependent PTS system for the transport of glucose and amplification of sugar-phosphorylating kinase gene; 3- Inactivation of PEP carboxylase; 4- Amplification of PEP synthase. Abbreviations, AA, aromatic amino acids; G3P, glyceraldehyde-3-P; and PEP, phosphoenolpyruvate.

in elucidating the function of different central pathways and suggesting useful strategies for redirecting carbon flow toward the biosynthesis of amino acids. For example, it has been shown that the pentose phosphate pathway (PPP) supports higher fluxes during the production of L-lysine compared to the production of L-glutamic acid in C. glutamicum (7,8). This was attributed to the higher requirements of reducing power (NADPH) in the production of L-lysine. Another example is improving aromatic amino acids and L-histidine production in C. glutamicum by increasing the availability of their precursor metabolites, erythrose 4-phos-phate and ribose 5-phosphate, respectively, as well as NADPH. This can be done by modifying the flux through the PPP, either by increasing the activity of transketolase (and providing more erythrose 4-phosphate for aromatic amino acids biosynthesis) or by decreasing the activity of transketolase (and providing more ribose 5-phosphate for L-histidine biosynthesis) as shown in Figure 5.1, strategy 1 (6). Both approaches have produced C. glutamicum strains with an increased capacity for making aromatic amino acids (9) and L-histidine (10). Figure 5.1 shows additional examples of engineering central metabolic pathways to increase the availability of precursor metabolites used to synthesize aromatic amino acids in C. glutamicum. In general, these strategies are based on increasing the availability of erythrose 4-phosphate (strategy 1, Figure 5.1), phosphoenolpyruvate (strategies 2, 3, and 4, Figure 5.1), and ribose-5-P (strategy 1,

Phosphoenolpyruvate + Erythrose-4-P l®

DAHP L-tyrosine

L-tryptophan i \

A Chorismate-Prephenate

Phosphoglycerate L-phenylalanine <— Phenylpyruvate (A)

Figure 5.2 Metabolic engineering of biosynthetic pathways to increase the synthesis of aromatic amino acids, threonine, and isoleucine in C. glutamicum. See Figure 5.1 for explanation of different types of lines/arrows. In some cases, feedback inhibition has been represented using round dotted lines. Examples illustrated here include amplification of a gene encoding a rate-limiting step, introduction of a heterologous enzyme subjected to a different regulatory mechanism, and redirection of metabolic flux in a branch point (Ikeda (6)). (A) Increasing the synthesis of aromatic amino acids, 1 - Overexpression of chorismate mutase increased L-phenylalanine production; 2- Overexpression of mutated (insensitive to L-phenylalanine) chorismate mutase-prephenate dehydratase from E. coli increased the production of L-phenylalanine; 3- Simultaneous amplification of chorismate mutase and prephenate dehydratase resulted in increased production of L-tyrosine and L-phenylalanine; 4- Coexpression of two enzymes catalyzing the initial steps in the biosynthesis of aromatic amino acids (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase) and L-serine (3-phosphoglycerate dehydrogenase) together with tryptophan-biosynthetic enzymes increased tryptophan production. (B) Increasing the synthesis of isoleucine and threonine, 1- Expression of L-isoleucine-insensitive E. coli threonine dehydratase (catabolic) enhanced isoleucine production; 2- Amplification of a threonine biosynthetic operon resulted in increased production of threonine in a lysine-producing C. glutamicum strain. Abbreviations, DAHP, 3-deoxy-D-arabino-heptulosonate-7-phosphate; I3GP, indole-3-glycerol-phosphate.

Figure 5.1) by inactivating the enzymes involved in their consumption and/or amplifying the enzymes involved in their production (for a review of these strategies, see Reference 6).

5.2.2 Modification of Biosynthetic Pathways

After engineering central metabolic pathways, a sufficient supply of precursor metabolites, energy, and reducing power is ensured, and efforts then need to be focused on engineering biosynthetic pathways that convert precursor metabolites into amino acids. Several strategies have been used to achieve this goal (6), and some of them are illustrated in Figure 5.2. For example, the gene that encodes a rate-limiting enzyme can be amplified, resulting in the release of a bottleneck. Ozaki et al. (11) and Ikeda et al. (12) used this strategy to improve the production of L-phenylalanine in C. glutamicum. Overexpression of the gene that encodes chorismate mutase in C. glutamicum K38 resulted in a 50% increase in the yield of L-phenylalanine (11). On the other hand, introducing heterologous enzymes subject to different regulatory mechanisms can also result in the release of a bottleneck. For example, overexpressing a mutated (insensitive to L-phenylalanine) E. coli gene that encoded the bifunctional enzyme chorismate mutase-prephenate dehydratase in C. glutamicum KY10694 led to a 35% increase in the production of L-phenylalanine (12). In addition, expressing the E. coli catabolic threonine dehydratase (insensitive to L-isoleucine) in C. glutamicum

L-aspartate

L-aspartate-semialdehyde

L-lysine L-threonine i >o|

2-oxobutanoate

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