Characteristics and Efficiency of Glutamine Production by Coupling of a Bacterial Glutamine Synthetase Reaction with the Alcoholic Fermentation System of Baker’s Yeast

Glutamine production with bacterial glutamine synthetase (GS) and the sugar-fermenting system of baker’s yeast for ATP regeneration was investigated by determining the product yield obtained with the energy source for ATP regeneration (i.e., glucose) for yeast fermentation. Fructose 1,6-bisphosphate was accumulated temporarily prior to the formation of glutamine in mixtures which consisted of dried yeast cells, GS, their substrate (glucose and glutamate and ammonia), inorganic phosphate, and cofactors. By an increase in the amounts of GS and inorganic phosphate, the amounts of glutamine formed increased to 19 to 54 g/liter, with a yield increase of 69 to 72% based on the energy source (glucose) for ATP regeneration. The analyses of sugar fermentation of the yeast in the glutamine-producing mixtures suggested that the apparent hydrolysis of ATP by a futile cycle(s) at the early stage of glycolysis in the yeast cells reduces the efficiency of ATP utilization. Inorganic phosphate inhibits phosphatase(s) and thus improves glutamine yield. However, the analyses of GS activity in the glutamine-producing mixtures suggested that the higher concentration of inorganic phosphate as well as the limited amount of ATP-ADP caused the low reactivity of GS in the glutamine-producing mixtures. A result suggestive of improved glutamine yield under the conditions with lower concentrations of inorganic phosphate was obtained by using a yeast mutant strain that had low assimilating ability for glycerol and ethanol. In the mutant, the activity of the enzymes involved in gluconeogenesis, especially fructose 1,6-bisphosphatase, was lower than that in the wild-type strain.Glutamine is one of the most important compounds in nitrogen metabolism; it is not only a constituent of proteins but is also a donor of the amino (amido) moiety in the biosynthesis of other amino acids, purines, pyrimidines, pyridine coenzymes, and complex carbohydrates. Glutamine is also used in the treatment of gastric ulcers and has been produced commercially by direct fermentation with certain bacteria (6–10).In recent years, enzymatic synthesis has come to rival direct fermentation as a means of producing amino acids. In the case of glutamine, however, the need for a stoichiometric supply of ATP for the endoergonic reaction of glutamine synthetase (GS) precludes the development of an economically valuable method, unless ATP can be regenerated and recycled.Processes for the production of various substances using dried yeast cells as an enzyme source were established by Tochikura and colleagues (2, 4, 16, 18–20). The processes are driven by the chemical energy of ATP released by the alcoholic fermentation by the yeast, which has been wasted in alcoholic brewing (17). Tochikura and colleagues also designed a process in which the yeast fermentation of sugar is combined with an endoergonic reaction catalyzed by an enzyme from a different microorganism (3). The results suggest that the process offers the possibility of producing many compounds at a high yield by using various biosynthetic reactions and high concentrations of substrates. Tochikura et al. introduced the general idea of coupled fermentation with energy transfer for the process; its principle is indicated in Fig. ​Fig.1,1, with glutamine production as an example. Open in a separate windowFIG. 1Scheme of glutamine production by the coupled fermentation with energy transfer method. ∗1, glycolytic pathway is abridged. ∗2, inorganic phosphate (P_i) is recycled.In the process of coupled fermentation with energy transfer, a catalytic amount of ATP is regenerated with the energy of sugar fermented by yeast, in the form of baker’s yeast (4, 16, 18, 19, 23). The energy-utilizing system for the synthesis can involve the enzyme(s) of yeast itself or those of other organisms. It should be noted that, from another point of view, the use of the energy-utilizing system results in ADP regeneration to complete the fermentation of glucose, and that, if there is no ADP regeneration, the yeast fermentation of sugar can proceed only as follows, in the presence of inorganic phosphate (the Harden-Young effect of inorganic phosphate [1]), 2 · glucose + 2 · inorganic phosphate → fructose 1,6-bisphosphate (FBP) + 2 · C₂H₅OH + 2 · CO₂ (Harden-Young equation), where ADP regeneration for the fermentation of 1 mol of glucose is carried out by the phosphorylation of another mole of glucose to FBP.We previously reported glutamine production, obtained by employing a combination of baker’s yeast cells and GS from Gluconobacter suboxydans, as the first application of the coupled fermentation with energy transfer method for the production of a nonphosphorylated compound (12, 13). In addition, we achieved high-yield glutamine production by using the Corynebacterium glutamicum (Micrococcus glutamicus) enzyme and larger amounts of the substrates (15). The maximum amounts of glutamine formed (23 to 25 g/liter) and the yield based on glutamate (50 to 100%) were to some extent satisfactory, but the yield based on the energy source (glucose) for ATP regeneration was not satisfactory (about 40% of the theoretical value; 2 mol of glutamine can be formed when 1 mol of glucose is consumed).In the present study, we examined the characteristics of glutamine production regarding product yield based on the energy source for ATP regeneration and regarding the reactivity of GS during glutamine production, which is closely related to the product yield. The results of preliminary attempts to improve glutamine production are also described. In these experiments, a yeast mutant which has a low assimilating ability for glycerol and/or ethanol was used.     

