Integrated development of fermentation and downstream processing or L-isoleucine production with Corynebacterium glutamicum

 Applying a genetic algorithm for the optimization of trace element composition in the medium for L-isoleucine production from glucose and DL-α-hydroxybutyric acid with Corynebacterium glutamicum resulted in a reduction of the byproduct L-valine. High L-isoleucine broth concentrations of 20 g/l within 72 h at an L-isoleucine/DL-α-hydroxy butyric acid yield of 70% (w/w) and an L-isoleucine/L-valine ratio of 100 were achieved, if closed-loop control of glucose and of DL-α-hydroxybutyric acid was applied. For the isolation of L-isoleucine from fermentation broth a specific downstream processing was developed and optimized up to semitechnical scale (ultrafiltration, reverse osmosis, first crystallization, active-carbon adsorption, electrodialysis, second crystallization). The economic model of this downstream processing, which was identified by coupling the mass balance and energy balance with the semi-empirical models of the unit operations, was used to quantify the isolation costs as a function of L-isoleucine concentration and L-isoleucine/L-valine ratio in the fermentation broth. A cost reduction for downstream processing from DM 55 to DM 25 (kg L-isoleucine)^-1 and an improvement of the L-isoleucine yield in downstream processing from 48% to 80% was achieved using this economic model as the objective function to be minimized by the fermentation process (scenario: production of 70 tonnes L-isoleucine/year). Received: 8 January 1996/Received revision: 22 April 1996/Accepted: 29 April 1996     

Studies on the the Isoleucine Fermentation

Accumulation of L-isoleucine and L-valine was studied on 14 genera, 47 species and 110 strains of aerobic bacteria using bacterial type cultures. A large amount of L-isoleucine and a small amount of L-valine accumulated when 1% of DL-α-aminobutyric acid was added to the culture medium. As a rule, facultative aerobes such as Aerobacter, Erwinia, Serratia and Bacillus showed good accumulation. In the absence of α-aminobutyric acid, powerful L-isoleucine accumulators produced a large amount of L-valine, although the accumulation of L- isoleucine was scarcely observed under that condition. In the presence of α-aminobutyric acid, the accumulation of L-valine was generally suppressed, but in several strains, on the contrary, the accumulation increased as well as that of L-isoleucine. When DL-threonine was used instead of α-aminobutyric acid, the amount of L-isoleucine accumulated was not as high as that with α-aminobutyric acid in almost all strains except Serratia marcescens. It was concluded that a distinct relationship between bacterial genera or species and accumulation of L-isoleucine did not exist, that is, powerful accumulators were limited to special strains, and that the addition of α-aminobutyric acid was necessary for the accumulation of a large amount of L-isoleucine.     

Feedback inhibition of homoserine dehydrogenase and threonine deaminase in the genusBifidobacterium

Feedback inhibition of crude and purified extracts of homoserine dehydrogenase and threonine deaminase activities in the genusBifidobacterium was studied. Homoserine dehydrogenase was partially or completely inhibited byl-threonine, and a marked inhibitory effect byl-isoleucine on threonine deaminase was observed. In the speciesBifidobacterium cuniculi high levels ofl-valine reversed the inhibitory effect ofl-isoleucine. The -aminobutyric acid-resistant mutant Ru 326/106 of the speciesB. ruminale, overproducer ofl-isoleucine, had a derepressed homoserine dehydrogenase and a lesser feedback inhibition byl-threonine. Homoserine dehydrogenase appeared to be in bifids specifically NAD dependent. The regulatory mechanisms of aspartate family amino acid biosynthesis in bifidobacteria was discussed.     

Living cell reaction process forl-isoleucine andl-valine production

Summary A new process (Living Cell Reaction Process) forl-isoleucine production using viable, non-growing cells ofBrevibacterium flavum AB-07 was optimised using ethanol as the energy source and -ketobutyric acid (-KB) as precursor.l-valine also could be produced from glucose at high yield by this process. This process differs from the usual fermentation method in that non-growing cells are used, and the production ofl-isoleucine andl-valine were carried out under conditions of repressed cell division and growth. Minimal medium missing the essential growth factor, biotin was employed as the reaction mixture for the production ofl-isoleucine andl-valine. The productivity ofl-isoleucine andl-valine were 200 mmol·l^–1 · day^–1 (molecular yield to -KB: 95%) and 300 mmol · l^–1 · day^–1 (molecular yield to glucose: 80%) respectively. The content ofl-isoleucine andl-valine in total amino acids produced in the each mixture were 97% and 96% respectively.     

