Tryptophan Metabolic Engineering Review

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MINIREVIEW

Metabolic Engineering for Microbial Production of Aromatic AminoAcids and Derived Compounds

Johannes Bongaerts, Marco Krmer, Ulrike Mller, Leon Raeven, and Marcel Wubbolts 1

To whom correspondence and reprint requests should be addressed.

x: +49.2461.690519. E-mail: [email protected].

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

table for the production of aromatic amino acids and derivatives

ereof requires control of a complicated network of metabolic

actions that partly act in parallel and frequently are in rapid equi-

rium. Engineering the regulatory circuits, the uptake of carbon,

e glycolytic pathway, the pentose phosphate pathway, and the

mmon aromatic amino acid pathway as well as amino acid

porters and exporters that have all been targeted to effect higher

oductivities of these compounds are discussed. 2001 Academic

ss

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

ino acids; PEP; E4P; DHS.

Amino acids are compounds of considerable industrialportance, which serve as feed and food additives, tasted aroma enhancers, pharmaceuticals or building blocksr drugs, dietary supplements, nutraceuticals and ingre-ents in cosmetics.The aromatic amino acids l-phenylalanine,l-tryptophand l-tyrosine and compounds derived thereof constitute ansiderable market volume. l-Trp is produced at a mul-le hundred-ton scale predominantly as a feed additive,spite the beneficial effects that have been ascribed inarma and food applications (see Table 1). This is duea number of casualties due to EMS (eosinophilia

yalgia syndrome), which have been associated withe consumption of impure, fermentatively produced l-Trp.Phe is produced predominantly for the production

the low-calorie sweetener aspartame using theutrasweet process. The DSM/Tosoh joint venture HSCoduces aspartame differently, using chemicallynthesized, racemic dl-phenylalanine in an enzymatic

enantio- and regioselective coupling process. Other appli-cations ofl-Phe are its use in infusion fluids, in food addi-tives, as intermediates for the synthesis of active compounds(Table 1) and as a flavor enhancer. l-Tyr is produced at asmall scale (Table 1) and is of use for the production of theanti-Parkinsons drug l-DOPA, for the treatment ofBasedows disease and as a dietary supplement. Commerciaproducers of the aromatic amino acids are listed in Table 1.

METABOLIC PATHWAYS TO AROMATIC

HYDROCARBONS

The common aromatic amino acid biosynthetic pathwayleading to the synthesis of the branch point compoundchorismate, and the three terminal pathways, which convertchorismate to l-Phe, l-Tyr and l-Trp are presented in Fig. 1(reviewed in Pittard, 1996). The committed step and mosttightly regulated reaction in the common aromatic aminoacid pathway is the condensation of phosphoenolpyruvate(PEP) and erythrose 4-phosphate (E4P) to d-arabino-heptulosonate 7-phosphate (DAHP) by DAHP synthaseThe pathway proceeds via a number of intermediates tochorismate, a branch point for the three aromatic amino

acids and for the routes to ubiquinone, menaquinonefolate, and enterochelin (Gibson and Gibson, 1964).A subsequent branch point occurs at the level of prephe-

nate, where the pathways toward l-Phe or l-Tyr diverge bythe action of the bifunctional enzymes chorismate mutase/prephenate dehydratase (toward l-Phe) and chorismatemutase/prephenate dehydrogenase (l-Tyr) (Hudson et al.,1984; Zhang et al., 1998; Turnbull and Morrison, 1990).

Anthranilate synthase-phosphoribosyl transferase com-plex (trpE, trpD) catalyzes the first two steps ofl-Trp bio-synthesis and is stimulated by chorismate (Romero et al.,

1995). l-Trp synthase (trpA) is an enzyme complex


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TABLE 1

Market of Aromatic Amino Acids

Listed price (USD/kg)

Use Main producers Market volume (tpa)a Feed/chemical Pharma

l-Tryptophan Feed additive, food additive, Ajinomoto Co., Mitsui 500600 4854 116133infusion liquids, injectables, Chemicals, Tanabe Seiyaku Co.,antidepressant, treatment of pellagra, Kyowa Hakko Kogyo,sleep induction (5-hydroxytryptophan, Archer Daniels Midland,serotonin), nutritional therapy Amino GmbH

l-Phenylalanine Aspartame precursor, infusion fluids, Nutrasweet Kelco, 1100012000 2024 3040diet aids, nutraceutical, intermediate Ajinomoto Co., Tanabefor synthesis of pharmaceuticals Seiyaku Co., Yoneyama(HIV protease inhibitor, Yakugin Kogyo Co.,anti-inflammatory drugs, rennin Daesang, Amino GmbH,inhibitors, etc.), flavor enhancer Rexim/Degussa

l-Tyrosine Raw material forl-DOPA Ajinomoto Co., Kyowa 150+ 1820 3435production, treatment of Hakko Kogyo, TanabeBasedows disease, dietary Seiyaku Co., Yoneyamasupplement Yakuhin Kogyo Co.,