基因工程酶法结合酵母能量耦联高效合成L-谷氨酰胺的研究

通过PCR方法从Bacillus subtilis基因组DNA中扩增出谷氨酰胺合成酶基因(glnA),克隆至表达载体pET28b, 经测序鉴定后转化大肠杆菌BL21(DE3), 用IPTG及乳糖诱导表达。 SDSPAGE分析表明,所表达的谷氨酰胺合成酶(glutamine synthetase ,简称GS)为可溶性蛋白,约占总菌蛋白的80％。利用表达的GS蛋白 N端的6×HisTag 对GS进行亲和层析,将获得的纯蛋白进行酶活性测定。结果表明,纯化的GS合成反应的最适温度为60℃,最适pH为6.5,Mn²⁺能明显提高GS的活性和稳定性。工程菌BL21(DE3)/pET28b-glnA粗提物中GS的比活是宿主菌本身的84倍。以谷氨酸、NH₄Cl和ATP为底物的转化实验表明谷氨酸的转化率达95％以上。 经筛选获得一株高效能量耦联酵母菌株,命名为YC001;通过能量耦联表明,该系统对谷氨酸的转化率高达80％,平均谷氨酰胺产量为22g/L。     

Glutamine Production by Coupling with Energy Transfer: Employing Yeast Cells and Gluconobacter Glutamine Synthetase

A glutamine production process was established by combining alcoholic fermentation of baker's yeast cells with glutamine synthetase from the bacterium Gluconobacter suboxydans. The maximum amount of glutamine formed under optimum conditions was about 20 mM in 3 hr with 80% yield based on glutamate, substrate. The fermentation proceeded in two steps: the accumulation of energy in a form of fructose 1,6-diphosphate (FDP) by yeast fermentation of sugar based on the Harden-Young effect and the fermentation of FDP coupled with glutamine synthetase reaction (an endergonic reaction) through an ATP-ADP system. The following factors were found to be important: (a) the ratio of the activities of yeast fermentation of sugar and glutamine synthetase, (b) effect of contaminating enzyme(s) in glutamine synthetase preparation, and (c) enzymatic properties of glutamine synthetase.     

Theanine production by coupled fermentation with energy transfer employing Pseudomonas taetrolens Y-30 glutamine synthetase and baker's yeast cells

