Metabolic Engineering 3, 289300 (2001)

doi:10.1006/mben.2001.0196, available online at http://www.idealibrary.com on

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

Metabolic engineering to design and construct microorganisms

suitable for the production of aromatic amino acids and derivatives
thereof requires control of a complicated network of metabolic
reactions that partly act in parallel and frequently are in rapid equilibrium. Engineering the regulatory circuits, the uptake of carbon,
the glycolytic pathway, the pentose phosphate pathway, and the
common aromatic amino acid pathway as well as amino acid
importers and exporters that have all been targeted to effect higher
productivities of these compounds are discussed. 2001 Academic

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

Key Words: L-Tyr; L-Phe; L-Trp; shikimate; chorismate; aromatic

amino acids; PEP; E4P; DHS.

Amino acids are compounds of considerable industrial

importance, which serve as feed and food additives, taste
and aroma enhancers, pharmaceuticals or building blocks
for drugs, dietary supplements, nutraceuticals and ingredients in cosmetics.
The aromatic amino acids l-phenylalanine, l-tryptophan
and l-tyrosine and compounds derived thereof constitute a
considerable market volume. l-Trp is produced at a multiple hundred-ton scale predominantly as a feed additive,
despite the beneficial effects that have been ascribed in
pharma and food applications (see Table 1). This is due
to a number of casualties due to EMS (eosinophilia
myalgia syndrome), which have been associated with
the consumption of impure, fermentatively produced l-Trp.
l-Phe is produced predominantly for the production
of the low-calorie sweetener aspartame using the
Nutrasweet process. The DSM/Tosoh joint venture HSC
produces aspartame differently, using chemically
synthesized, racemic dl-phenylalanine in an enzymatic

1
To whom correspondence and reprint requests should be addressed.
Fax: +49.2461.690519. E-mail: marcel.wubbolts@dsm.com.

METABOLIC PATHWAYS TO AROMATIC

HYDROCARBONS
The common aromatic amino acid biosynthetic pathway
leading to the synthesis of the branch point compound
chorismate, and the three terminal pathways, which convert
chorismate to l-Phe, l-Tyr and l-Trp are presented in Fig. 1
(reviewed in Pittard, 1996). The committed step and most
tightly regulated reaction in the common aromatic amino
acid pathway is the condensation of phosphoenolpyruvate
(PEP) and erythrose 4-phosphate (E4P) to d-arabinoheptulosonate 7-phosphate (DAHP) by DAHP synthase.
The pathway proceeds via a number of intermediates to
chorismate, a branch point for the three aromatic amino
acids and for the routes to ubiquinone, menaquinone,
folate, and enterochelin (Gibson and Gibson, 1964).
A subsequent branch point occurs at the level of prephenate, where the pathways toward l-Phe or l-Tyr diverge by
the action of the bifunctional enzymes chorismate mutase/
prephenate dehydratase (toward l-Phe) and chorismate
mutase/prephenate dehydrogenase (l-Tyr) (Hudson et al.,
1984; Zhang et al., 1998; Turnbull and Morrison, 1990).
Anthranilate synthase-phosphoribosyl transferase complex (trpE, trpD) catalyzes the first two steps of l-Trp biosynthesis and is stimulated by chorismate (Romero et al.,
1995). l-Trp synthase (trpA) is an enzyme complex

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Metabolic Engineering 3, 289300 (2001)

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TABLE 1
Market of Aromatic Amino Acids

Listed price (USD/kg)

Use

Main producers

l-Tryptophan

Feed additive, food additive,

infusion liquids, injectables,
antidepressant, treatment of pellagra,
sleep induction (5-hydroxytryptophan,
serotonin), nutritional therapy

Ajinomoto Co., Mitsui

Chemicals, Tanabe Seiyaku Co.,
Kyowa Hakko Kogyo,
Archer Daniels Midland,
Amino GmbH

l-Phenylalanine

Aspartame precursor, infusion fluids,

diet aids, nutraceutical, intermediate
for synthesis of pharmaceuticals
(HIV protease inhibitor,
anti-inflammatory drugs, rennin
inhibitors, etc.), flavor enhancer