Amino GmbH, Rexim/Degussa

tpa, metric tons per annum in 1997 (source: Chemical Economics Handbook, SRI International, 1999).

at catalyzes the last step and converts indole-3-glycerolosphate and l-serine via the formation of indole to l-Trp

d d-glyceraldehyde 3-phosphate.

REGULATION OF THE AROMATIC AMINO ACIDS

BIOSYNTHESIS AND TRANSPORT

Transcriptional regulation (Fig. 1) of aromatic aminoid biosynthesis and transport in Escherichia coli isediated by the polypeptide products oftyrR(Wallace andttard, 1969; Camakaris and Pittard, 1973) and trpRohen and Jacob, 1959).The TyrR protein modulates the expression of at least

ght unlinked operons. Seven of these operons aregulated in response to changes in the concentrations ofe three aromatic amino acids. Positive control by therR protein is exerted at two transporter encoding genes:r (for l-Trp) (Heatwole and Somerville, 1991; Sarserod Pittard, 1991) and tyrP(for l-Tyr) (Kasian et al., 1986).hereas both l-Tyr and l-Phe effect activation of ther gene (Heatwole and Somerville, 1991; Sarsero andttard, 1991) only l-Phe induces expression of the tyrPne (Kasian et al., 1986). Expression of aroFand aroL,repressed by TyrR, which binds to the TyrR box

(Pittard and Davidson, 1991; Andrews et al., 1991; Wilsonet al., 1994). Zhao et al.(2000) reported that TyrR protein

contains phosphatase activity, which is inhibited by l-Tyrand ATP. Binding of l-Tyr is the conformational triggerfor TyrR in Haemophilus influenzae, where ATP is a co-activator (Kristlet al., 2000). Detailed insights with regardto the TyrR operator complex have been publishedrecently (Sawyer et al., 2000; Howlett and Davidson2000).

The TrpR repressor ofE. coliregulates genes involved inl-Trp synthesis and transport, namely aroH, the trp operonandmtr,and regulates its own expression as well (Gunsalusand Yanofsky, 1980). Expression ofaroLis under the dual

control of both TrpR and TyrR (Heatwole and Somerville,1992) and regulation by TrpR, which is only significant inthe presence of TyrR, is greatest when TyrR is bound to allthree TyrR boxes (Lawley and Pittard, 1994). In addition toaroL, the mtrgene is regulated by TyrR and TrpR, and ithas been suggested these two proteins may interact at themtr operator sites (Sarsero et al., 1991; Yang et al., 1993). Inthis case, however, TrpR, is the dominant regulator andcooperative binding between TyrR and TrpR has not beenshown. The transcription of the gene pheAis regulated byattenuated 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 ared: , transcriptional and allosteric control exerted by the aromatic amino acid end products; , allosteric control only; , transcriptional

ntrol only. Abbreviations used: ANTA, anthranilate; aKG, a-ketoglutarate; CDRP, 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate; CHA,orismate; DAHP, 3-deoxy-d-arobino-heptulosonate 7-phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; EPSP, 5 enolpyruvoylshikite 3-phosphate; E4P, erythrose 4-phosphate; GA3P, glyceraldehyde 3-phosphate; HPP, 4-hydroxyphenlypyruvate, I3GP, indole 3-glycerol

osphate; 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; PEPosphoenolpyruvate; PPA, prephenate; PPY, phenylpyruvate; PRAA, phosphoribosyl anthranilate; PRPP, 5-phosphoribosyl-a-pyrophosphate; Pyruvate; SHIK, shikimate; S3P, shikimate 3-phosphate.

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In addition to regulation at the expression level, allos-ric inhibition of the committed reaction of DAHPnthasesand thebranchpoint enzymes chorismate mutase/ephenate dehydratase, chorismate mutase/prephenatehydrogenase and anthranilate synthase by the endoducts occurs (reviewed in Pittard, 1996). The onlyzyme that is inhibited by an intermediate in the common

omatic amino acids pathway is shikimate dehydro-nase, which is inhibited by shikimate (aroE, Fig. 1)hibiting linear mixed-type inhibition with a inhibitionnstant of 0.16 mM (Dell and Frost, 1993).