Theanine was formed from glutamic acid and ethylamine by coupling the reaction of glutamine synthetase (GS) of Pseudomonas taetrolens Y-30 with sugar fermentation of baker's yeast cells as an ATP-regeneration system. Theanine formation was stimulated by the addition of Mn2+ to the mixture for the coupling. The addition of Mg2+ was less effective. In a mixture containing a larger amount of yeast cells with a fixed level of GS, glucose (the energy source) was consumed rapidly, resulting in a decrease in the final yield of theanine. On the other hand, an increase in GS amounts increased theanine formation in a mixture with a fixed amount of yeast cells. High concentrations of ethylamine enhanced theanine formation whereas inhibited yeast fermentation of sugar and the two contrary effects of ethylamine caused a high yield of theanine based on glucose consumed. In an improved reaction mixture containing 200 mM sodium glutamate, 1,200 mM ethylamine, 300 mM glucose, 50 mM potassium phosphate buffer (pH 7.0), 5 mM MnCl2, 5 mM AMP, 100 units/ml GS, and 60 mg/ml yeast cells, approximately 170 mM theanine was formed in 48 h.     

Efficient production of 2-deoxyribose 5-phosphate from glucose and acetaldehyde by coupling of the alcoholic fermentation system of Baker's yeast and deoxyriboaldolase-expressing Escherichia coli

2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker's yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker's yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.     

Isolation of a New Tobacco Constituent, 5,6-Dihydro-5-hydroxy-3,6-epoxy-β-ionol,from Japanese Domestic Suifu Tobacco

Glutamine production was investigated by coupling of glutamine synthetase from Gluconobacter suboxydans with a sugar fermentation system of baker's yeast (energy generating system). Under the optimum condition, 22 mM glutamine was formed in 3 hr, and the yield was 92% based on the substrate glutamate. The first step of the process was the accumulation of fructose 1,6-diphosphate (FDP) as a reservoir of fermentation energy, in the presence of a high concentration of inorganic phosphate; and the second step was accomplished by coupling the degradation of FDP with glutamine synthetase reaction through an ADP-ATP system. The effects of enzyme concentration, additives in the reaction mixture and others on glutamine formation were investigated, and the importance of three factors was pointed out: (a) the ratio of activity of energy generating system to utilizing system, (b) contaminated enzyme(s) in the energy utilizing system and (c) the enzymatic properties of the energy utilizing system.     

谷氨酰胺高效酶法转化条件研究

谷氨酰胺合成酶是生物体氮代谢的中心酶之一,在消耗ATP的情况下,谷氨酰胺合成酶催化由谷氨酸和NH4+向谷氨酰胺的转化,Toch ikura提出了将酵母发酵与纯化酶结合生产谷氨酰胺(G ln)的方法,本实验通过建立酶法合成L-G ln与酵母酒精发酵的能量偶联体系,研究了在此偶联体系中各因素对谷氨酰胺酶转化效率的影响,为工业上利用酶法生产G ln提供理论依据。     

Antibacterial Activity of l-Lysine α-Oxidase against rec+ and rec− Strains of Bacillus subtilis

2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker’s yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker’s yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.     

过量无机盐及谷氨酸抑制重组菌核黄素发酵

在重组枯草芽孢杆菌24/pMX45核黄素发酵中,酵母粉促进核黄素合成,酵母抽提物抑制核黄素合成。分析显示,酵母抽提物的无机离子和游离氨基酸含量均高于酵母粉。在酵母粉基础发酵培养基中,添加各种无机离子和游离氨基酸,使其含量与酵母抽提物相同。摇瓶发酵结果表明:过量的无机离子和谷氨酸对核黄素合成有显著的抑制作用。酵母抽提物含有较高浓度的谷氨酸,是其抑制核黄素合成的主要原因。     

Analysis of the energy metabolism after incubation of Saccharomyces cerevisiae with sulfite or nitrite

