Production of phenylalanine ammonia-lyase (PAL): isolation and evaluation of yeast strains suitable for commercial production of l-phenylalanine

Summary Phenylalanine Ammonia-Lyase (PAL) containing microorganisms were isolated from a wide variety of natural habitats. The best 21 strains to emerge from the primary screen were screened for PAL activities in both directions using l-phenylalanine and t-cinnamate substrates. Twelve of the latter strains were compared for total cell production and PAL activity and 7 isolates were chosen for examination of the extent of PAL induction in various media. On the basis of these screens, isolate SPA 10 (identified as Rhodotorula rubra) was selected for further optimization. Growth was optimal at 28° C and pH 5.0, although cellular PAL activity was shown to be higher at sub-optimal temperatures (36° C) and pH (8.0) for growth. Synthesis of PAL was repressed when grown in the presence of various sugars and NH ₄ ⁺ ions. Manipulation of fermentation conditions enabled PAL synthesis to occur at maximum biomass levels, upon glucose exhaustion. PAL was rapidly inactivated within cells shortly after maximum synthesis was attained: feeding of d,l-isoleucine and low concentrations of d,l-phenylalanine, and shifting of fermentation temperature conferred catalyst stability for fermentations over 100 h. These results demonstrate the suitability and superiority of isolate SPA 10 for the commercial production of l-phenylalanine from trans-cinnamic acid.     

Biotransformation of phenylpyruvic acid to l-phenylalanine using a strain of Pseudomonas fluorescens ATCC 11250 with high transaminase activity

The rate of l-phenylalanine production from phenylpyruvic acid by whole cells of Pseudomonas fluorescens strain ATCC 11250 was greater than 3 g·l^-1 h^-1. Synthesis of transaminase was constitutive but activity was greatest in medium containing d- or l- phenylalanine as sole nitrogen source. Maximum conversion was observed at 34–40° C and at alkaline pH, with over six times initial rate of conversion at pH 12 than at pH 5. The optimum catalyst (cell) concentration was between 10–20 mg ml^-1 dry weight. The initial rate of conversion was directly proportional to phenylpyruvate concentration, up to 4%, but the conversion yield steadily decreased between 2% and 4% substrate concentration. The rate of conversion, as expected, increased as the concentration of glutamate increased. Whole cells were still capable of over 63% conversion after 40 days providing reactions were supplemented with pyridoxal phosphate. Immobilisation of cells in calcium alginate and operation of a packed bed bioreactor enabled the continuous production of l-phenylalanine in concentrations greater than 15 g·l^-1 after 60 days operation.     

Isobutene production by Rhodotorula minuta

Summary Isobutene production by Rhodotorula minuta IFO 1102 was studied. It was confirmed that the gas species produced by this yeast was isobutene from the result of analysis with a gas chromatograph mass spectrometer. Oxygen supply was essential to the microbial production of isobutene. The optimum pH was found to be approximately pH 6.0 and optimum temperature 25°–27° C. Isobutene production rate was maximal when l-leucine and l-phenylalanine in the medium were being uptaken by the yeast.The results from an investigation of the role of l-leucine and l-phenylalanine suggested that l-leucine was the precursor of isobutene and l-phenylalanine the inducer for the enzyme concerned with isobutene production.     

Biotransformation of alkyl and aryl carbonates: enantioselective hydrolysis

Summary N-(Benzyloxycarbonyl)-l-asparty-l-phenylalanine methyl ester, the precursor of the synthetic sweetener aspartame, was continuously synthesized in an immobilized thermolysin plug-flow type reactor at 25° C with the substrates (N-benzyloxycarbonyl-l-aspartic acid and l-phenylalanine methyl ester) dissolved in ethyl acetate. The immobilized enzyme was quite stable in ethyl acetate containing 2.5% 0.01 M 2-(N-morpholino)ethanesulphonic acid-NaOH buffer, pH 6.0, and 20 mM CaCl₂ with or without the substrate at 25° C. By periodically washing the column, we could conduct a continuous reaction for over 500 h with an average yield of 95% and a space velocity of 1.85 h ^–1.Offprint requests to: K. Nakanishi     

The differential allosteric regulation of two chorismate-mutase isoenzymes of Nicotiana silvestris