Nutrasweet Kelco,
Ajinomoto Co., Tanabe
Seiyaku Co., Yoneyama
Yakugin Kogyo Co.,
Daesang, Amino GmbH,
Rexim/Degussa

l-Tyrosine

Raw material for l-DOPA

production, treatment of
Basedows disease, dietary
supplement

Ajinomoto Co., Kyowa

Hakko Kogyo, Tanabe
Seiyaku Co., Yoneyama
Yakuhin Kogyo Co.,
Amino GmbH, Rexim/Degussa

Market volume (tpa) a

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).

that catalyzes the last step and converts indole-3-glycerol

phosphate and l-serine via the formation of indole to l-Trp
and d-glyceraldehyde 3-phosphate.
REGULATION OF THE AROMATIC AMINO ACIDS
BIOSYNTHESIS AND TRANSPORT
Transcriptional regulation (Fig. 1) of aromatic amino
acid biosynthesis and transport in Escherichia coli is
mediated by the polypeptide products of tyrR (Wallace and
Pittard, 1969; Camakaris and Pittard, 1973) and trpR
(Cohen and Jacob, 1959).
The TyrR protein modulates the expression of at least
eight unlinked operons. Seven of these operons are
regulated in response to changes in the concentrations of
the three aromatic amino acids. Positive control by the
TyrR protein is exerted at two transporter encoding genes:
mtr (for l-Trp) (Heatwole and Somerville, 1991; Sarsero
and Pittard, 1991) and tyrP (for l-Tyr) (Kasian et al., 1986).
Whereas both l-Tyr and l-Phe effect activation of the
mtr gene (Heatwole and Somerville, 1991; Sarsero and
Pittard, 1991) only l-Phe induces expression of the tyrP
gene (Kasian et al., 1986). Expression of aroF and aroL,
is repressed by TyrR, which binds to the TyrR box

(Pittard and Davidson, 1991; Andrews et al., 1991; Wilson

et al., 1994). Zhao et al. (2000) reported that TyrR protein
contains phosphatase activity, which is inhibited by l-Tyr
and ATP. Binding of l-Tyr is the conformational trigger
for TyrR in Haemophilus influenzae, where ATP is a coactivator (Kristl et al., 2000). Detailed insights with regard
to the TyrR operator complex have been published
recently (Sawyer et al., 2000; Howlett and Davidson,
2000).
The TrpR repressor of E. coli regulates genes involved in
l-Trp synthesis and transport, namely aroH, the trp operon,
and mtr, and regulates its own expression as well (Gunsalus
and Yanofsky, 1980). Expression of aroL is under the dual
control of both TrpR and TyrR (Heatwole and Somerville,
1992) and regulation by TrpR, which is only significant in
the presence of TyrR, is greatest when TyrR is bound to all
three TyrR boxes (Lawley and Pittard, 1994). In addition to
aroL, the mtr gene is regulated by TyrR and TrpR, and it
has been suggested these two proteins may interact at the
mtr operator sites (Sarsero et al., 1991; Yang et al., 1993). In
this case, however, TrpR, is the dominant regulator and
cooperative binding between TyrR and TrpR has not been
shown. The transcription of the gene pheA is regulated by
attenuated control (Hudson and Davidson, 1984).

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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).

METABOLIC ENGINEERING OF AROMATIC

AMINO ACID PRODUCTION
The precursors of the common aromatic amino acid
biosynthetic pathway, PEP and E4P (Fig. 1), derive from
central metabolism (Fig. 2); PEP is formed during

FIG. 2. Schematic overview of reactions in the central metabolism of

E. coli. Abbreviations used: PTS, phosphoenolpyruvate phosphotransferase system; G6P dh, glucose-6-phosphate dehydrogenase; Tkt,
transketolase; Tal, transaldolase; Pgi, phosphoglucose isomerase; Ppc,
PEP-carboxylase; Pyk, pyruvate kinase; Pyk, PEP carboxykinase; Pps,
PEP synthetase; G6P, glucose 6-phosphate; F6P, fructose 6-phosphate;
GA3P, glyceraldehyde 3-phosphate; PEP, phosphoenolpyruvate; OAA,
oxaloactetate; 6P-Gnt, 6-phosphogluconate; Rul5P, ribulose 5-phosphate; Rib5P, ribose 5-phosphate; Xul5P, xylulose 5-phosphate; Sed7P,
sedoheptulose 7-phosphate; E4P, erythrose 4-phosphate.