After addition of 5 mM sulfite or nitrite to glucose-metabolizing cells of Saccharomyces cerevisiae a rapid decrease of the ATP content and an inversely proportional increase in the level of inorganic phosphate was observed. The concentration of ADP shows only small and transient changes. Cells of the yeast mutant pet 936, lacking mitochondrial F₁ATPase, after addition of 5 mM sulfite or nitrite exhibit changes in ATP, ADP and inorganic phosphate very similar to those observed in wild type cells. They key enzyme of glucose degradation, glyceraldehyde-3-phosphate dehydrogenase was previously shown to be the most sulfiteor nitrite-sensitive enzyme of the glycolytic pathway. This enzyme shows the same sensitivity to sulfite or nitrite in cells of the mutant pet 936 as in wild type cells. It is concluded that the effects of sulfite or nitrite on ATP, ADP and inorganic phosphate are the result of inhibition of glyceraldehyde-3-phosphate dehydrogenase and not of inhibition of phosphorylation processes in the mitochondria. Levels of GTP, UTP and CTP show parallel changes to ATP. This is explained by the presence of very active nucleoside monophosphate kinases which cause a rapid exchange between the nucleoside phosphates. The effects of the sudden inhibition of glucose degradation by sulfite or nitrite on levels of ATP, ADP and inorganic phosphate are discussed in terms of the theory of Lynen (1942) on compensating phosphorylation and dephosphorylation in steady state glucose metabolizing yeast.Abbreviations ATP adenosine triphosphate - ADP adenosine diphosphate - AMP adenosine monophosphate - P_i inorganic orthophosphate Dedicated to Prof. Dr. Hans Grisebach on the occasion of his sixtieth birthday     

Neuronal Glutamine Utilization: Glutamine/Glutamate Homeostasis in Synaptosomes

The synaptosomal metabolism of glutamine was studied under in vitro conditions that simulate depolarization in vivo. With [2-15N]glutamine as precursor, the [glutamine]i was diminished in the presence of veratridine or 50 mM KCl, but the total amounts of [15N]glutamate and [15N]aspartate formed were either equal to those of control incubations (veratridine) or higher (50 mM [KCl]). This suggests that depolarization decreases glutamine uptake and independently augments glutaminase activity. Omission of sodium from the medium was associated with low internal levels of glutamine which indicates that influx occurs as a charged Na(+)-amino acid complex. It is postulated that a reduction in membrane potential and a collapse of the Na+ gradient decrease the driving forces for glutamine accumulation and thus inhibit its uptake and enhance its release under depolarizing conditions. Inorganic phosphate stimulated glutaminase activity, particularly in the presence of calcium. At 2 mM or lower [phosphate] in the medium, calcium inhibited glutamine utilization and the production of glutamate, aspartate, and ammonia from glutamine. At a high (10 mM) medium [phosphate], calcium stimulated glutamine catabolism. It is suggested that a veratridine-induced increase in intrasynaptosomal inorganic phosphate is responsible for the enhancement of flux through glutaminase; calcium affects glutaminase indirectly by modulating the level of free intramitochondrial [phosphate]. Because phosphate also lowers the Km of glutaminase for glutamine, augmentation of the amino acid breakdown may occur even when depolarization lowers [glutamine]i. Reducing the intrasynaptosomal glutamate to 26 nmol/mg of protein had little effect on glutamine catabolism, but raising the pH to 7.9 markedly increased formation of glutamate and aspartate. It is concluded that phosphate and H+ are the major physiologic regulators of glutaminase activity.     

Glutamine assimilation pathways in Neurospora crassa growing on glutamine as sole nitrogen and carbon source

Neurospora crassa wild-type is almost unable to grow on glutamine as sole nitrogen and carbon source but a GDH-; GS +/- double mutant strain, lacking NADP-dependent glutamate dehydrogenase and partially lacking glutamine synthetase did grow. Under these conditions, the double mutant had a higher chemical energy content than the wild-type. Enzyme assays and labelling experiments with glutamine indicated that in the double mutant glutamine was degraded to ammonium and to carbon skeletons by glutamate synthase, the catabolic (NADH-dependent) glutamate dehydrogenase and the glutamine transaminase-omega-amidase pathway.     