The reaction catalyzed by chorismate mutase (EC 5.4.99.5) is a crucial step for biosynthesis of two aromatic amino acids as well as for the synthesis of phenylpropanoid compounds. The regulatory properties of two chorismate-mutase isoenzymes expressed in Nicotiana silvestris Speg. et Comes are consistent with their differential roles in pathway flow routes ending with l-phenylalanine and l-tyrosine on one hand (isoenzyme CM-1), and ending with secondary metabolites on the other hand (isoenzyme CM-2). Isoenzyme CM-1 was very sensitive to allosteric control by all three aromatic amino acids. At pH 6.1, l-tryptophan was a potent allosteric activator (K _a =1.5 M), while feedback inhibition was effected by l-tyrosine (K _i =15 M) or by l-phenylalanine (K_i=15 M). At pH 6.1, all three effectors acted competitively, influencing the apparent K _m for chorismate. All three allosteric effectors protected isoenzyme CM-1 at pH 6.1 from thermal inactivation at 52° C. l-Tryptophan abolished the weak positive cooperativity of substrate binding found with isoenzyme CM-1 only at low pH. At pH 7.2, the allosteric effects of l-tyrosine and l-tryptophan were only modestly different, in striking contrast to results obtained with l-phenylalanine. At pH 7.2 (i) the K _i for l-phenylalanine was elevated over 30-fold to 500 M, (ii) the kinetics of inhibition became non-competitive, and (iii) l-phenylalanine now failed to protect isoenzyme CM-1 against thermal inactivation. l-Phenylalanine may act at different binding sites depending upon the intracellular pH milieu. In-vitro data indicated that the relative ability of allosteric activation to dominate over allosteric inhibition increases markedly with both pH and temperature. The second isoenzyme, CM-2, was inhibited competitively by caffeic acid (K _i =0.2 mM). Aromatic amino acids failed to affect CM-2 activity over a broad range of pH and temperature. Inhibition curves obtained in the presence of caffeic acid were sigmoid, yielding an interaction coefficient (from Hill plots) of n=1.8.Abbreviation DAHP synthase 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase     

Modification of l-phenylalanine ammonia-lyase in soybean cell suspension cultures by 2-aminooxyacetate and l-2-aminooxy-3-phenylpropionate

Suspension-cultured cells of soybean (Glycine max (L.) Merr. cv. Kanrich) produce large amounts of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), the first enzyme of phenylpropanoid metabolism, during growth. 2-Aminooxyacetic acid (AOA) and l-2-aminooxy-3-phenylpropionic acid (l-AOPP) inhibit the enzyme competitively in vitro and have been used for in vivo studies. The amount of extractable enzyme in the cells and their utilization of NO ₃ ^– and NH ₃ ⁺ are reduced upon the addition of AOA. When AOA was added at various times during growth, the appearance of additional enzyme activity was prevented but enzyme already formed was not inhibited. No evidence was obtained for the presence of an inhibitor in the extracts and AOA inhibition in vitro was readily reversible. It is conculded that AOA acts to inhibit the formation of PAL in suspension-cultured soy bean cells. In vitro inhibition of soybean PAL by l-AOPP could not be reversed; in contrast, the inhibition of maize (Zea mays L.) PAL was readily reversible. Added l-AOPP, which was rapidly taken up by the soybean cells, prevented the large increase in enzyme activity. Although PAL activity was blocked in the cultures, no appreciable increase in phenylalanine content could be detected in cell extracts. The response of soybean cell suspensions to l-AOPP addition thus differs from that of other tissues which in presence of l-AOPP show an increase in PAL activity and an accumulation of phenylalanine.Abbreviations AOA 2-aminooxyacetic acid - l-AOPP l-2-aminoxy-3-phenylpropionic acid - PAL l-phenylalanine ammonialyase (EC4.3.1.5)     

Production of l-phenylalanine from phenylpyruvate by Paracoccus denitrificans containing aminotransferase activity

Summary To establish an efficient production method for l-phenylalanine, the production of l-phenylalanine from phenylpyruvate by Paracoccus denitrificans pFPr-1 containing aminotransferase activity was investigated. By using intact cells, 0.74M l-phenylalanine was produced from 0.8M phenylpyruvate (conversion yield, 92.5%). Moreover, by using immobilized cells with -carrageenan, when the space velocity was 0.1 h^-1 at 30°C, 0.135 M l-phenylalanine was produced from 0.15 M phenylpyruvate (conversion yield, 90%). The half-life of the l-phenylalanine-forming activity of the column was estimated to be about 30 days at 30°C.     

