Metabolic Engineering 3, 289–300 (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 Krämer, Ulrike Müller, Leon Raeven, and Marcel Wubbolts 1
DSM Biotech GmbH, Karl-Heinz-Beckurts-Strasse 13, D-52428 Jülich, 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-Parkinson’s drug l-DOPA, for the treatment of
Basedow’s 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

289

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antidepressant. 1973) and trpR (Cohen and Jacob.. TrpR. treatment of pellagra.2001. nutritional therapy Ajinomoto Co. 289–300 (2001) doi:10. Sarsero and Pittard. anti-inflammatory drugs.. 1) of aromatic amino acid biosynthesis and transport in Escherichia coli is mediated by the polypeptide products of tyrR (Wallace and Pittard.. food additive. Rexim/Degussa l-Tyrosine Raw material for l-DOPA production. 290 . Tanabe Seiyaku Co. diet aids. rennin inhibitors. Positive control by the TyrR protein is exerted at two transporter encoding genes: mtr (for l-Trp) (Heatwole and Somerville..0196 Minireview TABLE 1 Market of Aromatic Amino Acids Listed price (USD/kg) Use Main producers l-Tryptophan Feed additive. 1984). coli regulates genes involved in l-Trp synthesis and transport. Amino GmbH. 1991. dietary supplement Ajinomoto Co. 1992) and regulation by TrpR. Andrews et al. In addition to aroL.... 1980). 1991. Tanabe Seiyaku Co. Expression of aroF and aroL. Rexim/Degussa a Market volume (tpa) a Feed/chemical Pharma 500–600 48–54 116–133 11000–12000 20–24 30–40 150+ 18–20 34–35 tpa. Amino GmbH l-Phenylalanine Aspartame precursor. is the dominant regulator and cooperative binding between TyrR and TrpR has not been shown. 1994). sleep induction (5-hydroxytryptophan. Seven of these operons are regulated in response to changes in the concentrations of the three aromatic amino acids. 1991) only l-Phe induces expression of the tyrP gene (Kasian et al. The TrpR repressor of E. Mitsui Chemicals. (2000) reported that TyrR protein contains phosphatase activity. is repressed by TyrR. etc.. 1993). Wilson et al. and it has been suggested these two proteins may interact at the mtr operator sites (Sarsero et al.Metabolic Engineering 3. 1991.. nutraceutical. 1959).. Camakaris and Pittard. where ATP is a coactivator (Kristl et al. 1986). infusion liquids. 1991) and tyrP (for l-Tyr) (Kasian et al. Archer Daniels Midland. 1994). In this case. and mtr. flavor enhancer Nutrasweet Kelco. The TyrR protein modulates the expression of at least eight unlinked operons. Kyowa Hakko Kogyo. 2000). Kyowa Hakko Kogyo. Yang et al.. Binding of l-Tyr is the conformational trigger for TyrR in Haemophilus influenzae. 1986). 1969.. 1999). which is only significant in the presence of TyrR. however. REGULATION OF THE AROMATIC AMINO ACIDS BIOSYNTHESIS AND TRANSPORT Transcriptional regulation (Fig. intermediate for synthesis of pharmaceuticals (HIV protease inhibitor. metric tons per annum in 1997 (source: Chemical Economics Handbook. 2000. Sarsero and Pittard. injectables. which binds to the TyrR box (Pittard and Davidson.. Tanabe Seiyaku Co. 2000). 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. and regulates its own expression as well (Gunsalus and Yanofsky.. which is inhibited by l-Tyr and ATP. SRI International. infusion fluids. Zhao et al.. Whereas both l-Tyr and l-Phe effect activation of the mtr gene (Heatwole and Somerville. The transcription of the gene pheA is regulated by attenuated control (Hudson and Davidson. Ajinomoto Co. Yoneyama Yakugin Kogyo Co.. Expression of aroL is under the dual control of both TrpR and TyrR (Heatwole and Somerville. Yoneyama Yakuhin Kogyo Co. Amino GmbH. the trp operon. Detailed insights with regard to the TyrR operator complex have been published recently (Sawyer et al. is greatest when TyrR is bound to all three TyrR boxes (Lawley and Pittard. namely aroH. the mtr gene is regulated by TyrR and TrpR. 1991.1006/mben. Howlett and Davidson. treatment of Basedow’s disease. Daesang. serotonin). 1991.).

