Metabolic Engineering for Microbial Production of Aromatic Amino Acids and Derived Compounds

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.

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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: [email protected].

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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, 289–300 (2001) doi:10.1006/mben.2001.0196

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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 Basedow’s disease, dietary supplement

Ajinomoto Co., Kyowa Hakko Kogyo, Tanabe Seiyaku Co., Yoneyama Yakuhin Kogyo Co., Amino GmbH, 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, 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 (Förberg 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 (Förberg 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 (Báez 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 (Krämer, 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 (Krämer et al., 1999; Krämer, 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 (Krämer 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 Å 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 (Förberg and Häggström, 1987). With nongrowing cells, the maximum theoretical yield of l-Phe on glucose was reached in batch cultures after depletion of l-Tyr (Förberg et al., 1988). Tyrosine-limited, glucose fed-batch cultures improved l-Phe production by applying proper feed rates for l-Tyr and glucose (Förberg and Häggström, 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 10–50% 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 enzyme’s 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 für Bildung und Forschung (BMBF, Grant 0311644) is gratefully acknowledged.

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