METABOLIC ENGINEERING OF AROMATIC

AMINO ACID PRODUCTION

The precursors of the common aromatic amino acidosynthetic pathway, PEP and E4P (Fig. 1), derive from

ntral metabolism (Fig. 2); PEP is formed during

FIG. 2. Schematic overview of reactions in the central metabolism ofcoli. Abbreviations used: PTS, phosphoenolpyruvate phosphotrans-ase system; G6P dh, glucose-6-phosphate dehydrogenase; Tkt,nsketolase; Tal, transaldolase; Pgi, phosphoglucose isomerase; Ppc,P-carboxylase; Pyk, pyruvate kinase; Pyk, PEP carboxykinase; Pps,P synthetase; G6P, glucose 6-phosphate; F6P, fructose 6-phosphate;

A3P, glyceraldehyde 3-phosphate; PEP, phosphoenolpyruvate; OAA,aloactetate; 6P-Gnt, 6-phosphogluconate; Rul5P, ribulose 5-phos-ate; Rib5P, ribose 5-phosphate; Xul5P, xylulose 5-phosphate; Sed7P,

oheptulose 7-phosphate; E4P, erythrose 4-phosphate.

glycolysis and the pentose phosphate pathway suppliesE4P. To improve the production of aromatic compoundsthe optimization of both the specific biosynthetic pathwayand the carbon flux from central carbon metabolism hasto be addressed (reviewed in Berry, 1996; Frost andDraths, 1995; Liaoet al., 1994).

ENGINEERING CENTRAL CARBON METABOLISM

Increase of E4P Supply

The key enzymes of the nonoxidative pentose phosphatepathway are transketolase and transaldolase. Thesecatalyze reactions that lead to fructose 6-phosphate andglyceraldehyde 3-phosphate linking the pathway toglycolysis and on the other hand to E4P, the precursor ofthe aromatic amino acid biosynthesis. To increase the

availability of E4P in E. coli, the tktA gene encodingtransketolase has been overexpressed in an E. coli strainthat accumulates DAH(P) due to an inactive aroBgene,3-dehydroquinate synthase (Draths and Frost, 1990Draths et al., 1992). Having high DAHP synthase activityby overproducing a feed back resistant DAHP synthase(aroGfbr) and transketolase resulted in additional twofoldincrease of carbon flow from glucose into aromatic bio-synthesis (Draths et al., 1992). With xylose as substrate noincrease in DAH(P) production by overexpression oftktAwas observed (Patnaiket al., 1995). This effect may be due

to sufficient supply of E4P from the high flux through thepentose phosphate pathway under these growth condi-tions. The overproduction of transketolase also raised theproduction of aromatic amino acids in Corynebacterium

glutamicum (Ikeda et al., 1999). It appeared from l-Trpproducing E. coli that transketolase gene overexpressionimposes a metabolic burden leading to retarded growthand segregation of the plasmids (Ikeda and Katsumata,1999; Kim et al., 2000). Minimizing the tktA expressionlevels resulted in stable maintenance of the plasmids.

The impact of transaldolase on the flux into the aroma-

tics pathway was analyzed as well (Lu and Liao, 1997;Sprenger et al., 1998a). Overexpression of talB, signifi-cantly increased the formation of DAH(P) (Lu and Liao,1997) and l-Phe (Sprenger et al., 1998a) from glucoseAdditional overexpression of tktA increased the flux intothe aromatic pathway of an E. coli l-Phe productionstrain (Sprenger et al., 1998a) but not in the DAHP pro-ducing strain (Lu and Liao, 1997). From experiments withPEP synthase expression combined with tktA and talB,respectively, it was concluded that transketolase is moreeffective 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 hasen made by deleting the pgi gene that encodes theosphoglucose isomerase (Mascarenhas et al., 1991).ithout pgiactivity, glycolysis is blocked and the carbonx is diverted into the pentose phosphate pathway. In an

Trp producing E. colistrain, the pgideletion resulted inalmost twofold more efficient conversion of glucose to

Trp, but reduces the growth rate.