5 enolpyruvoylshikimate 3-phosphate. Pathway of aromatic amino acid biosynthesis and its regulation in E. 3-deoxy-d-arobino-heptulosonate 7-phosphate.2001. 4-hydroxyphenlypyruvate. pyruvate. prephenate. IND. coli. —. DHQ. PRAA. 3-dehydroquinate. l-Phe. l-Tyr. a-ketoglutarate. PEP.Metabolic Engineering 3. shikimate 3-phosphate.0196 Minireview FIG. l-Trp. E4P. 5-phosphoribosyl-a-pyrophosphate. transcriptional control only. 1. anthranilate. glyceraldehyde 3-phosphate. aKG. allosteric control only.1006/mben. GA3P. SHIK. l-phenylalanine. CHA. I3GP. l-Glu. 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate. l-Gln. PRPP. PPY. Abbreviations used: ANTA. EPSP. 291 . phosphoribosyl anthranilate. different types of lines are used: – – –. phosphoenolpyruvate. indole 3-glycerolphosphate. indole. · · · . DHS. 289–300 (2001) doi:10. l-tryptophan. To indicate the type of regulation. l-tyrosine. erythrose 4-phosphate. phenylpyruvate. chorismate. PPA. shikimate. l-Ser. l-glutamine. l-serine. l-glutamate. transcriptional and allosteric control exerted by the aromatic amino acid end products. Pyr. HPP. 3-dehydroshikimate. CDRP. S3P. DAHP.

2).. Rib5P. This effect may be due to sufficient supply of E4P from the high flux through the pentose phosphate pathway under these growth conditions. 1998a) from glucose. 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. 1996. respectively. PEP synthetase. coli strain that accumulates DAH(P) due to an inactive aroB gene. 1990. PEP and E4P (Fig. Tal. 1996). Pyk. the tktA gene encoding transketolase has been overexpressed in an E. glycolysis and the pentose phosphate pathway supplies E4P. allosteric inhibition of the committed reaction of DAHP synthases and the branch point enzymes chorismate mutase/ prephenate dehydratase. glucose 6-phosphate. significantly increased the formation of DAH(P) (Lu and Liao. Draths et al.0196 Minireview In addition to regulation at the expression level. With xylose as substrate no increase in DAH(P) production by overexpression of tktA was observed (Patnaik et al. Pyk.Metabolic Engineering 3.. coli. coli. PEP is formed during FIG. G6P dh. Kim et al. 1997). ribose 5-phosphate. GA3P. The only enzyme that is inhibited by an intermediate in the common aromatic amino acids pathway is shikimate dehydrogenase.16 mM (Dell and Frost. 6P-Gnt.. Abbreviations used: PTS. 1994). Frost and Draths. 1992). 2. fructose 6-phosphate. glyceraldehyde 3-phosphate. It appeared from l-Trp producing E. To increase the availability of E4P in E.. 292 .. 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. 1995. phosphoenolpyruvate phosphotransferase system... coli l-Phe production strain (Sprenger et al. Sed7P. 1) exhibiting linear mixed-type inhibition with a inhibition constant of 0. Overexpression of talB. it was concluded that transketolase is more effective in directing the carbon flux to the aromatic pathway than transaldolase (Liao et al. Pgi. which is inhibited by shikimate (aroE. The overproduction of transketolase also raised the production of aromatic amino acids in Corynebacterium glutamicum (Ikeda et al. Pps. Liao et al. METABOLIC ENGINEERING OF AROMATIC AMINO ACID PRODUCTION The precursors of the common aromatic amino acid biosynthetic pathway. E4P. From experiments with PEP synthase expression combined with tktA and talB. 1999. 2000). transketolase. OAA. PEP-carboxylase. 1997. derive from central metabolism (Fig. glucose-6-phosphate dehydrogenase. xylulose 5-phosphate. 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. F6P. 289–300 (2001) doi:10. Schematic overview of reactions in the central metabolism of E. 3-dehydroquinate synthase (Draths and Frost.2001. 1997) and l-Phe (Sprenger et al. pyruvate kinase. Sprenger et al. sedoheptulose 7-phosphate. ENGINEERING CENTRAL CARBON METABOLISM Increase of E4P Supply The key enzymes of the nonoxidative pentose phosphate pathway are transketolase and transaldolase. PEP. oxaloactetate. Tkt. the precursor of the aromatic amino acid biosynthesis. 