glycolysis and the pentose phosphate pathway supplies

E4P. To improve the production of aromatic compounds
the optimization of both the specific biosynthetic pathway
and the carbon flux from central carbon metabolism has
to be addressed (reviewed in Berry, 1996; Frost and
Draths, 1995; Liao et al., 1994).

ENGINEERING CENTRAL CARBON METABOLISM

Increase of E4P Supply
The key enzymes of the nonoxidative pentose phosphate
pathway are transketolase and transaldolase. These
catalyze reactions that lead to fructose 6-phosphate and
glyceraldehyde 3-phosphate linking the pathway to
glycolysis and on the other hand to E4P, the precursor of
the aromatic amino acid biosynthesis. To increase the
availability of E4P in E. coli, the tktA gene encoding
transketolase has been overexpressed in an E. coli strain
that accumulates DAH(P) due to an inactive aroB gene,
3-dehydroquinate synthase (Draths and Frost, 1990;
Draths et al., 1992). Having high DAHP synthase activity
by overproducing a feed back resistant DAHP synthase
(aroG fbr) and transketolase resulted in additional twofold
increase of carbon flow from glucose into aromatic biosynthesis (Draths et al., 1992). With xylose as substrate no
increase in DAH(P) production by overexpression of tktA
was observed (Patnaik et al., 1995). This effect may be due
to sufficient supply of E4P from the high flux through the
pentose phosphate pathway under these growth conditions. The overproduction of transketolase also raised the
production of aromatic amino acids in Corynebacterium
glutamicum (Ikeda et al., 1999). It appeared from l-Trp
producing E. coli that transketolase gene overexpression
imposes a metabolic burden leading to retarded growth
and segregation of the plasmids (Ikeda and Katsumata,
1999; Kim et al., 2000). Minimizing the tktA expression
levels resulted in stable maintenance of the plasmids.
The impact of transaldolase on the flux into the aromatics pathway was analyzed as well (Lu and Liao, 1997;
Sprenger et al., 1998a). Overexpression of talB, significantly increased the formation of DAH(P) (Lu and Liao,
1997) and l-Phe (Sprenger et al., 1998a) from glucose.
Additional overexpression of tktA increased the flux into
the aromatic pathway of an E. coli l-Phe production
strain (Sprenger et al., 1998a) but not in the DAHP producing strain (Lu and Liao, 1997). From experiments with
PEP synthase expression combined with tktA and talB,
respectively, it was concluded that transketolase is more
effective in directing the carbon flux to the aromatic
pathway than transaldolase (Liao et al., 1996).

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A different attempt to improve the supply with E4P has