crease of PEP Supply

Phosphoenolpyruvate is a key intermediate involved inveral cellular processes (reviewed in Valle et al., 1996). Inld-type E. colithe major PEP consumer is the phospho-

ansferase system, PTS, responsible for uptake andosphorylation of glucose at the same time (reviewed instma et al., 1996). Other PEP consuming enzymes areosphoenolpyruvate carboxylase, Ppc, and the pyruvate

nases, PykA and PykF. Additionally, there are PEP-rming reactions, such as phosphoenolpyruvate synthase,s, and phosphoenolpyruvate carboxykinase, Pck, that

t in gluconeogenesis (Fig. 2).The maximum theoretical molar yield for DAHPnthesis from glucose is 0.43 mol/mol. This yield can beubled if either pyruvate formed during glucose uptake is

cycled to PEP or glucose uptake and phosphorylationcomes PEP-independent (Frberg et al., 1988; Patnaikd Liao, 1994). An approach to avoid PEP consumptionring substrate uptake is to use a non-PTS carbon

urce, such as xylose, reaching a maximum theoreticaleld for DAH(P) of 0.71 mol/mol (Patnaik et al., 1995).nce the synthesis of l-Phe requires an additional mole-le of PEP, a theoretical yield of 0.3 mol/mol on glucose

as calculated, and 0.6 mol/mol without loss of PEPrberget al., 1988).Using stoichiometric pathway analysis, a novel pathwayrecycle pyruvate was proposed (Liao et al., 1996). Thispothetical cycle was considered to consist of PEPrboxykinase (pck) and the glyoxylate shunt, but couldt be proven by further investigations. It was however

t excluded that some pyruvate recycling might occur vias route in DAH(P) production experiments.By inactivation of the PEP carboxylase in an l-Pheoducing strain of E. coli, the formation of l-Phe wasnificantly increased, but the production of the un-

anted by-products acetate and pyruvate increased as wellMiller et al., 1987). Moreover, the growth of the

c-negative strain was reduced twofold and the additiona C4-dicarboxylate, such as succinate, is required for

owth. In a DAH(P) producing strain the deletion of thec gene did not lead to any positive effect (Patnaik and

ao, 1994). This discrepancy was explained with the

different conditions used, nongrowth versus growth, andthe phenotypic differences between the host strains.

The two pyruvate kinases ofE. colirepresent anotherPEP consuming activity. Inactivation of either gene causedhardly any effect, but simultaneous inactivation of bothgenes significantly increased carbon flow from glucose intoDAH(P) (Berry, 1996; Gosset et al., 1996) and l-Phe

(Grinter, 1998). Combined with growth on non-PTS sub-strates (e.g., maltose, lactose) the increase was even higher,but growth was poor (Grinter, 1998).

Pyruvate produced via the PTS is lost for the aromaticpathway because pyruvate is not recycled to PEP underglycolytic conditions. By overexpression of the gene ppsthat encodes PEP synthase pyruvate is converted back intoPEP and the carbon flux was successfully directed towardDAH(P) production (Patnaik and Liao, 1994). This posi-tive pps effect was only significant with concomitantoverexpression of a feedback-deregulated DAHP synthase

and transketolase gene tktA, suggesting that the concen-tration of E4P is the first limiting substrate for DAHPsynthase, followed by PEP (Liao et al., 1996). Instead ofchanneling PTS-derived pyruvate back into PEP, the PTScan 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 theoreticalyields with high level of DAHP synthase activity alone,i.e., no further increase of the yield by transketolase orPEP synthase overexpression was observed (Patnaik et al.,1995).

From a PTS-negative E. coli mutant, a glucose-positiverevertant was isolated (Flores et al., 1996). This strainchanneled glucose via galactose permease (galP), into thecell and grew on glucose with rates comparable to wildtype. In strains that at the same time overproduced DAHPsynthase, an increase of DAH(P) excretion into themedium was observed (Flores et al., 1996; Gosset et al.,1996; Berry, 1996). Recently, the effect of PTS inactivationand GalP dependent glucose transport has been furtheranalyzed in isogenic strains with a block after the firstintermediate of the aromatic amino acid pathway (Bez

et al., 2001). The DAH(P) yield on glucose increased sig-nificantly corresponding to 83% of the maximum theo-retical yield. Independently, Chen at al. also constructed a

pts-negative E. colistrain that uses the galactose permease,GalP, for glucose uptake (Chen et al., 1997), howeverneither the PEP pool was increased nor l-Phe productionwas enhanced in the non-PTS strain. Stoichiometricanalysis confirmed the before mentioned positive effect ofGalP on the theoretical yield of l-Phe, but regarding thetheoretical energy yield GalP has a major disadvantagethe GalP system requires higher amounts of ATP to

phosphorylate glucose.