1995). Rul5P.1006/mben.. coli that transketolase gene overexpression imposes a metabolic burden leading to retarded growth and segregation of the plasmids (Ikeda and Katsumata. 1998a) but not in the DAHP producing strain (Lu and Liao. 1996). 1999). Minimizing the tktA expression levels resulted in stable maintenance of the plasmids. ribulose 5-phosphate. erythrose 4-phosphate. PEP carboxykinase. The impact of transaldolase on the flux into the aromatics pathway was analyzed as well (Lu and Liao. Ppc. 6-phosphogluconate. phosphoenolpyruvate. phosphoglucose isomerase. 1992). Xul5P. chorismate mutase/prephenate dehydrogenase and anthranilate synthase by the end products occurs (reviewed in Pittard.. 1993). Fig. 1). Additional overexpression of tktA increased the flux into the aromatic pathway of an E. transaldolase. G6P.. 1998a).

g.. suggesting that the concentration of E4P is the first limiting substrate for DAHP synthase. an increase of DAH(P) excretion into the medium was observed (Flores et al. This positive pps effect was only significant with concomitant overexpression of a feedback-deregulated DAHP synthase and transketolase gene tktA. lactose) the increase was even higher. 1994). there are PEPforming reactions. reaching a maximum theoretical yield for DAH(P) of 0. 1996). such as phosphoenolpyruvate synthase. but reduces the growth rate. Since the synthesis of l-Phe requires an additional molecule of PEP. The DAH(P) yield on glucose increased significantly corresponding to 83% of the maximum theoretical yield. and phosphoenolpyruvate carboxykinase. however neither the PEP pool was increased nor l-Phe production was enhanced in the non-PTS strain. 1991).0196 Minireview 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. but growth was poor (Grinter. such as xylose. and the phenotypic differences between the host strains.. 1998). 1988. is required for growth. Patnaik et al. coli represent another PEP consuming activity... Moreover. the PTS can be avoided using non-PTS sugars such as xylose (Frost and Draths. the pgi deletion resulted in an almost twofold more efficient conversion of glucose to l-Trp.. 1997). In a DAH(P) producing strain the deletion of the ppc gene did not lead to any positive effect (Patnaik and Liao. 2001). nongrowth versus growth. 1995). the growth of the ppc-negative strain was reduced twofold and the addition of a C4 -dicarboxylate. Combined with growth on non-PTS substrates (e.6 mol/mol without loss of PEP (Förberg et al. Instead of channeling PTS-derived pyruvate back into PEP..3 mol/mol on glucose was calculated. This yield can be doubled if either pyruvate formed during glucose uptake is recycled to PEP or glucose uptake and phosphorylation becomes PEP-independent (Förberg et al. 1994). a theoretical yield of 0.. 1996). Recently. Using stoichiometric pathway analysis.. Ppc. 1987). Patnaik and Liao. 1994). but could not be proven by further investigations. Increase of PEP Supply Phosphoenolpyruvate is a key intermediate involved in several cellular processes (reviewed in Valle et al.. the formation of l-Phe was significantly increased. Pyruvate produced via the PTS is lost for the aromatic pathway because pyruvate is not recycled to PEP under glycolytic conditions. coli strain that uses the galactose permease. also constructed a pts-negative E. 1995). a glucose-positive revertant was isolated (Flores et al. Additionally. GalP. and 0. into the cell and grew on glucose with rates comparable to wild type. 2).2001. 