been made by deleting the pgi gene that encodes the
phosphoglucose isomerase (Mascarenhas et al., 1991).
Without pgi activity, glycolysis is blocked and the carbon
flux is diverted into the pentose phosphate pathway. In an
l-Trp producing E. coli strain, the pgi deletion resulted in
an almost twofold more efficient conversion of glucose to
l-Trp, but reduces the growth rate.
Increase of PEP Supply
Phosphoenolpyruvate is a key intermediate involved in
several cellular processes (reviewed in Valle et al., 1996). In
wild-type E. coli the major PEP consumer is the phosphotransferase system, PTS, responsible for uptake and
phosphorylation of glucose at the same time (reviewed in
Postma et al., 1996). Other PEP consuming enzymes are
phosphoenolpyruvate carboxylase, Ppc, and the pyruvate
kinases, PykA and PykF. Additionally, there are PEPforming reactions, such as phosphoenolpyruvate synthase,
Pps, and phosphoenolpyruvate carboxykinase, Pck, that
act in gluconeogenesis (Fig. 2).
The maximum theoretical molar yield for DAHP
synthesis from glucose is 0.43 mol/mol. This yield can be
doubled if either pyruvate formed during glucose uptake is
recycled to PEP or glucose uptake and phosphorylation
becomes PEP-independent (Frberg et al., 1988; Patnaik
and Liao, 1994). An approach to avoid PEP consumption
during substrate uptake is to use a non-PTS carbon
source, such as xylose, reaching a maximum theoretical
yield for DAH(P) of 0.71 mol/mol (Patnaik et al., 1995).
Since the synthesis of l-Phe requires an additional molecule of PEP, a theoretical yield of 0.3 mol/mol on glucose
was calculated, and 0.6 mol/mol without loss of PEP
(Frberg et al., 1988).
Using stoichiometric pathway analysis, a novel pathway
to recycle pyruvate was proposed (Liao et al., 1996). This
hypothetical cycle was considered to consist of PEP
carboxykinase (pck) and the glyoxylate shunt, but could
not be proven by further investigations. It was however
not excluded that some pyruvate recycling might occur via
this route in DAH(P) production experiments.
By inactivation of the PEP carboxylase in an l-Phe
producing strain of E. coli, the formation of l-Phe was
significantly increased, but the production of the unwanted by-products acetate and pyruvate increased as well
(Miller et al., 1987). Moreover, the growth of the
ppc-negative strain was reduced twofold and the addition
of a C4 -dicarboxylate, such as succinate, is required for
growth. In a DAH(P) producing strain the deletion of the
ppc gene did not lead to any positive effect (Patnaik and
Liao, 1994). This discrepancy was explained with the

different conditions used, nongrowth versus growth, and

the phenotypic differences between the host strains.
The two pyruvate kinases of E. coli represent another
PEP consuming activity. Inactivation of either gene caused
hardly any effect, but simultaneous inactivation of both
genes significantly increased carbon flow from glucose into
DAH(P) (Berry, 1996; Gosset et al., 1996) and l-Phe
(Grinter, 1998). Combined with growth on non-PTS substrates (e.g., maltose, lactose) the increase was even higher,
but growth was poor (Grinter, 1998).
Pyruvate produced via the PTS is lost for the aromatic
pathway because pyruvate is not recycled to PEP under
glycolytic conditions. By overexpression of the gene pps
that encodes PEP synthase pyruvate is converted back into
PEP and the carbon flux was successfully directed toward
DAH(P) production (Patnaik and Liao, 1994). This positive pps effect was only significant with concomitant
overexpression of a feedback-deregulated DAHP synthase
and transketolase gene tktA, suggesting that the concentration of E4P is the first limiting substrate for DAHP
synthase, followed by PEP (Liao et al., 1996). Instead of
channeling PTS-derived pyruvate back into PEP, the PTS
can be avoided using non-PTS sugars such as xylose
(Frost and Draths, 1995; Patnaik et al., 1995). DAH(P)
production from xylose results in maximum theoretical
yields with high level of DAHP synthase activity alone,
i.e., no further increase of the yield by transketolase or
PEP synthase overexpression was observed (Patnaik et al.,
1995).
From a PTS-negative E. coli mutant, a glucose-positive
revertant was isolated (Flores et al., 1996). This strain
channeled glucose via galactose permease (galP), into the
cell and grew on glucose with rates comparable to wild
type. In strains that at the same time overproduced DAHP
synthase, an increase of DAH(P) excretion into the
medium was observed (Flores et al., 1996; Gosset et al.,
1996; Berry, 1996). Recently, the effect of PTS inactivation
and GalP dependent glucose transport has been further
analyzed in isogenic strains with a block after the first
intermediate of the aromatic amino acid pathway (Bez
et al., 2001). The DAH(P) yield on glucose increased significantly corresponding to 83% of the maximum theoretical yield. Independently, Chen at al. also constructed a
pts-negative E. coli strain that uses the galactose permease,
GalP, for glucose uptake (Chen et al., 1997), however
neither the PEP pool was increased nor l-Phe production
was enhanced in the non-PTS strain. Stoichiometric
analysis confirmed the before mentioned positive effect of
GalP on the theoretical yield of l-Phe, but regarding the
theoretical energy yield GalP has a major disadvantage:
the GalP system requires higher amounts of ATP to
phosphorylate glucose.