The fermentation process for l-phenylalanine production using an auxotrophic regulatory mutant of Escherichia coli

Summary A process for l-phenylalanine production was studied using a tyrosine auxotrophic regulatory mutant of Escherichia coli, resistant to both -2-thienyl-dl-alanine and p-fluoro-dl-phenylalanine. Fermentations were carried out in a 30-1 fermentor with intermittent feeding of glucose plus phosphate. The mutant accumulated l-phenylalanine in the fermentation broth up to 15 g/l at pH 7.0 and 33°C. Column chromatography on a strong cation exchanger was employed as the most effective step in the purification of l-phenyl-alanine from the broth. This step brought about 4-fold concentration of the product with 96% recovery.     

Purification,characterization and regulation of a monomeric l-phenylalanine dehydrogenase from the facultative methylotroph Nocardia sp. 239

In Nocardia sp. 239 d-phenylalanine is converted into l-phenylalanine by an inducible amino acid racemase. The further catabolism of this amino acid involves an NAD-dependent l-phenylalanine dehydrogenase. This enzyme was detected only in cells grown on l- or d-phenylalanine and in batch cultures highest activities were obtained at relatively low amino acid concentrations in the medium. The presence of additional carbon- or nitrogen sources invariably resulted in decreased enzyme levels. From experiments with phenylalanine-limited continuous cultures it appeared that the rate of synthesis of the enzyme increased with increasing growth rates. The regulation of phenylalanine dehydrogenase synthesis was studied in more detail during growth of the organism on mixtures of methanol and l-phenylalanine. Highest rates of l-phenylalanine dehydrogenase production were observed with increasing ratios of l-phenylalanine/methanol in the feed of chemostat cultures. Characteristic properties of the enzyme were investigated following its (partial) purification from l- and d-phenylalanine-grown cells. This resulted in the isolation of enzymes with identical properties. The native enzyme had a molecular weight of 42 000 and consisted of a single subunit; it showed activity with l-phenylalanine, phenylpyruvate, 4-hydroxyphenyl-pyruvate, indole-3-pyruvate and -ketoisocaproate, but not with imidazolepyruvate, d-phenylalanine and other l-amino acids tested. Maximum activities with phenylpyruvate (310 mol min^-1 mg^-1 of purified protein) were observed at pH 10 and 53°C. Sorbitol and glycerol stabilized the enzyme.Abbreviations RuMP ribulose monophosphate - HPS hexulose-6-phosphate synthase - HPT hexulose-6-phosphate isomerase - FPLC fast protein liquid chromatography     

Isolation and characterization of acetamidocinnamate acylase,a new enzyme suitable for production of l-phenylalanine

Summary A new acylase catalyzing the deacetylation of acetamidocinnamic acid (ACA) was found in strains of Brevibacterium sp. Such strains could be isolated from soil samples by their ability to grow on ACA as well as on l-phenylalanine. A 110-fold enrichment of the enzyme with an over-all yield of 48% was obtained in 4 steps resulting in an electrophoretically pure preparation of 28.6 U·mg^-1. Important enzymological data concerning the application of the enzyme are: K _M (ACA) 0.45 mM, pH-optimum 7.5, heat stability up to 52°C, molecular weight of 50.000 Dalton, two subunits. Deacetylation of ACA resulted in phenylpyruvate via the unstable enamine-imine derivative. Coupling the acylase with l-phenylalanine dehydrogenase proved to be an alternative route for l-phenylalanine production avoiding substrate inhibition by phenylpyruvate and its instability. The substrate specifity of ACA-acylase revealed that the enzyme probably acts as a dipeptidase in its biological function.Abbreviations ACA acetamidocinnamate, acetamidocinnamic acid - FPLC fast protein liquid chromatography - pheDH l-Phenylalanine dehydrogenase - HicDH Hydroxyisocaproate dehydrogenase - OD optical density - BSA bovine serum albumin - FDH formate dehydrogenase Dedicated to Professor Dr. H. J. Rehm on the occasion of his 60th birthday     

Enzymatic production of l-phenylalanine from the racemic mixture of d,l-phenyllactate