1995). but simultaneous inactivation of both genes significantly increased carbon flow from glucose into DAH(P) (Berry. no further increase of the yield by transketolase or PEP synthase overexpression was observed (Patnaik et al. 1988).. but regarding the theoretical energy yield GalP has a major disadvantage: the GalP system requires higher amounts of ATP to phosphorylate glucose. In strains that at the same time overproduced DAHP synthase. but the production of the unwanted by-products acetate and pyruvate increased as well (Miller et al. a novel pathway to recycle pyruvate was proposed (Liao et al. and the pyruvate kinases.. responsible for uptake and phosphorylation of glucose at the same time (reviewed in Postma et al. Stoichiometric analysis confirmed the before mentioned positive effect of GalP on the theoretical yield of l-Phe.. Other PEP consuming enzymes are phosphoenolpyruvate carboxylase. Gosset et al..71 mol/mol (Patnaik et al. 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. It was however not excluded that some pyruvate recycling might occur via this route in DAH(P) production experiments. This discrepancy was explained with the different conditions used.. The two pyruvate kinases of E. Berry... followed by PEP (Liao et al. coli strain. 1996. coli mutant. Without pgi activity. Pps. The maximum theoretical molar yield for DAHP synthesis from glucose is 0. 1998). In an l-Trp producing E. i. 1996). In wild-type E. Inactivation of either gene caused hardly any effect.43 mol/mol. Pck. This hypothetical cycle was considered to consist of PEP carboxykinase (pck) and the glyoxylate shunt. 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 (Báez et al. By inactivation of the PEP carboxylase in an l-Phe producing strain of E. 1996. glycolysis is blocked and the carbon flux is diverted into the pentose phosphate pathway. 1996). DAH(P) production from xylose results in maximum theoretical yields with high level of DAHP synthase activity alone. This strain channeled glucose via galactose permease (galP). 1996) and l-Phe (Grinter.Metabolic Engineering 3. PTS. 289–300 (2001) doi:10. 1996). 293 . 1996). An approach to avoid PEP consumption during substrate uptake is to use a non-PTS carbon source.e. 1996. coli. PykA and PykF... coli the major PEP consumer is the phosphotransferase system. such as succinate. From a PTS-negative E. Chen at al. maltose.1006/mben. Gosset et al. Independently. for glucose uptake (Chen et al. that act in gluconeogenesis (Fig.. 1995.

1006/mben. 1999.. PEP independent glucose uptake systems was suggested to save PEP for the biosynthesis of aromatics (Frost and Draths. In aromatic amino acid producing strains. The l-Trp-feedback inhibited DAHPsynthase (aroH) has only a marginal contribution to the total DAHP-synthase activity.2001. 2000). 1994.. 2001)... 1990). devoid of DHQ synthase activity. 1998. 289–300 (2001) doi:10. 1990) and Gln152 Ile mutations (Edwards et al. a positive effect of glf and glk expression on l-Phe production was shown in PTS-positive as well as in PTS-negative strains. Glucose is taken up via the facilitator into the cell. 1996). 1995).. coli.. 1997). an Asn8 Lys substitution in AroF from E. A combined approach. coli (Krämer et al. The disruption of the csrA gene. avoiding PEP consumption by PTS and enhancing the flux through the pentose phosphate pathway thereby increasing the availability of E4P. 1995)... 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. coli and a twofold increase of l-Phe was determined (Tatarko and Romeo. resulted in an increase of DAH(P) excretion (Krämer.0196 Minireview The use of heterologous. 1999). In the latter case the glf gene was chromosomally integrated into the pts region to disrupt the PTS genes (Sprenger et al.. 1976) and by substituting Ser330 or deleting amino acid residues downstream from this residue (Tonouchi et al. 1975..Metabolic Engineering 3. De Boer and Dijkhuizen. coli mutant (Snoep et al. 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. 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. The interaction of l-Phe with the regulatory domains of chorismate mutase prephenate dehydratase has been investigated in more detail (Zhang et al. 1988).. Weisser et al. the production of l-Phe could be increased (Krämer et al. where it is oxidized to gluconate and subsequently phosphorylated to gluconate 6-phosphate. ENGINEERING OF THE AROMATIC AMINO ACIDS PATHWAY Alleviation of Feedback Inhibition In wild-type E.. 