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The use of heterologous, PEP independent glucosetake systems was suggested to save PEP for the

osynthesis of aromatics (Frost and Draths, 1995). It hadready been shown that expression of the Zymomonasobilis glucose facilitator (glf) and glucokinase (glk)stored glucose uptake and phosphorylation in a glucosegative E. colimutant (Snoep et al., 1994; Weisser et al.,

95). The expression of both Zymomonas genes glfandk in a PTS-negative E. coli, devoid of DHQ synthasetivity, resulted in an increase of DAH(P) excretionrmer, 2000). Likewise, a positive effect of glfand glkpression on l-Phe production was shown in PTS-posi-e as well as in PTS-negative strains. In the latter case

e glfgene was chromosomally integrated into the ptsgion to disrupt the PTS genes (Sprenger et al., 1998b).A combined approach, avoiding PEP consumption by

TS and enhancing the flux through the pentoseosphate pathway thereby increasing the availability of

4P, was performed by substituting the PTS with theucose facilitator and by expression of glucosehydrogenase gene gdh from Bacillus megateriumand theuconate kinase gene gntK from E. coli (Krmer et al.,99; Krmer, 2000). Glucose is taken up via the facilita-r into the cell, where it is oxidized to gluconate andbsequently phosphorylated to gluconate 6-phosphate,hich is an intermediate of the oxidative pentoseosphate pathway. With this new pathway, the produc-n ofl-Phe could be increased (Krmer et al., 1999).A new approach called global metabolic engineering

as introduced, when a global regulatory network wasanipulated that controls carbon flux through the centralrbon pathways (Tatarko and Romeo, 2001). Thesruption of the csrA gene, carbon storage regulator,

mong other things influences the regulation of severalzymes that participate in PEP metabolism resulting inelevation of the intracellular PEP pool (Sabnis et al.,

95). This ability to increase PEP was translated to anPhe producing E. coliand a twofold increase of l-Pheas determined (Tatarko and Romeo, 2001).

ENGINEERING OF THE AROMATIC AMINOACIDS PATHWAY

leviation of Feedback Inhibition

In wild-type E. coligrown on minimal media, the l-Tyr-edback inhibited DAHP synthase (aroF) contributes% and the l-Phe-feedback inhibited DAHP synthase

roG) contributes 80% of the total enzyme activity (Tribeal., 1976). The l-Trp-feedback inhibited DAHP-nthase (aroH) has only a marginal contribution to the

tal DAHP-synthase activity. In aromatic amino acid

producing strains, DAHP synthase activity is stronglyreduced as a result of feed back control by the end pro-ducts. Regulation at the transcriptional level is alleviatedby placing the regulated genes behind promoters that arenot controlled by TrpR/TyrR or by deletion of theregulators (LaDucaet al., 1999; Berry, 1996).

To overcome allosteric inhibition of aromatic amino

acid pathway reactions, amino acid analogues have beensuccessfully used to isolate feedback inhibition resistantmutants (Hagino and Nakayama, 1974; Shiio et al., 1975;Ray et al., 1988; De Boer and Dijkhuizen, 1990). Anumber of l-Tyr feedback inhibition resistant DAHPsynthase mutants have been characterized: Pro148Leu(Weaver and Herrmann, 1990) and Gln152Ile mutations(Edwards et al., 1987) of the E. coli aroF gene productresulted in a tyrosine-feedback resistant phenotype. At theN-terminal end, an Asn8Lys substitution in AroF fromE. coliled to an l-Tyr-insensitive DAHP synthase as well

(Jossek et al., 2001). In Corynebacterium l-Tyr feedback-insensitive DAHP synthase mutants Ser187Cys, Ser187Tyrand Ser187Phe were obtained, whereas Ser187Ala showed nosignificant effect (Liaoet al., 2001).

Feedback inhibition by l-Phe is suppressed by aLeu76Val mutation in the aroGgene product and muta-tions in AroG between residues 146-150 affected inhibitionby l-Phe (Kikuchi et al., 1997). In the crystal structure ofthe AroG from E. coli, mutations that reduce feedbackinhibition cluster around a cavity near the twofold axis ofthe tight dimeric structure at approximately 15 from the

active site (Shumilin et al., 1999). Eight other feedbackresistant DAHP synthase mutants ofaroFand aroGhavebeen described (Tonouchi et al., 1997). Mutagenesis wasalso used to identify residues and regions of the AroHpolypeptide essential for catalytic activity and l-Trpfeedback regulation (Ray et al., 1988). Feedback resistantchorismate mutase prephenate dehydratase mutants fromE. coli have been made by modifying Trp226 and Trp338(Gethinget al., 1976) and by substituting Ser330 or deletingamino acid residues downstream from this residue(Tonouchiet al., 1997). Mutations in codons 304 to 310 of

the pheA gene exhibited almost complete resistance tofeedback inhibition even at very high l-Phe concentrations(Nelms et al., 1992). The interaction of l-Phe with theregulatory domains of chorismate mutase prephenatedehydratase has been investigated in more detail (Zhanget al., 1998; Pohnert et al., 1999). A feedback resistantmutant of coryneform bacteria prephenate dehydratasewas obtained (Ozakiet al., 1985).

