Biosynthesis and Production of L-Lysine

The processes for amino acid production can be roughly divided into enzymatic, extractive, chemical, and fermentative methods. Most amino acids were initially obtained by extractive methods from protein-rich resources. The main problems with this method are product purity, satisfying the demand for the amino acid, the availability of the raw materials, and environmental concerns about the process as odor or waste treatment can be an issue. Chemical processes are also not predominant (except for glycine and d,l-methionine), primarily due to the high costs of the optical resolution to generate L-forms from the racemic mixtures. The use of biocatalysis, for example, for production of L-cysteine is mainly limited by the cost of the substrates used. Fermentation is the most applied method for commercial production of L-amino acids, and is also used for production of L-lysine. 

Two different routes for the biosynthesis of L-lysine are known in microorganisms. The a-aminoadipate route (AA), of which two variants are known, which start from 2-oxoglutarate and acetyl-CoA [23]. The three variants of the diaminopime-late route (DAP), which starts from L-aspartate, can be distinguished by their use of either succinylated or acetylated intermediates or by direct reductive amination of tetrahydropicolinate [24]. 

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The intermediate of L-lysine biosynthesis, L-aspartyl-semialdehyde, is also a precursor for the biosynthesis of L-threonine, L-isoleucine, and L-methionine. Homoserine dehydrogenase catalyses NADPH-dependent reduction of L-aspartyl-semialdehyde to L-homoserine. Flux from L-aspartyl-semialdehyde toward L-homoserine could be reduced by introducing alleles for less active homoserine dehydrogenase variants. In addition, L-lysine production increased as the prevailing threonine concentrations in such horn mutants were too low for feedback inhibition of aspartokinase [67, 68]. 

In 1958, Kinoshita and Nakayama of Kyowa Hakko Kogyo Co. Ltd. reported that the auxotrophic mutant of Corynebacterium glutamicum, which lacks homoserine dehydrogenase and is defective in L-homoserine (or i-threonine plus i-methionine) biosynthesis, produced L-lysine in the culture medium (Kinoshita et al. 1958). This was the first report on production of an amino acid by an auxotrophic mutant. Subsequently, amino acid production by auxotrophic mutants expanded greatly. Then, the mutants with the L-threonine- or L-methio-nine-sensitive phenotype due to the mutation in homoserine dehydrogenase (low activity) were also found to produce appreciable amounts of L-lysine in the culture medium (Tosaka and Takinami 1986). Furthermore, a lysine analogue (S-aminoethylcysteine)-resistant mutant was obtained as an L-lysine producer. In this strain, aspartokinase was insensitive to feedback inhibition (Tosaka and Takinami 1986). This is the first demonstration of amino acid production by an analogue-resistant mutant. 

FIGURE 22-15 Biosynthesis of six essential amino acids from oxalo-acetate and pyruvate in bacteria methionine, threonine, lysine, isoleucine, valine, and leucine. Here, and in other multistep pathways, the enzymes are listed in the key. Note that L,L-a,e-diaminopimelate, the product of step (HI), is symmetric. The carbons derived from pyruvate (and the amino group derived from glutamate) are not traced beyond this point, because subsequent reactions may place them at either end of the lysine molecule. 

Nomura et al. (1987a) attempted to minimize product inhibitory effect on the aspartate kinase step in lysine biosynthesis and enhance L-lysine production from Brevibacterium Jlavum QL-5 using a combined ED-F system. However, lysine production was not statistically different from that obtained in diffusion dialysis fermentation and about 20% greater than that achieved during conventional fermentation, thus making practically ineffective such a use of ED. 

The enzyme, i.e. lysine decarboxylase, that is required for the conversion of lysine into cadaverine, and thus the first step of alkaloid biosynthesis, has been isolated from chloroplasts of L. polyphyllus,28 Like the majority of amino-acid decarboxylases, this enzyme is dependent on pyridoxal 5 phosphate. Its activity was found not to be affected by the presence or absence of quinolizidine alkaloids. Control of the enzyme by simple product feedback inhibition therefore seems unlikely. The operational parameters of this enzyme resemble those of the 17-oxosparteine synthase. Co-operation between the two enzymes would explain why cadaverine is almost undetectable in vivo. 

