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MINIREVIEW
Metabolic Engineering for Microbial Production of Aromatic Amino
Acids and Derived Compounds
Johannes Bongaerts, Marco Krmer, Ulrike Mller, Leon Raeven, and Marcel Wubbolts 1
DSM Biotech GmbH, Karl-Heinz-Beckurts-Strasse 13, D-52428 Jlich, Germany
Received July 26, 2001; accepted July 27, 2001
enantio- and regioselective coupling process. Other applications of l-Phe are its use in infusion fluids, in food additives, as intermediates for the synthesis of active compounds
(Table 1) and as a flavor enhancer. l-Tyr is produced at a
small scale (Table 1) and is of use for the production of the
anti-Parkinsons drug l-DOPA, for the treatment of
Basedows disease and as a dietary supplement. Commercial
producers of the aromatic amino acids are listed in Table 1.
Press
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To whom correspondence and reprint requests should be addressed.
Fax: +49.2461.690519. E-mail: marcel.wubbolts@dsm.com.
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Copyright 2001 by Academic Press
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TABLE 1
Market of Aromatic Amino Acids
Main producers
l-Tryptophan
l-Phenylalanine
Nutrasweet Kelco,
Ajinomoto Co., Tanabe
Seiyaku Co., Yoneyama
Yakugin Kogyo Co.,
Daesang, Amino GmbH,
Rexim/Degussa
l-Tyrosine
Feed/chemical
Pharma
500600
4854
116133
1100012000
2024
3040
150+
1820
3435
tpa, metric tons per annum in 1997 (source: Chemical Economics Handbook, SRI International, 1999).
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FIG. 1. Pathway of aromatic amino acid biosynthesis and its regulation in E. coli. To indicate the type of regulation, different types of lines are
used: , transcriptional and allosteric control exerted by the aromatic amino acid end products; , allosteric control only; , transcriptional
control only. Abbreviations used: ANTA, anthranilate; aKG, a-ketoglutarate; CDRP, 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate; CHA,
chorismate; DAHP, 3-deoxy-d-arobino-heptulosonate 7-phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; EPSP, 5 enolpyruvoylshikimate 3-phosphate; E4P, erythrose 4-phosphate; GA3P, glyceraldehyde 3-phosphate; HPP, 4-hydroxyphenlypyruvate, I3GP, indole 3-glycerolphosphate; IND, indole; l-Gln, l-glutamine; l-Glu, l-glutamate; l-Phe, l-phenylalanine; l-Ser, l-serine; l-Trp, l-tryptophan; l-Tyr, l-tyrosine; PEP,
phosphoenolpyruvate; PPA, prephenate; PPY, phenylpyruvate; PRAA, phosphoribosyl anthranilate; PRPP, 5-phosphoribosyl-a-pyrophosphate; Pyr,
pyruvate; SHIK, shikimate; S3P, shikimate 3-phosphate.
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In addition to regulation at the expression level, allosteric inhibition of the committed reaction of DAHP
synthases and the branch point enzymes chorismate mutase/
prephenate dehydratase, chorismate mutase/prephenate
dehydrogenase and anthranilate synthase by the end
products occurs (reviewed in Pittard, 1996). The only
enzyme that is inhibited by an intermediate in the common
aromatic amino acids pathway is shikimate dehydrogenase, which is inhibited by shikimate (aroE, Fig. 1)
exhibiting linear mixed-type inhibition with a inhibition
constant of 0.16 mM (Dell and Frost, 1993).
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E4P, the tktA gene was also introduced and the yield
increased from 0.18 to 0.27 mol/mol (Gibson et al., 2001).
Production of shikimate was also carried out by a
classically screened B. subtilis strain (Iomantas et al.,
2000). As opposed to E. coli, a Bacillus strain carrying
only one defect allele of shikimate kinase produced
shikimate and dehydroshikimate as a side-product in relative high amounts. Increasing the activity of shikimate
dehydrogenase by introducing the corresponding gene
from Bacillus amyloliquefaciens successfully improved the
shikimate production of the strain (Iomantas et al., 2000).
subsequently. An example is the inhibition of the transketolase from Saccharomyces cerevisiae by p-hydroxyphenylpyruvate, the penultimate intermediate in the biosynthesis of l-Tyr (Solovjeva and Kochetov, 1999).
Although various metabolic engineering approaches
increase the yield, many of these in turn slow down the
growth or just deteriorate the performance of a production process. Therefore, the transfer of promising results
from lab-scale experiments to industrial processes is still
difficult.
ACKNOWLEDGMENT
Production of d-Phenylalanine
The fermentative production of d-phenylalanine was
performed with an E. coli strain lacking all three transaminase genes responsible for l-phenylalanine formation
from phenylpyruvate. The carbon flux through the aromatic amino acid pathway toward phenylpyruvate was
increased by expressing the DAHP synthase gene aroH
and a feedback resistant chorismate mutase/prephenate
dehydratase, pheA fbr. To produce d-phenylalanine, a respective d-aminotransferase together with an alanine
racemase was expressed (Fotheringham et al., 1998).
CONCLUSIONS
Metabolic engineering of the central metabolism in
order to improve the biosynthesis of aromatics is almost
restricted to E. coli, and less work has been done with C.
glutamicum. By now, several modifications proved to be
valuable, but most impressing results were obtained when
approaches were combined to show a synergistic effect:
Patnaik and Liao (1994) attained a near theoretical yield
of DAH(P) by overexpressing transketolase together with
PEP synthase, and Gosset et al. (1996) used a PTS-negative glucose + mutant, additionally inactivated both pyruvate kinases and amplified the transketolase taken
together a 20-fold increase in carbon flow into DAH(P)
was achieved.
In general, changes in central pathways have the strongest effects when the impact is determined as carbon flux
into DAHP, the first intermediate of the aromatic biosynthesis pathway. However, to produce end products,
aromatic amino acids or compounds derived from
chorismate, one must keep in mind that an extra molecule
of PEP enters the pathway later, which can influence the
balance of precursor supply. Unexpected interconnections
between the central metabolism and the biosynthesis
pathway may be detected, which have to be circumvented
Financial support from the BioRegio program of the Bundesministerium fr Bildung und Forschung (BMBF, Grant 0311644) is gratefully
acknowledged.
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