Factors enhancing l-valine production by the growth-limited l-isoleucine auxotrophic strain Corynebacterium glutamicum ΔilvA ΔpanB ilvNM13 (pECKAilvBNC)

Cell growth limitation is known to be an important condition that enhances l-valine synthesis in Corynebacterium glutamicum recombinant strains with l-isoleucine auxotrophy. To identify whether it is the limited availability of l-isoleucine itself or the l-isoleucine limitation-induced rel-dependent ppGpp-mediated stringent response that is essential for the enhancement of l-valine synthesis in growth-limited C. glutamicum cells, we deleted the rel gene, thereby constructing a relaxed (rel ⁻ ) C. glutamicum ΔilvA ΔpanB Δrel ilvNM13 (pECKAilvBNC) strain. Variations in enzyme activity and l-valine synthesis in rel ⁺ and rel ⁻ strains under conditions of l-isoleucine excess and limitation were investigated. A sharp increase in acetohydroxy acid synthase (AHAS) activity, a slight increase in acetohydroxyacid isomeroreductase (AHAIR) activity, and a dramatic increase in l-valine synthesis were observed in both rel ⁺ and rel ⁻ cells exposed to l-isoleucine limitation. Although the positive effect of induction of the stringent response on AHAS and AHAIR upregulation in cells was not confirmed, we found the stringent response to be beneficial for maintaining increased AHAS, dihydroxyacid dehydratase, and transaminase B activity and l-valine synthesis in cells during the stationary growth phase.     

Influence of l-isoleucine and pantothenate auxotrophy for l-valine formation in Corynebacterium glutamicum revisited by metabolome analyses

The effect of different amounts of supplemented l-isoleucine and pantothenate has been analysed with the auxotrophic strain Corynebacterium glutamicum ΔilvA ΔpanB, showing that the final biomass concentration of this preliminary l-valine production strain can be controlled by the amount of added l-isoleucine. One gramme cell dry weight is formed from 48 μmol l-isoleucine. Different amounts of available pantothenate affect the intracellular pyruvate concentration. By limiting pantothenate supplementation from 0.8 to 0.1 μM, a 35-fold increase of cytoplasmic pyruvate up to 14.2 mM can be observed, resulting in the increased formation of l-valine, l-alanine and organic acids in the presence of low pantothenate concentrations. These findings can be used to redirect the carbon flux from glycolysis via pyruvate to the TCA cycle towards the desired product l-valine.     

Enzymic synthesis ofL-Lysine fromDL-α-Amino-ε-caprolactam by new microbial strains

The production of L-lysine fromDL-α-amino-ε-caprolactam (DL-ACL) by new strains producingL-α-amino-ε-caprolactamase and aminocaprolactam racemase is described. Optimal conditions for hydrolysis ofL-ACL byCryptococcus sp. and for racemization of ACL by cells of a strain isolated in nature and identified asPseudomonas sp. were determined. Synthesis ofL-α-amino-ε-caprolactamase is induced byDL-ACL orL-lysine with the same effectivity. A positive effect of phosphates (potassium salts) on reduction of the induction lag was detected, the synthesis of this enzyme was found to be repressed by glucose and some possibilities of the reversion of this repressive effect were demonstrated. Under conditions optimal for the production of both enzymes a quantitative theoretical conversion of 10 % aqueousDL-ACL toL-lysine by a mixture of native cells in a mass ratio of 1: 2 (producer of ACL-hydrolase to producer of ACL-racemase) occurred in 8 h at 40 °C and pH 8.0     

Some physiological functions of the L-leucine dehydrogenase in Bacillus subtilis

Mutants of Bacillus subtilis constitutive for L-leucine dehydrogenase synthesis were selected. Using these mutants we could determine two functional roles for the L-leucine dehydrogenase. This enzyme liberates ammonium ions from branched chain amino acids when supplied as the sole nitrogen source. Another function is to synthesize from L-isoleucine, L-leucine, and L-valine the branched chain -keto acids which are precursors of branched chain fatty acid biosynthesis. These results together with the inducibility of the enzyme suggest that the L-leucine dehydrogenase has primarily a catabolic rather than an anabolic function in the metabolism of Bacillus subtilis.     

Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during fermentation of α-ketobutyrate to l-isoleucine

Summary Corynebacterium glutamicum ATCC 13 032 produces 13 g/l l-isoleucine from 200 mM -ketobutyrate as a synthetic precursor. In fed batch cultures up to 19 g/l l-isoleucine is formed. For optimal conversion the addition of 0.3 mM l-valine plus 0.3 mM l-leucine to the fermentation medium is required. The affinity constants for the acetohydroxy acid synthase (AHAS) were determined. (This enzyme directs the flow of -ketobutyrate plus pyruvate towards l-isoleucine and that of two moles of pyruvate to l-valine and l-leucine, respectively.) For -ketobutyrate the K _m is 4.8×10^-3 M, and V _max 0.58 U/mg, for pyruvate the K _m is 8.4×10^-3 M, and V _max 0.37 U/mg. Due to these characteristics the presence of high -ketobutyrate concentrations apparently results in a l-valine, l-leucine deficiency. This in turn leads to a derepression of the AHAS synthesis from 0.03 U/mg to 0.29 U/mg and high l-isoleucine production is favoured. The derepression of the AHAS synthesis induced by the l-valine, l-leucine shortage was directly proven with a l-valine, l-leucine, l-isoleucine auxotrophic mutant where the starvation of each amino acid resulted in an increased AHAS level. This is in accordance with the fact that only one AHAS enzyme could be verified by chromatographic and electrophoretic separations as being responsible for the synthesis of all three branched-chain amino-acids.     