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. 2001). 1999).. 1997). A new approach called ‘‘global metabolic engineering’’ was introduced. coli led to an l-Tyr-insensitive DAHP synthase as well (Jossek et al. A number of l-Tyr feedback inhibition resistant DAHP synthase mutants have been characterized: Pro148 Leu (Weaver and Herrmann. This is the result from allosteric effects associated with the 294 . 1974. coli aroF gene product resulted in a tyrosine-feedback resistant phenotype. DAHP synthase activity is strongly reduced as a result of feed back control by the end products. Ray et al. 1999).. 2000). With this new pathway. 1976). To overcome allosteric inhibition of aromatic amino acid pathway reactions... Ser187 Tyr. 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. In Corynebacterium l-Tyr feedbackinsensitive DAHP synthase mutants Ser187 Cys. 1992). Pohnert et al. 1997). In the crystal structure of the AroG from E. coli grown on minimal media. 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. The anthranilate synthase-phosphoribosyl transferase enzyme complex which catalyzes the first two steps in of l-Trp biosynthesis is feedback inhibited by l-Trp. 1995). 1999. Eight other feedback resistant DAHP synthase mutants of aroF and aroG have been described (Tonouchi et al... 1985). 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. which is an intermediate of the oxidative pentose phosphate pathway. This ability to increase PEP was translated to an l-Phe producing E. Feedback resistant chorismate mutase prephenate dehydratase mutants from E. whereas Ser187 Ala showed no significant effect (Liao et al. carbon storage regulator. At the N-terminal end. 2001).. when a global regulatory network was manipulated that controls carbon flux through the central carbon pathways (Tatarko and Romeo. A feedback resistant mutant of coryneform bacteria prephenate dehydratase was obtained (Ozaki et al.. and Ser187 Phe were obtained. Shiio et al. Krämer.. coli have been made by modifying Trp226 and Trp338 (Gething et al. amino acid analogues have been successfully used to isolate feedback inhibition resistant mutants (Hagino and Nakayama. Likewise. Berry. coli. 1988. mutations that reduce feedback inhibition cluster around a cavity near the twofold axis of the tight dimeric structure at approximately 15 Å from the active site (Shumilin et al.. 1987) of the E. The expression of both Zymomonas genes glf and glk in a PTS-negative E. 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.. 1998b). 2001).

resulting in a 10–50% increase of the titer of l-Tyr (Ikeda et al. also engineered E.b) generated a collection of trpE mutants. coli strains auxotrophic for all three aromatic amino acids. when additionally equipped with a plasmid encoding chorismate mutase and prephenate dehydratase. Additional process development using this strain improved the l-Phe fermentation process significantly. The genetic engineering of an l-Trp producing mutant of C. 1985. C. Engineering l-Trp Production Metabolic engineering for production of l-Trp. 1999). Ikeda and Katsumata. the maximum theoretical yield of l-Phe on glucose was reached in batch cultures after depletion of l-Tyr (Förberg et al.. These were mostly strains of E. glutamicum. the shikimate kinase gene was introduced and the impact of the expression of shikimate kinase on l-Tyr production was investigated.23 g/g could be reached (Backman et al. Engineering l-Phe Production Microbial production of l-phenylalanine has been focused mainly on E.. aroF fbr..0196 Minireview binding of one molecule of inhibitor to each of the TrpE subunits of the complex in the case of S. 1990. coli in an l-Phe producing C. pheA fbr was constructed (Förberg and Häggström.Metabolic Engineering 3. glutamicum. 