L-threonine biosynthesis

L-Threonine is one of the three major amino acids produced by fermentation processes [45]. Currently, more than 4,000 tons of L-threonine are produced annually by fermentation [46]. In this section, we examine the L-threonine biosynthetic pathway and its regulation, and discuss how the carbon flux can be maximized towards L-threonine biosynthesis by metabolic engineering. The detailed description on L-threonine biosynthetic pathways and regulations involved is shown in Fig. 1. 

As mentioned earlier, L-threonine production can be enhanced by engineering the export or uptake system. An efficient L-threonine producer strain of E. coli KY10935, which was derived from the wild-type strain by multiple rounds of random mutation and selection, was able to produce 100 g L-1 L-threonine after 77 h cultivation [53]. In this strain, the two key enzymes in the L-threonine biosynthesis (homoserine dehydrogenase and homoserine kinase) were identified to be still inhibited by much lower intracellular concentrations of L-threonine than... 

L-threonine biosynthesis involves centeral metabolism including glycolysis, pentose phosphate pathway, TCA cycle and anaplerotic pathways between glycolysis and TCA cycle, prior to its terminal pathway (Fig. 14.1). 

Many kinds of amino acids (eg, L-lysine, L-omithine, t-phenylalanine, L-threonine, L-tyrosine, L-valine) are accumulated by auxotrophic mutant strains (which are altered to require some growth factors such as vitamins and amino acids) (Table 6, Primary mutation) (22). In these mutants, the formation of regulatory effector(s) on the amino acid biosynthesis is genetically blocked and the concentration of the effector(s) is kept low enough to release the regulation and iaduce the overproduction of the corresponding amino acid and its accumulation outside the cells (22). 

The study of the biosynthesis of D-mannoproteins in other fungi is less developed. It has already been mentioned that the participation of Man-P-Dol as the glycosyl donor in O-glycosylation was demonstrated in Neuro-spora crassa97 and in Fusarium solani f. pisi.56 In the latter, the lipid acceptor was identified as C90- to C110-dolichols. In Hansenula species, O-glycosyla-tion follows the same pathway that is D-mannose linked to L-serine or L-threonine is incorporated by way of Man-P-Dol, and further elongation... 

The first step in valine biosynthesis is a condensation between pyruvate and active acetaldehyde (probably hy-droxyethyl thiamine pyrophosphate) to yield a-acetolactate. The enzyme acetohydroxy acid synthase usually has a requirement for FAD, which, in contrast to most flavopro-teins, is rather loosely bound to the protein. The very same enzyme transfers the acetaldehyde group to a-ketobutyrate to yield a-aceto-a-hydroxybutyrate, an isoleucine precursor. Unlike pyruvate, the a-ketobutyrate is not a key intermediate of the central metabolic routes rather it is produced for a highly specific purpose by the action of a deaminase on L-threonine as shown in figure 21.10. 

M Offenzeller, G Santer, K Totschnig, Z Su, H Moser, R Traber, E Schneider-Scherzer. Biosynthesis of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine of cyclosporin A enzymatic analysis of the reaction sequence including identification of the methylation precursor in a polyketide pathway. Biochemistry 35 8401-8412, 1996. 

Evidence for such a modular pathway has been provided from studies into the biosynthesis of the polyketide backbone 77 of (41 )-4-[(E)-2-butenyl]-4-methyl-L-threonine 78 which is incorporated into cyclosporin A in Tolypo-cladium niveum [117]. The proposed biosynthesis of 77 is presented in Scheme 29. In vitro studies using a cell extract have verified unambiguously that the biosynthetic mechanism is processive, that the first PKS free intermediate is the tetraketide 79, and that methylation unequivocally occurs at the stage of the enzyme bound 3-oxo-4-hexenoic acid thioester 80 which is the triketide product from the second elongation cycle. These and other results indicate that the methyl transferase activity is inherent in the second module of the putative PKS. 

Further experiments with threonine and isoleucine have been carried out in Senecio magnificus Both [2- " C]- and [6- " C]-isoleucine were incorporated into senecic acid (21) and degradation gave a pattern of incorporation consistent with biosynthesis from two isoleucine units (Scheme 5). [ /- " CJ-L-Threonine was equally well incorporated into senecic acid, which was shown to contain essentially no activity at C-2 and C-8, consistent with the way in which threonine condenses with pyruvic acid to give isoleucine the route from threonine to senecic acid is illustrated (Scheme 6). In these three feeding experiments the incorporation of radioactivity into the main alkaloidal component of this plant, retronecine, was very low. 

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