Breeding anl-isoleucine producer by protoplast fusion ofCorynebacterium glutamicum

Summary Corynebacterium glutamicum R-18 is a strain forl-isoleucine production. Polyethylene glycol (PEG)-induced protoplast fusion was applied to improve the strain of thisl-isoleucine producer. Strain R-18 was fused with anl-lysine producerC. glutamicum S-37, becausel-isoleucine andl-lysine are synthesized from a common intermediate, aspartate--semialdehyde. Two thousand fusants were checked for their phenotypes. Most of the fusants accumulatedl-lysine, and only 0.9% of the fusants accumulatedl-isoleucine. Two strains, F-28 and F-91, were selected and cultivated in production medium. Fusant F-28 accumulated 12.1 g/l ofl-isoleucine and 4.8 g/l ofl-lysine, and fusant F-91 accumulated 4.8 g/l ofl-isoleucine and 13.0 g/l ofl-lysine, while the parental strains R-18 and S-37 accumulated 9.5 g/l ofl-isoleucine and 26.8 g/l ofl-lysine, respectively. Sugar consumption activity was improved by protoplast fusion, and thel-isoleucine production rate of F-28 was 2.4 times higher than that of R-18.     

Utilization ofL- andDL-cystine by the fungusMicrosporum gypseum

Growth of the fungusMicrosporum gypseum and utilization of cystine during this growth was studied in a glucose-arginine medium containing either sodium sulphate, andL-cystine orDL-cystine. Replacement of sulphate withL-cystine brought about no significant changes in the growth of the microorganism. Utilization ofL-cystine as a source of carbon and nitrogen was rapid and complete and excess sulphur was excreted into the medium in the form of sulphate. Similarly excreted were also minute amounts of sulphite which immediately reacted with the remaining cystine to formS. sulphocysteine. Growth ofM. gypseum in a medium withDL-cystine was slow. Although this substance was not utilized as readily asL-cystine, its utilization was still complete and excess sulphur was similarly excreted in the form of sulphate and sulphite. The initial step in the utilization of theD-isomer is probably its extracellular deamination.     

l-leucine dehydrogenase fromBacillus cereus

Summary An improved method for the production ofl-leucine dehydrogenase is described employing a mutant with a constitutive enzyme and a fed-batch cultivation technique yielding high cell concentrations. Purification ofl-leucine dehydrogenase to homogeneity was carried out starting with 30 kgBacillus cereus cells by heat treatment at 63°C, followed by two liquid-liquid extraction steps and three conventional column chromatographies. Crystals have been obtained from the 95-fold purified enzyme. The molecular weight of the native enzyme was determined by sedimentation equilibrium and gel filtration studies to be 310 000 containing eight identical subunits with a molecular weight of 39 000. The sedimentation coefficient was estimated to 11.65 S. Branched-chain amino acids likel-leucine,l-valine orl-isoleucine are deaminated by the NAD-dependent enzyme. In the reverse reaction a variety of 2-ketoacids, especially 2-ketoisocaproate, 2-ketoisovalerate and 2-keto-3-methyl-valerate, were reductive aminated to the correspondingl-amino acids in the presence of 0.9 M ammonia. The amino acid composition for the subunit ofl-leucine dehydrogenase is presented.     

Effects of a Feedback-Resistant Aspartokinase III Gene on L-Isoleucine Production in Escherichia coli K-12

An L-isoleucine-overproducing recombinant strain of E. coli, TVD5, was also found to overproduce L-valine. The L-isoleucine productivity of TVD5 was markedly decreased by addition of L-lysine to the medium. Introduction of a gene encoding feedback-resistant aspartokinase III increased L-isoleucine productivity and decreased L-valine by-production. The resulting strain accumulated 12 g/l L-isoleucine from 40 g/l glucose, and suppression of L-isoleucine productivity by L-lysine was relieved.     