1990). coli for l-Phe production. 1999). glutamicum to produce l-Tyr or l-Phe has been discussed above (Ikeda and Katsumata. which displayed varying degrees of resistance to feedback inhibition. With nongrowing cells.. 1993). B... C. coli. 1990). were summarized by Fotheringham et al. into a temperature-controllable expression vector (Sugimoto et al. Metabolic engineering of C. 1993). 1995).2001.. pheA fbr. but equipped with a plasmid encoding the wild-type aroF gene and a feedback resistant chorismate mutase/ prephenate dehydratase. lactofermentum. coli is given in de Boer and Dijkhuizen (1990). 1988).8 g/L at the optimal temperature. 1987. E. Ikeda et al. Metabolic engineering of the central carbon metabolism was performed in l-Tyr producing strains of C. Konstantinov and Yoshida. 1997). 1996). By doing so. Sugimoto et al. Engineering l-Tyr Production To obtain l-Tyr overproducers. coli strains. 1987). which include mechanisms for l-Phe export. glutamicum strain resulted in a significant increase of the productivity (Ikeda et al. glucose fed-batch cultures improved l-Phe production by applying proper feed rates for l-Tyr and glucose (Förberg and Häggström. (1994) and Grinter (1998). reaching a titer of 46 g/L and a productivity of 0.. most attention has been focused on screening for regulatory and auxotrophic mutants. and E. chorismate mutase and prephenate dehydratase. Bacillus subtilis or various coryneform bacteria (Maiti et al. the carbon flow was altered to produce up to 26 g/L l-Phe (Ikeda and Katsumata. An overview of analog resistant mutants of B. 1987). 1990)... 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. typhimurium. Takagi et al. Classic methods have been applied to screen for auxotrophs and mutants with feedback deregulated key enzymes. 1987). 1992). glutamicum and Brevibacterium strains (De Boer and Dijkhuizen. coli cells carrying this plasmid were analyzed with regard to the effects of PEP carboxylase deficiency on the l-Phe yield.1006/mben.. 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. which was both triggered by the market potential of l-Trp and 295 . coli strain carrying this plasmid had a maximal l-Phe production of 16. cloned the genes encoding feedback resistant forms of DAHP synthase. Backman et al. 289–300 (2001) doi:10. increased l-Phe accumulation about 50% (Ozaki et al. pheA fbr (Miller et al. 1991. flavum. To overcome a key-limiting step of the aromatic amino acid pathway. glutamicum (Ikeda et al.5° C (Sugimoto et al. 1990). aroF. Katsumata and Ikeda. Ikeda et al. 1988). Caligiuri and Bauerle (1991a. 1992. 1992. Heterologous expression of a feedback resistant mutant of chorismate mutase/ prephenate dehydratase from E. coli. Additionally improved strains have been generated by metabolic engineering of an l-Phe auxotrophic Brevibacterium lactofermentum (Ito et al. By application of recombinant DNA technology additional improvements of these l-Tyr producing strains have been made (Ito et al. Ikeda and Katsumata. An l-Trp producing Corynebacterium strain was made suitable for l-Tyr or l-Phe production by introducing feedback resistant variants of DAHP synthase. by combining the genes on one plasmid.. and chorismate mutase/prephenate dehydratase. Approaches to increase availability of E4P were carried out by overexpression of the homologous transketolase in an l-Tyr producing strain of C. 1992. Tyrosine-limited. glutamicum resulted in an l-Phe producing strain that. 1992). Backman and Balakrishnan.. 1999. 38. An l-Tyr auxotrophic E.. Miller et al... used a plasmid carrying a wild-type DAHP synthase gene.85 g/L · h (Konstantinov et al. and a feedback resistant chorismate mutase/prephenate dehydratase gene.. An overview of the efforts at the Nutrasweet Company to obtain l-phenylalanine producing E. E. 1987).