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The use of heterologous, PEP independent glucose

uptake systems was suggested to save PEP for the
biosynthesis of aromatics (Frost and Draths, 1995). It had
already been shown that expression of the Zymomonas
mobilis glucose facilitator (glf ) and glucokinase (glk)
restored glucose uptake and phosphorylation in a glucose
negative E. coli mutant (Snoep et al., 1994; Weisser et al.,
1995). The expression of both Zymomonas genes glf and
glk in a PTS-negative E. coli, devoid of DHQ synthase
activity, resulted in an increase of DAH(P) excretion
(Krmer, 2000). Likewise, a positive effect of glf and glk
expression on l-Phe production was shown in PTS-positive as well as in PTS-negative strains. In the latter case
the glf gene was chromosomally integrated into the pts
region to disrupt the PTS genes (Sprenger et al., 1998b).
A combined approach, avoiding PEP consumption by
PTS and enhancing the flux through the pentose
phosphate pathway thereby increasing the availability of
E4P, was performed by substituting the PTS with the
glucose facilitator and by expression of glucose
dehydrogenase gene gdh from Bacillus megaterium and the
gluconate kinase gene gntK from E. coli (Krmer et al.,
1999; Krmer, 2000). Glucose is taken up via the facilitator into the cell, where it is oxidized to gluconate and
subsequently phosphorylated to gluconate 6-phosphate,
which is an intermediate of the oxidative pentose
phosphate pathway. With this new pathway, the production of l-Phe could be increased (Krmer et al., 1999).
A new approach called global metabolic engineering
was introduced, when a global regulatory network was
manipulated that controls carbon flux through the central
carbon pathways (Tatarko and Romeo, 2001). The
disruption of the csrA gene, carbon storage regulator,
among other things influences the regulation of several
enzymes that participate in PEP metabolism resulting in
an elevation of the intracellular PEP pool (Sabnis et al.,
1995). This ability to increase PEP was translated to an
l-Phe producing E. coli and a twofold increase of l-Phe
was determined (Tatarko and Romeo, 2001).
ENGINEERING OF THE AROMATIC AMINO
ACIDS PATHWAY
Alleviation of Feedback Inhibition
In wild-type E. coli grown on minimal media, the l-Tyrfeedback inhibited DAHP synthase (aroF) contributes
20% and the l-Phe-feedback inhibited DAHP synthase
(aroG) contributes 80% of the total enzyme activity (Tribe
et al., 1976). The l-Trp-feedback inhibited DAHPsynthase (aroH) has only a marginal contribution to the
total DAHP-synthase activity. In aromatic amino acid