Summary The production of l-phenylalanine from the racemate d,l-phenyllactate in an enzyme membrane reactor has been examined. In a first step the racemate is dehydrogenated to the prochiral intermediate phenylpyruvate by the enzymes d-and l-hydroxyisocaproate dehydrogenase. In a second step phenylpyruvate is reductively aminated to l-phenylalanine by l-phenylalanine dehydrogenase. Both steps are dependent on coenzyme, the first one requires NAD, the second one NADH in stoichiometric amounts; in this way the coenzyme is regenerated and only required catalytically. The coenzyme is covalently bound to polyethylene glyco-20 000 and can thus be retained in the reactor analogously to the three enzymes. In order to optimize the continuous production of l-phenylalanine from d,l-phenyllactate, models of the reaction kinetics and of the reactor system have been set up. By means of the reactor model, we can calculate the optimum ratio of the three enzymes, the optimum coenzyme concentration and the optimum phenylpyruvate concentration in the feed.In this process, at a substrate concentration of 50 mM d,l-phenyllactate we reached a spacetime-yield of 28 g l-Phe/(l*d).Abbreviations PEG polyethylene glycol - d-HicDH d-hydroxyisocaproate dehydrogenase - l-HicDH l-hydroxyisocaproate dehydrogenase - PheDH l-phenylalanine dehydrogenase - V _max maximum velocity - K _M Michaelis-Menten constant - K _l inhibition constant - R₁ reaction rate of the d-HicDH forward reaction - R₂ reaction rate of the d-HicDH reverse reaction - R₃ reaction rate of the l-HicDH forward reaction - R₄ reaction rate of the l-HicDH reverse reaction - R₅ reaction rate of the PheDH forward reaction - R₆ reaction rate of the PheDH reverse reaction - d-PLac d-phenyllactate - l-PLac l-phenyllactate - PPy phenylpyruvate - l-Phe l-phenylalanine - NH₄ ammonium - residence time     

Isolation,identification and characterisation of a soil bacterium producing an enzyme with l-phenylalanine oxidase activity

A gram-positive, mesophilic bacterium which assimilated l-phenylalanine but which failed to utilise l-tyrosine was isolated from soil. The isolate, identified as a strain of Bacillus carotarum, converted l-phenylalanine to phenylpyruvate with the initial step catalysed by an inducible, intracellular enzyme which possessed l-phenylalanine oxidase activity. Phenylalanine oxidase has not been previously reported in Gram-positive bacteria, although there are a few examples of non-specific l-amino acid oxidases with activity towards l-phenylalanine. The isolate grew abundantly on complex media but failed to synthesise significant amounts of the enzyme in the absence of l-phenylalanine. The highest enzyme levels were achieved in a chemically defined minimal salts medium containing the amino acid at 10 g/l as the primary carbon and energy source.     

l-Phenylalanine dehydrogenase from Brevibacterium sp. for production of l-phenylalanine by reductive amination of phenylpyruvate

Summary For production of l-phenylalanine the reductive amination of phenylpyruvate, catalyzed by phenylalanine-dehydrogenase was examined. To reach high levels and a sufficient stability of the inducible intracellular enzyme, growth conditions of Brevibacterium sp. are optimized. For continuous production of l-phenylalanine in an enzyme membrane reactor, the kinetic parameters of the partially purified enzyme are determined.In continuous production a space time yield of 37.4 g l-Phe l^-1 d^-1 can be reached.By means of the measured kinetic parameters and simultaneous calculation of the mass balances of all reaction components the behaviour of the reactor can be simulated. For certain conditions the multi-enzyme-system shows multiple steady-states.Abbreviations l-phe l-phenylalanine - phepy phenylpyruvate - PEG polyethylenglycol - pheDH l-phenylalanine dehydrogenase     

Phenylalanine ammmonia-lyase activity in permeabilised yeast cells (Rhodotorula glutinis)

Summary Whole cells of the yeast Rhodotorula glutinis IFO 0559 had low phenylalanine ammonia-lyase (PAL) activity due to a limited membrane permeability barrier for phenylalanine. Permeabilization with detergents and organic solvents increased cellular PAL activity significantly and of these cetyltrimethylammonium bromide gives the maximum increase (6-fold). PAL activity of such detergent permeabilized cells could be used for the synthesis and degradation of l-phenylalanine.     