The anthranilate synthase-phosphoribosyl transferaseenzyme complex which catalyzes the first two steps in ofl-Trp biosynthesis is feedback inhibited by l-Trp. This

is the result from allosteric effects associated with the


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nding of one molecule of inhibitor to each of the TrpEbunits of the complex in the case of S. typhimurium.

aligiuri and Bauerle (1991a,b) generated a collection ofpEmutants, which displayed varying degrees of resis-nce to feedback inhibition.

gineeringl-Phe Production

Microbial production of l-phenylalanine has beencused mainly on E. coli, C. glutamicum and Brevibac-ium strains (De Boer and Dijkhuizen, 1990). Classic

ethods have been applied to screen for auxotrophs andutants with feedback deregulated key enzymes. Anerview of analog resistant mutants ofB. lactofermentum,flavum, C. glutamicum, and E. coli is given in de Boer

d Dijkhuizen (1990).Sugimoto et al. cloned the genes encoding feedbacksistant forms of DAHP synthase, aroFfbr, and choris-ate mutase/prephenate dehydratase, pheA fbr, into a

mperature-controllable expression vector (Sugimotoal., 1987). An l-Tyr auxotrophic E. coli strain carryings plasmid had a maximal l-Phe production of 16.8 g/Lthe optimal temperature, 38.5 C (Sugimoto et al.,

87). Additional process development using this strain im-oved the l-Phe fermentation process significantly, reach-g a titer of 46 g/L and a productivity of 0.85 g/Lhonstantinov et al., 1991; Konstantinov and Yoshida,92; Takagiet al., 1996).Miller et al.used a plasmid carrying a wild-type DAHPnthase gene, aroF, and a feedback resistant chorismate

utase/prephenate dehydratase gene, pheAfbr

(Milleral., 1987; Backman and Balakrishnan, 1988). E. colils carrying this plasmid were analyzed with regard to the

fects of PEP carboxylase deficiency on the l-Phe yield.coli strains auxotrophic for all three aromatic aminoids, but equipped with a plasmid encoding the wild-typeoFgene and a feedback resistant chorismate mutase/ephenate dehydratase, pheAfbr was constructed (Frbergd Hggstrm, 1987). With nongrowing cells, theaximum theoretical yield of l-Phe on glucose wasached in batch cultures after depletion ofl-Tyr (Frberg

al., 1988). Tyrosine-limited, glucose fed-batch culturesproved l-Phe production by applying proper feed ratesr l-Tyr and glucose (Frberg and Hggstrm, 1987).ckman et al. also engineered E. coli for l-Phe produc-n, based on the aroFWT and pheAfbr genes and devel-ed an efficient fermentation process and within 36 h aal l-Phe titer of 50 g/L with a yield on glucose of

23 g/g could be reached (Backman et al., 1990). Anerview of the efforts at the Nutrasweet Company totain l-phenylalanine producing E. coli strains, which

clude mechanisms for l-Phe export, were summarized by

otheringhamet al.(1994) and Grinter (1998).

Metabolic engineering of C. glutamicum resulted in anl-Phe producing strain that, when additionally equippedwith a plasmid encoding chorismate mutase and prephe-nate dehydratase, increased l-Phe accumulation about50% (Ozaki et al., 1985; Ikeda and Katsumata, 1992Ikeda et al., 1993). An l-Trp producing Corynebacteriumstrain was made suitable for l-Tyr or l-Phe production

by introducing feedback resistant variants of DAHPsynthase, chorismate mutase and prephenate dehydratase,by combining the genes on one plasmid. By doing so, thecarbon flow was altered to produce up to 26 g/L l-Phe(Ikeda and Katsumata, 1992). Heterologous expression ofa feedback resistant mutant of chorismate mutase/prephenate dehydratase fromE. coliin an l-Phe producingC. glutamicum strain resulted in a significant increase ofthe productivity (Ikedaet al., 1993).