producing strains, DAHP synthase activity is strongly

reduced as a result of feed back control by the end products. Regulation at the transcriptional level is alleviated
by placing the regulated genes behind promoters that are
not controlled by TrpR/TyrR or by deletion of the
regulators (LaDuca et al., 1999; Berry, 1996).
To overcome allosteric inhibition of aromatic amino
acid pathway reactions, amino acid analogues have been
successfully used to isolate feedback inhibition resistant
mutants (Hagino and Nakayama, 1974; Shiio et al., 1975;
Ray et al., 1988; De Boer and Dijkhuizen, 1990). A
number of l-Tyr feedback inhibition resistant DAHP
synthase mutants have been characterized: Pro148 Leu
(Weaver and Herrmann, 1990) and Gln152 Ile mutations
(Edwards et al., 1987) of the E. coli aroF gene product
resulted in a tyrosine-feedback resistant phenotype. At the
N-terminal end, an Asn8 Lys substitution in AroF from
E. coli led to an l-Tyr-insensitive DAHP synthase as well
(Jossek et al., 2001). In Corynebacterium l-Tyr feedbackinsensitive DAHP synthase mutants Ser187 Cys, Ser187 Tyr,
and Ser187 Phe were obtained, whereas Ser187 Ala showed no
significant effect (Liao et al., 2001).
Feedback inhibition by l-Phe is suppressed by a
Leu76 Val mutation in the aroG gene product and mutations in AroG between residues 146-150 affected inhibition
by l-Phe (Kikuchi et al., 1997). In the crystal structure of
the AroG from E. coli, mutations that reduce feedback
inhibition cluster around a cavity near the twofold axis of
the tight dimeric structure at approximately 15 from the
active site (Shumilin et al., 1999). Eight other feedback
resistant DAHP synthase mutants of aroF and aroG have
been described (Tonouchi et al., 1997). Mutagenesis was
also used to identify residues and regions of the AroH
polypeptide essential for catalytic activity and l-Trp
feedback regulation (Ray et al., 1988). Feedback resistant
chorismate mutase prephenate dehydratase mutants from
E. coli have been made by modifying Trp226 and Trp338
(Gething et al., 1976) and by substituting Ser330 or deleting
amino acid residues downstream from this residue
(Tonouchi et al., 1997). Mutations in codons 304 to 310 of
the pheA gene exhibited almost complete resistance to
feedback inhibition even at very high l-Phe concentrations
(Nelms et al., 1992). The interaction of l-Phe with the
regulatory domains of chorismate mutase prephenate
dehydratase has been investigated in more detail (Zhang
et al., 1998; Pohnert et al., 1999). A feedback resistant
mutant of coryneform bacteria prephenate dehydratase
was obtained (Ozaki et al., 1985).
The anthranilate synthase-phosphoribosyl transferase
enzyme complex which catalyzes the first two steps in of
l-Trp biosynthesis is feedback inhibited by l-Trp. This
is the result from allosteric effects associated with the

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binding of one molecule of inhibitor to each of the TrpE

subunits of the complex in the case of S. typhimurium.
Caligiuri and Bauerle (1991a,b) generated a collection of
trpE mutants, which displayed varying degrees of resistance to feedback inhibition.
Engineering l-Phe Production
Microbial production of l-phenylalanine has been
focused mainly on E. coli, C. glutamicum and Brevibacterium strains (De Boer and Dijkhuizen, 1990). Classic
methods have been applied to screen for auxotrophs and
mutants with feedback deregulated key enzymes. An
overview of analog resistant mutants of B. lactofermentum,
B. flavum, C. glutamicum, and E. coli is given in de Boer
and Dijkhuizen (1990).
Sugimoto et al. cloned the genes encoding feedback
resistant forms of DAHP synthase, aroF fbr, and chorismate mutase/prephenate dehydratase, pheA fbr, into a
temperature-controllable expression vector (Sugimoto
et al., 1987). An l-Tyr auxotrophic E. coli strain carrying
this plasmid had a maximal l-Phe production of 16.8 g/L
at the optimal temperature, 38.5 C (Sugimoto et al.,
1987). Additional process development using this strain improved the l-Phe fermentation process significantly, reaching a titer of 46 g/L and a productivity of 0.85 g/L h
(Konstantinov et al., 1991; Konstantinov and Yoshida,
1992; Takagi et al., 1996).
Miller et al. used a plasmid carrying a wild-type DAHP
synthase gene, aroF, and a feedback resistant chorismate
mutase/prephenate dehydratase gene, pheA fbr (Miller
et al., 1987; Backman and Balakrishnan, 1988). E. coli
cells carrying this plasmid were analyzed with regard to the
effects of PEP carboxylase deficiency on the l-Phe yield.
E. coli strains auxotrophic for all three aromatic amino
acids, but equipped with a plasmid encoding the wild-type
aroF gene and a feedback resistant chorismate mutase/
prephenate dehydratase, pheA fbr was constructed (Frberg
and Hggstrm, 1987). With nongrowing cells, the
maximum theoretical yield of l-Phe on glucose was
reached in batch cultures after depletion of l-Tyr (Frberg
et al., 1988). Tyrosine-limited, glucose fed-batch cultures
improved l-Phe production by applying proper feed rates
for l-Tyr and glucose (Frberg and Hggstrm, 1987).
Backman et al. also engineered E. coli for l-Phe production, based on the aroF WT and pheA fbr genes and developed an efficient fermentation process and within 36 h a
final l-Phe titer of 50 g/L with a yield on glucose of
0.23 g/g could be reached (Backman et al., 1990). An
overview of the efforts at the Nutrasweet Company to
obtain l-phenylalanine producing E. coli strains, which
include mechanisms for l-Phe export, were summarized by
Fotheringham et al. (1994) and Grinter (1998).