Repeated batch and continuous syntheses of N-(benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester with immobilized thermolysin

Summary N-(Benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester was synthesized from N-(benzyloxycarbonyl)-l-phenylalanine and l-phenylalanine methyl ester in an aqueous solution (aqueous phasic reaction), in an aqueous/organic biphasic system (biphasic reaction), and in an organic solvent (organic phasic reaction) with immobilized thermolysin. In the aqueous phasic reaction with thermolysin immobilized on Amberlite XAD-7, the whole product was trapped inside the support; extraction with ethyl acetate was needed to recover the product, and the equilibrium yield was low (about 65%). With the biphasic and organic phasic reactions with ethyl acetate as an organic solvent, the yield was around 95%. Because of the high yield and feasibility of operation, repeated batch and continuous reactions were done in the biphasic and organic phasic systems, respectively. The half-lives of the activity for the immobilized enzyme used in the biphasic system at 40°C by repeated batch operation and in a plug flow reactor fed with substrate dissolved in ethyl acetate at 40°C and 30°C were estimated to be about 200 h (67 batches), 420 h, and 1100 h, respectively.     

Optimization of reaction conditions and stabilization of phenylalanine ammonia lyase-containing Rhodotorula glutinis cells during bioconversion of trans-cinnamic acid to L-phenylalanine

Studies were performed to elucidate the optimal reaction conditions (pH, temperature, ammonia concentration and biocatalyst loading) for bioconversion of trans-cinnamic acid (t-CA) to L-phenylalanine (L-Phe) by L-phenylalanine ammonia lyase (PAL) containing Rhodotorula glutinis cells. All treatments with permeabilizing agents stimulated L-Phe production and also enhanced instability of the catalyst, except Triton X-100 which gave a superior (56%) increase in conversion as compared to the control and a significant stabilization of PAL enzyme. Inclusion of several activity modifiers and stabilizer additives in reaction mixtures were shown to enhance the yield of L-Phe and maintained PAL stability over several successive incubations during the bioconversion process. Maximum stabilization of PAL and enhancement of L-Phe production was achieved with addition of 20% polyhydric alcohol (glycerol). The production of L-Phe continued to the fifth cycle and the total yield increased 2.3 times compared to the yield produced by the control (without glycerol addition) during the repeated batch process. Reducing agents such as 2-mercaptoethanol and thioglycolic acid were added to the bioconversion mixture in order to reduce the effects of oxygen on PAL catalyst life. Production of L-Phe by addition of 400 mgL(-1) of thioglycolic acid was maximized over the control by 55%. When both 20% glycerol and 400 mgL(-1) thioglycolic acid were simultaneously present in the reaction mixture, reuseability and stability of biocatalyst (PAL) were extended to eight consecutive cycles and conversion rate and overall productivity of L-Phe were higher than that of the control. These results may lead to improvements in the production of the essential amino acid L-Phe.     

A mutant leucine aminopeptidase from <Emphasis Type="Italic">Streptomyces cinnamoneus</Emphasis> with enhanced <Emphasis Type="SmallCaps">l</Emphasis>-aspartyl <Emphasis Type="SmallCaps">l</Emphasis>-amino acid methyl ester synthetic activity

l-Aspartyl l-amino acid methyl ester was synthesized using a mutant of a thermostable leucine aminopeptidase from Streptomyces cinnamoneus, D198 K SSAP, obtained in previously. A peptide of high-intensity sweetener, l-aspartyl-l-phenylalanine methyl ester, was selected as a model for demonstrating the synthesis of l-aspartyl l-amino acid methyl ester. The hydrolytic activities of D198 K SSAP toward l-aspartyl-l-phenylalanine and its methyl ester were, respectively, 74-fold and fourfold higher than those of wild type. Similarly, the initial rate of the enzyme for l-aspartyl-l-phenylalanine methyl ester synthesis was over fivefold higher than that of wild-type SSAP in 90% methanol (v/v) in a one-pot reaction. Furthermore, other l-aspartyl l-amino acid methyl esters were synthesized efficiently using D198 K SSAP. Results show that the substitution of Asp198 of SSAP with Lys is effective for synthesizing l-aspartyl l-amino acid methyl ester.     