Engineeringl

-Tyr Production

To obtain l-Tyr overproducers, most attention has beenfocused on screening for regulatory and auxotrophicmutants. These were mostly strains of E. coli, Bacillussubtilisor various coryneform bacteria (Maiti et al., 1995)By application of recombinant DNA technology addi-tional improvements of these l-Tyr producing strains havebeen made (Ito et al., 1990; Ikeda and Katsumata, 1992;Ikedaet al., 1999).

Additionally improved strains have been generated bymetabolic engineering of an l-Phe auxotrophic Brevibac-terium lactofermentum (Ito et al., 1990). To overcome akey-limiting step of the aromatic amino acid pathway, theshikimate kinase gene was introduced and the impact ofthe expression of shikimate kinase on l-Tyr productionwas investigated. The engineered strain demonstrated afive to 10-fold increase in the enzyme activity and a signi-ficant increase of l-Tyr titer (Ito et al., 1990). The geneticengineering of an l-Trp producing mutant of C. glutami-cum 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. glutami-cum (Ikeda et al., 1999; Katsumata and Ikeda, 1997)Approaches to increase availability of E4P were carriedout by overexpression of the homologous transketolase inan l-Tyr producing strain ofC. glutamicum,resulting in a1050% increase of the titer ofl-Tyr (Ikedaet al., 1999).

Engineeringl-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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the efforts to develop a fermentative route to the bluee indigo from indole (Ensley et al., 1983), has primarilyen carried out in E. coli (reviewed in Berry, 1996),

orynebacterium(Ikeda and Katsumata, 1999; Katsumatad Ikeda, 1993) and Bacillus(Kurahashiet al., 1984).To obtain l-Trp-producing strains, alterations in thentral carbon metabolism and the common aromatic

mino acid pathway, including its regulation, were carriedt as described above. In addition, overexpression of thenes encoding the tryptophan branch of the aromatic

mino acid pathway was performed (Berry, 1996).Deletion of the pheA and tyrA genes to prevent thensumption of chorismate would have resulted in l-Phed l-Tyr auxotrophies, thereby increasing productionsts. Such deletions were not required in strains thaterexpressed the trpE gene, since anthranilate synthases a higher affinity for chorismate than PheA or TyrBopheide et al., 1972; Hudson et al., 1983; Baker and

awford, 1966), resulting in only little loss of carbon toPhe and l-Tyr (LaDuca et al., 1999). Removal of theaAgene that encodes tryptophanase, which catalyzes thenversion of l-Trp to indole and pyruvate, was effec-ely used to prevent product loss (Aiba et al., 1982). The

verse reaction of tryptophanase has been used to convertruvate producing Enterobacter aerogenes into l-Trpoduction strains (Yokota et al., 1989). Interestingly,

ansport mutants of Corynebacteriumthat were impairedl-Trp uptake were shown to be more effective in pro-ction, which was ascribed to changes in intracellular

ncentrations resulting in a change of regulation. Amilar effect was reached by addition of nonionictergents, that resulted in l-Trp precipitation and provedore productive (Azumaet al., 1993).

ENGINEERING PRODUCTION OF DERIVED

COMPOUNDS AND INTERMEDIATES

Dehydroshikimic Acid Production

3-Dehydroshikimic acid (DHS) is an intermediate in theomatic amino acid pathway and was shown to serve as aitable starting compound for the renewable productiona variety of industrial chemicals, ranging from catechol,nillic acid to adipic acid (Li et al., 1999). In addition,HS can be used as a potent antioxidant (Richman et al.,96).Fermentative production of DHS from glucose wascomplished by engineered shikimate dehydrogenaseroE)-deficient E. coli mutants, in which a gene codingr a feedback resistant DAHP synthase (aroFfbr ) and a

cond copy of the aroBgene, encoding DHQ synthase,

were introduced. Production of DHS was associated withthe formation of dehydroquinate and gallic acid, whichcould be products of abiotic conversion reactions(Richman et al., 1996). Gallic acid production could alsobe due to formation of protocatechuic acid by DHSdehydratase, followed by a hydroxylation step (Li andFrost, 1999). To increase the availability of E4P, tktA

was introduced into the DHS producing strain, whichresulted in an increase of the DHS titer and the yield to0.3 mol/mol on glucose (Liet al., 1999). By using pentosesugars an improvement of DHS yield was observed rela-tive to the use of glucose (Li and Frost, 1999). Over-expressing the transketolase gene resulted in an increasedyield 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 suffi-cient, but PEP supply was limited for carbon flux into thearomatic amino acid pathway.