Metabolic engineering of C. glutamicum resulted in an

l-Phe producing strain that, when additionally equipped
with a plasmid encoding chorismate mutase and prephenate dehydratase, increased l-Phe accumulation about
50% (Ozaki et al., 1985; Ikeda and Katsumata, 1992;
Ikeda et al., 1993). An l-Trp producing Corynebacterium
strain was made suitable for l-Tyr or l-Phe production
by introducing feedback resistant variants of DAHP
synthase, chorismate mutase and prephenate dehydratase,
by combining the genes on one plasmid. By doing so, the
carbon flow was altered to produce up to 26 g/L l-Phe
(Ikeda and Katsumata, 1992). Heterologous expression of
a feedback resistant mutant of chorismate mutase/
prephenate dehydratase from E. coli in an l-Phe producing
C. glutamicum strain resulted in a significant increase of
the productivity (Ikeda et al., 1993).
Engineering l-Tyr Production
To obtain l-Tyr overproducers, most attention has been
focused on screening for regulatory and auxotrophic
mutants. These were mostly strains of E. coli, Bacillus
subtilis or various coryneform bacteria (Maiti et al., 1995).
By application of recombinant DNA technology additional improvements of these l-Tyr producing strains have
been made (Ito et al., 1990; Ikeda and Katsumata, 1992;
Ikeda et al., 1999).
Additionally improved strains have been generated by
metabolic engineering of an l-Phe auxotrophic Brevibacterium lactofermentum (Ito et al., 1990). To overcome a
key-limiting step of the aromatic amino acid pathway, the
shikimate kinase gene was introduced and the impact of
the expression of shikimate kinase on l-Tyr production
was investigated. The engineered strain demonstrated a
five to 10-fold increase in the enzyme activity and a significant increase of l-Tyr titer (Ito et al., 1990). The genetic
engineering of an l-Trp producing mutant of C. glutamicum to produce l-Tyr or l-Phe has been discussed above
(Ikeda and Katsumata, 1992).
Metabolic engineering of the central carbon metabolism
was performed in l-Tyr producing strains of C. glutamicum (Ikeda et al., 1999; Katsumata and Ikeda, 1997).
Approaches to increase availability of E4P were carried
out by overexpression of the homologous transketolase in
an l-Tyr producing strain of C. glutamicum, resulting in a
1050% increase of the titer of l-Tyr (Ikeda et al., 1999).
Engineering l-Trp Production
Metabolic engineering for production of l-Trp, which
was both triggered by the market potential of l-Trp and

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by the efforts to develop a fermentative route to the blue

dye indigo from indole (Ensley et al., 1983), has primarily
been carried out in E. coli (reviewed in Berry, 1996),
Corynebacterium (Ikeda and Katsumata, 1999; Katsumata
and Ikeda, 1993) and Bacillus (Kurahashi et al., 1984).
To obtain l-Trp-producing strains, alterations in the
central carbon metabolism and the common aromatic
amino acid pathway, including its regulation, were carried
out as described above. In addition, overexpression of the
genes encoding the tryptophan branch of the aromatic
amino acid pathway was performed (Berry, 1996).
Deletion of the pheA and tyrA genes to prevent the
consumption of chorismate would have resulted in l-Phe
and l-Tyr auxotrophies, thereby increasing production
costs. Such deletions were not required in strains that
overexpressed the trpE gene, since anthranilate synthase
has a higher affinity for chorismate than PheA or TyrB
(Dopheide et al., 1972; Hudson et al., 1983; Baker and
Crawford, 1966), resulting in only little loss of carbon to
l-Phe and l-Tyr (LaDuca et al., 1999). Removal of the
tnaA gene that encodes tryptophanase, which catalyzes the
conversion of l-Trp to indole and pyruvate, was effectively used to prevent product loss (Aiba et al., 1982). The
reverse reaction of tryptophanase has been used to convert
pyruvate producing Enterobacter aerogenes into l-Trp
production strains (Yokota et al., 1989). Interestingly,
transport mutants of Corynebacterium that were impaired
in l-Trp uptake were shown to be more effective in production, which was ascribed to changes in intracellular
concentrations resulting in a change of regulation. A
similar effect was reached by addition of nonionic
detergents, that resulted in l-Trp precipitation and proved
more productive (Azuma et al., 1993).