Computational Structural Analysis and Kinetic Studies of a Cytosolic Glutamine Synthetase from Camellia sinensis (L.) O. Kuntze

Structural analysis of a cytosolic glutamine synthetase from Camellia sinensis (CsGS) has been conducted employing computational techniques. This was conducted to compare its structural aspects with other known structures of GS. The disordered residues and their distribution in CsGS are in close comparison to earlier reported GS. The 3-D structure of CsGS also showed high degree of similarity with the only known crystal structure of GS from Zea mays. The K _m values observed with recombinant CsGS for all the three substrates are higher compared to rice, Arabidopsis, maize and human. This suggests lower affinity of CsGS for substrates. Further, kinetic mechanism of CsGS catalysis was investigated using initial velocity analysis and product inhibition studies. Initial velocity data eliminate the possibility of ping-pong mechanism and favor the random mechanism of catalysis. Through product inhibition studies, ADP was found to be a competitive inhibitor with respect to ATP and noncompetitive inhibitor versus both glutamate and ammonium. While, glutamine and inorganic phosphate were found to be non-competitive inhibitors of ATP, glutamate and ammonia. Taken together, these observations are consistent with a random catalysis mechanism for the CsGS where the binding order of certain substrates is kinetically preferred by the enzyme.     

Developmentally Regulated Expression of the Gene Family for Cytosolic Glutamine Synthetase in Pisum sativum

In Pisum sativum, two classes of genes encode distinct isoforms of cytosolic glutamine synthetase (GS). The first class comprises two nearly identical or “twin” GS genes (GS341 and GS132), while the second comprises a single GS gene (GS299) distinct in both coding and noncoding regions from the “twin” GS genes. Gene-specific analyses were used to monitor the individual contribution of each gene for cytosolic GS during root nodule development and in cotyledons during germination, two contexts where large amounts of ammonia must be assimilated by GS for nitrogen transport. mRNAs corresponding to all three genes for cytosolic GS were shown to accumulate coordinately during a time course of nodule development. All the GS mRNAs also accumulate to wild-type levels in mutant nodules formed by a nifD⁻ strain of Rhizobium leguminosarum indicating that induced GS expression in pea root nodules does not depend on the production of ammonia. Distinct patterns of expression for the two classes of GS genes were observed in certain mutant root nodules and most dramatically in cotyledons of germinating seedlings. The different patterns of expression between the two classes of genes for cytosolic GS suggests that their distinct gene products may serve nonoverlapping functions during pea development.     

Molecular Mechanisms of Glutamine Synthetase Mutations that Lead to Clinically Relevant Pathologies

Glutamine synthetase (GS) catalyzes ATP-dependent ligation of ammonia and glutamate to glutamine. Two mutations of human GS (R324C and R341C) were connected to congenital glutamine deficiency with severe brain malformations resulting in neonatal death. Another GS mutation (R324S) was identified in a neurologically compromised patient. However, the molecular mechanisms underlying the impairment of GS activity by these mutations have remained elusive. Molecular dynamics simulations, free energy calculations, and rigidity analyses suggest that all three mutations influence the first step of GS catalytic cycle. The R324S and R324C mutations deteriorate GS catalytic activity due to loss of direct interactions with ATP. As to R324S, indirect, water-mediated interactions reduce this effect, which may explain the suggested higher GS residual activity. The R341C mutation weakens ATP binding by destabilizing the interacting residue R340 in the apo state of GS. Additionally, the mutation is predicted to result in a significant destabilization of helix H8, which should negatively affect glutamate binding. This prediction was tested in HEK293 cells overexpressing GS by dot-blot analysis: Structural stability of H8 was impaired through mutation of amino acids interacting with R341, as indicated by a loss of masking of an epitope in the glutamate binding pocket for a monoclonal anti-GS antibody by L-methionine-S-sulfoximine; in contrast, cells transfected with wild type GS showed the masking. Our analyses reveal complex molecular effects underlying impaired GS catalytic activity in three clinically relevant mutants. Our findings could stimulate the development of ATP binding-enhancing molecules by which the R324S mutant can be repaired extrinsically.     