The effect of methyl jasmonate on accumulation of 2-phenylethylamine and putrescine in seedlings of common buckwheat (<Emphasis Type="Italic">Fagopyrum esculentum</Emphasis>)

The effects of exogenously applied methyl jasmonate on content of biogenic amines: putrescine, spermidine, tyramine, cadaverine and 2-phenylethylamine in seedlings of common buckwheat (Fagopyrum esculentum Moench) were investigated. The studies have shown that methyl jasmonate stimulates the conversion of l-phenylalanine into 2-phenylethylamine and increases the endogenous levels of putrescine in hypocotyls and cotyledons of buckwheat seedlings. Simultaneous feeding the seedlings with l-phenylalanine and methyl jasmonate has indicated that conversion of l-phenylalanine into 2-phenylethylamine can be one of possible reasons, caused by the methyl jasmonate suppression of anthocyanins synthesis in hypocotyls. To our knowledge, the stimulation of conversion of l-phenylalanine into 2-phenylethylamine by methyl jasmonate, as found in the present study, is described for the first time in higher plants.     

L-Phenylalanine ammonia-lyase fromPhaseolus vulgaris: Modulation of the levels of active enzyme bytrans-cinnamic acid

The extractable activity ofl-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in cell suspension cultures of bean (Phaseolus vulgaris) is greatly induced following exposure to an elicitor preparation from the cell walls of the phytopathogenic fungusColletotrichum lindemuthianum. Following exogenous application oftrans-cinnamic acid (the product of the PAL reaction) to elicitor-induced cells, the activity of the enzyme rapidly declines. Loss of enzyme activity is accompanied by inhibition of the rate of synthesis of PAL subunits, as determined by [³⁵S]methionine pulse-labelling followed by specific immunoprecipitation; this is insufficient to account for the rapid loss of PAL enzyme activity. Pulse-chase and immune blotting experiments indicate that cinnamic acid does not affect the rate of degradation of enzyme subunits, but rather mediates inactivation of the enzyme. A non-dialysable factor from cinnamicacid-treated bean cells stimulates removal of PAL activity from enzyme extracts in vitro; this effect is dependent on the presence of cinnamic acid. Such loss of enzyme activity in vitro is accompanied by an apparent loss or reduction of the dehydroalanine residue of the enzyme's active site, as detected by active-site-specific tritiation, although levels of immunoprecipitable enzyme subunits do not decrease. Furthermore, cinnamic-acid-mediated loss of enzyme activity in vivo is accompanied, in pulse-chase experiments, by a greater relative loss of³⁵S-labelled enzyme subunits precipitated by an immobilised active-site affinity ligand than of subunits precipitated with anti-immunoglobulin G. It is therefore suggested that a possible mechanism for cinnamic-acid-mediated removal of PAL activity may involve modification of the dehydroalanine residue of the enzyme's active site.Abbreviations AOPP l--aminoxy--phenylpropionic acid - CA trans-cinnamic acid - PAGE polyacrylamide gel electrophoresis - PAL l-phenylalanine ammonia-lyase - SDS sodium dodecyl sulphate     

Molecular breeding of a Brevibacterium lactofermentum l-phenylalanine producer using a cloned prephenate dehydratase gene

Summary The prephenate dehydratase gene was cloned from a mutant of Brevibacterium lactofermentum, AJ11957 that produced enzyme free from feedback inhibition. The recombinant plasmids pPH11 and pPH14 complemented a phenylalanine auxotroph of B. lactofermentum, A-15, provided the transformant with the desensitized enzyme and caused an increased level of the enzyme compared to that of a wild strain. Plasmid pPH14 was introduced into l-phenylalanine producers genetically induced from B. lactofermentum; MF358 and FP-1 excreting l-tyrosine and anthranilate, respectively, as by-products. Both transformants predominantly accumulated l-phenylalanine at the expense of by-product formation. Co-existence of pPH14 and pTAR16, a recombinant plasmid expressing desensitized 3-deoxy-d-arabino-hepturosonate-7-phosphate synthase had a marked effect on further improvement in l-phenylalanine productivity, accompanied by an increase in the corresponding enzyme activity. The parent, MF358, accumulating 5.5 g/l l-phenylalanine, 6.8 g/l l-tyrosine and 0.3 g/l anthranilate turned into a potent l-phenylalanine producer producing 18.2 g/l l-phenylalanine and 1.0 g/l l-tyrosine by-product. Offprint requests to: Hisao Ito