ranging from catechol. 1996). 2001) leading to an increased availability of PEP. 1983). 1999). A similar effect was reached by addition of nonionic detergents. which catalyzes the conversion of l-Trp to indole and pyruvate. Corynebacterium (Ikeda and Katsumata. resulting in only little loss of carbon to l-Phe and l-Tyr (LaDuca et al.. 1999). which could be products of abiotic conversion reactions (Richman et al. thereby increasing production costs.Metabolic Engineering 3. 1972. Approaches to further improve shikimic acid production by E. 1982). overexpression of the genes encoding the tryptophan branch of the aromatic amino acid pathway was performed (Berry. The PTS of a shikimate producing strain was substituted by glf/glk (Gibson et al. 1966).. Deletion of the pheA and tyrA genes to prevent the consumption of chorismate would have resulted in l-Phe and l-Tyr auxotrophies. by increasing the availability of PEP and E4P have also been made (Gibson et al. In addition. which resulted in an increase of the DHS titer and the yield to 0. 1996).. were introduced. To increase availability of 296 . aroB was combined with the gene coding for a feedback resistant DAHP synthase aroF fbr. 1993) and Bacillus (Kurahashi et al. shikimic acid is a suitable starting compound for the synthesis of neuramidase inhibitors for the treatment of influenza (Zhang. which was ascribed to changes in intracellular concentrations resulting in a change of regulation. encoding DHQ synthase. 1999). Reducing shikimate re-uptake by adding a nonmetabolizable d-glucose analogue could drastically reduce the formation of quinic acid (Draths et al. alterations in the central carbon metabolism and the common aromatic amino acid pathway.. Frost et al.. coli strains (Draths et al. 1998). since anthranilate synthase has a higher affinity for chorismate than PheA or TyrB (Dopheide et al. presumably caused by the equilibria of initially synthesized shikimate via dehydroshikimate to quinic acid (Draths et al. arabinose and glucose was utilized (Li and Frost.. including its regulation. coli mutants.1006/mben. Fermentative production of DHS from glucose was accomplished by engineered shikimate dehydrogenase (aroE)-deficient E. an additional gene coding for shikimate dehydrogenase.. 1999)... Furthermore. as compensation for the enzyme’s feedback inhibition by shikimate. Gallic acid production could also be due to formation of protocatechuic acid by DHS dehydratase. The reverse reaction of tryptophanase has been used to convert pyruvate producing Enterobacter aerogenes into l-Trp production strains (Yokota et al. 1999. followed by a hydroxylation step (Li and Frost.... were carried out as described above. To increase the availability of E4P. Such deletions were not required in strains that overexpressed the trpE gene. The fermentative production of shikimate was associated with the formation of quinic acid as a side product. 1999.. 1999). coli. 1999). Baker and Crawford.. was effectively used to prevent product loss (Aiba et al. 1999).. 1999).. but PEP supply was limited for carbon flux into the aromatic amino acid pathway. DHS can be used as a potent antioxidant (Richman et al. 1999).. Shikimate is also interesting as a starting compound for combinatorial libraries (Tan et al. 1996). To obtain l-Trp-producing strains. was introduced (Draths et al. To circumvent polar effects caused by aroK disruption and to overcome the rate limiting DHQ synthase step. 1999.. In addition. Hudson et al. has primarily been carried out in E.3 mol/mol on glucose (Li et al. 1989). Katsumata and Ikeda. Interestingly. Production of DHS was associated with the formation of dehydroquinate and gallic acid. By using pentose sugars an improvement of DHS yield was observed relative to the use of glucose (Li and Frost. that resulted in l-Trp precipitation and proved more productive (Azuma et al. Removal of the tnaA gene that encodes tryptophanase.0196 Minireview by the efforts to develop a fermentative route to the blue dye indigo from indole (Ensley et al... Frost et al. tktA was introduced into the DHS producing strain. This could be interpreted that E4P availability was sufficient. 1993). 1984). 1999. 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. 1999). A classical approach to obtain shikimate producing strains has been described using Citrobacter freudii (Shirai et al. Shikimate Production Because of three chiral centers in the molecule. coli (reviewed in Berry. 1983. vanillic acid to adipic acid (Li et al. when a mixture of xylose. 1999). which carried disrupted aroL and aroK genes.. 1996).. in which a gene coding for a feedback resistant DAHP synthase (aroF fbr ) and a second copy of the aroB gene. 1999). transport mutants of Corynebacterium that were impaired in l-Trp uptake were shown to be more effective in production.. 289–300 (2001) doi:10. 2001). Frost et al.2001. Overexpressing the transketolase gene resulted in an increased yield of DHS on xylose only. Microbial production of shikimate was drastically improved by metabolic engineered E.

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