Gatto et al m characterized the mechanism of L-pipecolic acid formation by cyclodeaminase RapL from L-lysine within rapamycin biosynthesis, which is a hybrid NRP—polyketide antibiotic (Figure 25(a)). RapL was characterized by biochemical assays to require cofactor nicotinamide adenine dinucleotide (NAD+) and an oxidative cyclodeamination reaction mechanism corresponding to ornithine cyclodeamination was proposed based on ESI-FTMS analysis of RapL reaction products (Figure 25(b)). 

Figure 25 L-Pipecolic acid formation by cyclodeaminase RapL in rapamycin biosynthesis, (a) Rapamycin and incorporated pipecolic acid moiety, (b) Proposed oxidative cyclodeamination mechanism of pipecolic acid formation from L-lysine. (c) RapL activity assays and exact ESI-FTMS analysis of derivatized reaction products revealing mechanistic insights such as a-H retainment and loss of e-N.

Nevertheless, an exception to the general rule of retention has recently been discovered in the form of mcfo-a,e-diaminopimelate decarboxylase from Bacillus sphaerkus (259). This PLP-dependent enzyme, which catalyzes the final step in lysine biosynthesis, is the only known amino acid decarboxylase to operate on an a-carbon having the D-configuiation (264). Inversion of configuration was demonstrated for the enzyme from Bacillus sphaerkus by conducting the decarboxylation reaction in H20 solvent and isolating as product (6I7)-l-[6-2H]lysine [Eq. (50)] ... 

Corynebacterium actively excretes amino acids through its cell wall membrane and does not degrade L-lysine due to the lack of lysine-decarboxylase. For 60 years all these characteristics have made this microbe the species of choice in L-lysine production. In addition it demonstrates the potential of natural biosynthesis pathways for commercial purposes. In contrast Escherichia coli entered the field of industrial amino acid fermentation not because of comparable advantages provided by nature but because of the availability of effective tools for genetic engineering. In the early 1980s such methods were state of the art for Escherichia coli but were only on an infant level for Corynebacterium. Developing industrial strains based on Escherichia coli, which at that time was not broadly covered by intellectual property (IP), provided room to build new IP in the field of amino acid fermentation. 

The biochemistry and molecular biology of quinolizidine alkaloid biosynthesis have not been fully characterized. Quinolizidine alkaloids are formed from lysine via lysine decarboxylase (LDC), where cadaverine is the first detectable intermediate (Scheme 6). Biosynthesis of the quinolizidine ring is thought to arise from the cyclization of cadaverine units via an enzyme-bound intermediate 176). LDC and the quinolizidine skeleton-forming enzyme have been detected in chlorop-lasts of L. polyphyllus 177). Once the quinolizidine skeleton has been formed, it is modified by dehydrogenation, hydroxylation, or esterification to generate the diverse array of alkaloid products. 

The effect of addition of amino acids to medium 3 (which already contains aspartate, arginine, and histidine as nitrogen sources) was studied by Cheng et al. (76). Of 18 amino acids tested at 1 g/liter, only L-lysine showed a marked stimulation of both volumetric and specific rapamycin production. Its effect was maximal at 10 g/liter, the highest concentration examined. Lysine stimulation is probably due to its conversion to pipccolic acid, a precursor of rapamycin (58). Suppression of rapamycin production was observed with L-methionine and L-phenylalanine. Since methionine is a rapamycin precursor (20), it is peculiar that it interferes with rapamycin formation. However, for some unknown reason, methionine often suppresses formation of antibiotics for which it acts as precursor (77-80). One possibility for phenylalanine suppression of rapamycin formation was considered to be feed-back inhibition of shikimic add formation, since shikimate is a precursor of rapamycin (20), However, in a recent study (81) demonstrating stimulation of rapamycin biosynthesis by exogenous shikimate (57 mM), it was also shown that shikimate does not reverse phenylalanine interference. 

L-lysine is an alkali-amino acid that belongs to the aspartate branch in the biosynthesis of amino acids. It is an essential amino acid in animal nutrition. Many plant products used for livestock feed, such as wheat and com, are deficient in L-lysine, and thus, it must be added as a supplement in the form of soybean meal. The content of lysine in such products in comparison with soybean meal is shown in Table 2. 

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