Cloning and Expression of Pseudomonas taetrolens Y-30 Gene Encoding Glutamine Synthetase: An Enzyme Available for Theanine Production by Coupled Fermentation with Energy Transfer

Glutamine synthetase (GS) of Pseudomonas taetrolens Y-30 can form theanine from glutamic acid and ethylamine in a mixture where yeast fermentation of sugar is coupled for ATP regeneration (coupled fermentation with energy transfer). From a genomic DNA library of P. taetrolens Y-30, a clone containing 6 kbp insertional DNA fragment was selected by the PCR screening technique with specific oligonucleotide primers for the GS gene. The fragment had an open reading frame of the GS gene encoding a protein of 468 amino acids (molecular mass, 52 kDa). The deduced amino acid sequence showed a significant homology with that of P. syringae pv. tomato GS (97%), and all the amino acid residues were fully conserved, which concern with catalytic activity in other bacterial GS. A tyrosine residue for adenylylation of GS was also found, and in vivo adenylylation was confirmed in P. taetrolens Y-30. The isolated GS gene was ligated into an expression vector (pET21a), and expressed in Escherichia coli AD494 (DE3). The enzyme productivity in the expression system was 30-fold higher than that in P. taetrolens Y-30. Recombinant GS had the same properties as those of unnadenylylated intrinsic GS, and formed theanine in the mixture of coupled fermentation with energy transfer.     

Prolonged cell-free protein synthesis using dual energy sources: Combined use of creatine phosphate and glucose for the efficient supply of ATP and retarded accumulation of phosphate

The accumulation of inorganic phosphate inhibits protein synthesis in cell-free protein synthesis reactions that are energized by high-energy-phosphate-containing compounds. This study developed a new scheme for supplying energy using dual energy sources to enhance the regeneration of ATP and lower the rate of phosphate accumulation. In the proposed scheme, where creatine phosphate (CP) and glucose were simultaneously used as the energy sources, the phosphate released from the CP was subsequently used in the glycolytic pathway for the utilization of the glucose, which enhanced the ATP supply and reduced the rate of inorganic phosphate accumulation. When tested against different proteins, the developed method produced 2-3 times more protein than the conventional ATP regeneration methods using single energy sources.     

Cloning and expression of Methylovorus mays No. 9 gene encoding gamma-glutamylmethylamide synthetase: an enzyme usable in theanine formation by coupling with the alcoholic fermentation system of baker's yeast

Gamma-glutamylmethylamide synthetase (GMAS), found in an obligate methylotroph, Methylovorus mays No. 9, can form theanine from glutamic acid and ethylamine in a mixture in which yeast sugar fermentation is coupled for ATP regeneration. The internal and N-terminal amino acid sequences of GMAS had certain similarities to putative glutamine synthetase type III (GS III) of Methylobacillus flagellatus KT. From the M. mays No. 9 genomic DNA library, a clone containing a 6.5-kbp insertional DNA fragment was selected by the PCR screening technique with oligonucleotide primers specific for the GMAS gene. The fragment had an open reading frame of the GMAS gene encoding a protein of 444 amino acids (molecular mass, 49 kDa). The deduced amino acid sequence showed significant identity with that of Met. flagellatus KT GS III (78%). The isolated gene was ligated into an expression vector (pET21a) and expressed in Escherichia coli AD494 (DE3). Enzyme productivity in the expression system was about 23-fold higher than that in M. mays No. 9. Recombinant GMAS had the same properties as intrinsic GMAS, and it formed theanine by coupling the reaction with the ATP-regeneration system of yeast sugar fermentation.     

Highly efficient bioconversion of glucose into fructose diphosphate with fed-batch-grown Saccharomyces cerevisiae cells

Summary One of the methods commonly used for manufacturing fructose 1,6-diphosphate is based on the enzymatic phosphorylation of glucose with inorganic phosphate using permeabilized brewer's yeast cells. Our results demonstrate that a substantial improvement in the yield of bioconversion can be achieved using fed-batch-grown Saccharomyces cerevisiae cells. Under an appropriate glucose and phosphate to cell ratio the efficiency of bioconversion reaches 70% of the theoretical value. Offprint requests to: C. Compagno