ENGINEERING PRODUCTION OF DERIVED

COMPOUNDS AND INTERMEDIATES
3-Dehydroshikimic Acid Production
3-Dehydroshikimic acid (DHS) is an intermediate in the
aromatic amino acid pathway and was shown to serve as a
suitable starting compound for the renewable production
of a variety of industrial chemicals, ranging from catechol,
vanillic acid to adipic acid (Li et al., 1999). In addition,
DHS can be used as a potent antioxidant (Richman et al.,
1996).
Fermentative production of DHS from glucose was
accomplished by engineered shikimate dehydrogenase
(aroE)-deficient E. coli mutants, in which a gene coding
for a feedback resistant DAHP synthase (aroF fbr ) and a
second copy of the aroB gene, encoding DHQ synthase,

were introduced. Production of DHS was associated with

the formation of dehydroquinate and gallic acid, which
could be products of abiotic conversion reactions
(Richman et al., 1996). Gallic acid production could also
be due to formation of protocatechuic acid by DHS
dehydratase, followed by a hydroxylation step (Li and
Frost, 1999). To increase the availability of E4P, tktA
was introduced into the DHS producing strain, which
resulted in an increase of the DHS titer and the yield to
0.3 mol/mol on glucose (Li et al., 1999). By using pentose
sugars an improvement of DHS yield was observed relative to the use of glucose (Li and Frost, 1999). Overexpressing the transketolase gene resulted in an increased
yield of DHS on xylose only, when a mixture of xylose,
arabinose and glucose was utilized (Li and Frost, 1999).
This could be interpreted that E4P availability was sufficient, but PEP supply was limited for carbon flux into the
aromatic amino acid pathway.
Shikimate Production
Because of three chiral centers in the molecule, shikimic
acid is a suitable starting compound for the synthesis of
neuramidase inhibitors for the treatment of influenza
(Zhang, 1998). Shikimate is also interesting as a starting
compound for combinatorial libraries (Tan et al., 1999).
A classical approach to obtain shikimate producing
strains has been described using Citrobacter freudii (Shirai
et al., 1999). Microbial production of shikimate was
drastically improved by metabolic engineered E. coli
strains (Draths et al., 1999; Frost et al., 1999), which
carried disrupted aroL and aroK genes. To circumvent
polar effects caused by aroK disruption and to overcome
the rate limiting DHQ synthase step, aroB was combined
with the gene coding for a feedback resistant DAHP
synthase aroF fbr. Furthermore, an additional gene coding
for shikimate dehydrogenase, as compensation for the
enzymes feedback inhibition by shikimate, was introduced (Draths et al., 1999). The fermentative production
of shikimate was associated with the formation of
quinic acid as a side product, presumably caused by the
equilibria of initially synthesized shikimate via dehydroshikimate to quinic acid (Draths et al., 1999; Frost
et al., 1999). Reducing shikimate re-uptake by adding a
nonmetabolizable d-glucose analogue could drastically
reduce the formation of quinic acid (Draths et al., 1999;
Frost et al., 1999). Approaches to further improve shikimic acid production by E. coli, by increasing the availability of PEP and E4P have also been made (Gibson et al.,
2001). The PTS of a shikimate producing strain was substituted by glf/glk (Gibson et al., 2001) leading to an
increased availability of PEP. To increase availability of

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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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