Shikimate Production

Because of three chiral centers in the molecule, shikimicacid is a suitable starting compound for the synthesis ofneuramidase inhibitors for the treatment of influenza(Zhang, 1998). Shikimate is also interesting as a startingcompound for combinatorial libraries (Tan et al., 1999).

A classical approach to obtain shikimate producingstrains has been described using Citrobacter freudii(Shiraiet al., 1999). Microbial production of shikimate was

drastically improved by metabolic engineered E. colistrains (Draths et al., 1999; Frost et al., 1999), whichcarried disrupted aroL and aroK genes. To circumventpolar effects caused by aroKdisruption and to overcomethe rate limiting DHQ synthase step, aroBwas combinedwith the gene coding for a feedback resistant DAHPsynthase aroFfbr. Furthermore, an additional gene codingfor shikimate dehydrogenase, as compensation for theenzymes feedback inhibition by shikimate, was intro-duced (Draths et al., 1999). The fermentative productionof shikimate was associated with the formation of

quinic acid as a side product, presumably caused by theequilibria of initially synthesized shikimate via dehydro-shikimate to quinic acid (Draths et al., 1999; Frostet al., 1999). Reducing shikimate re-uptake by adding anonmetabolizable d-glucose analogue could drasticallyreduce the formation of quinic acid (Draths et al., 1999;Frost et al., 1999). Approaches to further improve shiki-mic acid production by E. coli, by increasing the availa-bility of PEP and E4P have also been made (Gibson et al.,2001). The PTS of a shikimate producing strain was sub-stituted by glf/glk (Gibson et al., 2001) leading to an

increased availability of PEP. To increase availability of


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4P, the tktA gene was also introduced and the yieldcreased from 0.18 to 0.27 mol/mol (Gibsonet al., 2001).Production of shikimate was also carried out by a

assically screened B. subtilis strain (Iomantas et al.,00). As opposed to E. coli, a Bacillus strain carryingly one defect allele of shikimate kinase producedikimate and dehydroshikimate as a side-product in rela-

e high amounts. Increasing the activity of shikimatehydrogenase by introducing the corresponding gene

om Bacillus amyloliquefaciens successfully improved theikimate production of the strain (Iomantas et al., 2000).

oduction ofd-Phenylalanine

The fermentative production of d-phenylalanine wasrformed with an E. coli strain lacking all three trans-

minase genes responsible for l-phenylalanine formation

om phenylpyruvate. The carbon flux through the aro-atic amino acid pathway toward phenylpyruvate wascreased by expressing the DAHP synthase gene aroHd a feedback resistant chorismate mutase/prephenatehydratase, pheA fbr. To produce d-phenylalanine, a re-ective d-aminotransferase together with an alaninecemase was expressed (Fotheringhamet al., 1998).

CONCLUSIONS

Metabolic engineering of the central metabolism inder to improve the biosynthesis of aromatics is almoststricted to E. coli, and less work has been done with C.utamicum. By now, several modifications proved to beluable, but most impressing results were obtained whenproaches were combined to show a synergistic effect:tnaik and Liao (1994) attained a near theoretical yieldDAH(P) by overexpressing transketolase together with

EP synthase, and Gosset et al. (1996) used a PTS-nega-e glucose+ mutant, additionally inactivated both pyru-te kinases and amplified the transketolase taken

gether a 20-fold increase in carbon flow into DAH(P)as achieved.In general, changes in central pathways have the stron-st effects when the impact is determined as carbon fluxto DAHP, the first intermediate of the aromatic bio-nthesis pathway. However, to produce end products,omatic amino acids or compounds derived fromorismate, one must keep in mind that an extra moleculePEP enters the pathway later, which can influence thelance of precursor supply. Unexpected interconnectionstween the central metabolism and the biosynthesis

thway may be detected, which have to be circumvented

subsequently. An example is the inhibition of the trans-ketolase from Saccharomyces cerevisiae by p-hydroxy-phenylpyruvate, the penultimate intermediate in the bio-synthesis ofl-Tyr (Solovjeva and Kochetov, 1999).

Although various metabolic engineering approachesincrease the yield, many of these in turn slow down thegrowth or just deteriorate the performance of a produc-

tion process. Therefore, the transfer of promising resultsfrom lab-scale experiments to industrial processes is stilldifficult.

ACKNOWLEDGMENT

Financial support from the BioRegio program of the Bundesminis-terium fr Bildung und Forschung (BMBF, Grant 0311644) is gratefullyacknowledged.

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