Susceptibility behaviour and specific heat anomaly in single crystals of alanine and valine

Magnetization of single crystals ofD-,L-alanine andD-valine were measured as a function of temperature using the SQUID magnetometer. An obvious lambda transition at 270 ± 1 K was shown in the specific heat measurement of alanine and valine enantiomers by differential scanning calorimetry (DSC). Magnetic transition temperature seems coincident with that of lambda transition. Temperature dependence of the X-ray powder diffraction forD-valine showed no crystal lattice change under the temperature cooling down from 293 K to 123 K. We propose that the difference of theX T curve between theD-alanine andL-alanine is attributable to the variation of intramolecular geometry of chirality density in the presence of an external magnetic field. The chirality characteristics of alanine and valine enantiomers by the specific heat and susceptibility behaviour are discussed.     

Microbial Conversion of DL-5-Substituted Hydantoins to the Corresponding L-Amino Acids by Pseudomonas sp. Strain NS671

A bacterial strain, NS671, which converts DL-5-(2-methylthioethyl)hydantoin stereospecifically to L-methionine, was isolated from soil and was classified into the genus Pseudomonas. With growing cells of Pseudomonas sp. strain NS671, DL-5-(2-methylthioethyl)hydantoin was effectively converted to L-methionine. Under adequate conditions, 34g of L-methionine per liter was produced with a molar yield of 93% from DL-5-(2-methylthioethyl)hydantoin added successively. In addition to L-methionine, other amino acids such as L-valine, L-leucine, L-isoleucine, and L-phenylalanine were also produced from the corresponding 5- substituted hydantoins, but these L-amino acids produced were partially consumed by strain NS671. The hydantoinase, by which 5-substituted hydantoin rings are opened, was ATP-dependent. The N-carbamylamino acid amidohydrolase was found to be strictly L-specific, and its activity was inhibited by high concentration of ATP.     

Influence of amino acids on the biosynthesis of cyclosporin A by Tolypocladium inflatum

 An indigenously isolated strain of Tolypocladium inflatum, when grown as a suspension culture in semi-synthetic and synthetic media, produced cyclosporin A. Biosynthesis of this well-known immunosuppressive agent was found to be influenced heavily by the external addition of the amino acid constituents of the molecule. In synthetic media, L-leucine and L-valine were found to act as strong inducers of drug production. L-Valine increased the specific production of cyclosporin A by 75% in semi-synthetic medium and by ten times in synthetic medium compared to an unsupplemented control culture. D-Valine had no stimulating effect on the production. The presence of amino acids in the exponential growth phase ensured optimal production, as was indicated in the experiment in which L-valine was added at different times; 4 g/l was the optimum concentration of exogenous L-valine. On the other hand, exogenous sarcosine and L-methionine tended to diminish drug production. Received: 23 October 1995/Received revision: 23 January 1996/Accepted: 29 January 1996     

Engineering the homoserine dehydrogenase and threonine dehydratase control points to analyse flux towards L-isoleucine in Corynebacterium glutamicum

 The synthesis of L-isoleucine by Corynebacterium glutamicum involves 11 reaction steps, with at least 5 of them regulated in activity or expression. Using gene replacement we constructed a vector-free C. glutamicum strain having feedback-resistant aspartate kinase and feedback-resistant homoserine dehydrogenase activity. Isogenic strains carrying in addition one or several copies of feedback-resistant threonine dehydratase were made and their product accumulations compared. With strain SM1, with high threonine dehydratase activity, accumulation of 50 mM L-isoleucine was achieved, whereas with the parent strain only 4 mM L-isoleucine was obtained. Applying a closed-loop control fed-batch strategy to strain SM1 a final titre of 138 mM L-isoleucine was achieved with an integral molar yield of 0.11 mol/mol, and a maximal specific productivity of 0.28 mmol (g h)^-1. This shows that high L-isoleucine yields can be obtained in the presence of one copy of feedback-resistant homoserine dehydrogenase by applying the appropriate fermentation strategy. In addition, the specific profiles of 2-oxoglutarate and pyruvate accumulation during fermentation revealed a major transition of the metabolism of C. glutamicum during the fermentation process. Received: 16 October 1995/Received revision: 21 December 1995/Accepted: 8 January 1996     

Valine production from hydrocarbon byMicrococcus varians

A bacterium isolated from Assam (India) soil was found to accumulateL-valine in the growth medium and was identified asMicrococcus varians. The strain grew and accumulated valine in a purely synthetic medium, but supplementation with either casamino acids or yeast extract or with both, improved the yield. The entire fermentation period could be divided into a growth phase and a production (phase which could be prolonged by adjustment of pH to neutral range. Among the different hydrocarbon and nitrogen sources tested straight run gas-oil and ammonium sulphate, respectively, were found most suitable. Antibiotics inhibited growth but stimulated extracellular valine accumulation. Vitamins stimulated growth and valine yield and an inoculum level of 10 % was found to be optimal. The yield ofL-valine under optimal conditions was 2.95 g/L.     

Chiral Selection in Oligoadenylate Formation in the Presence of a Metal ion Catalyst or Poly(U) Template

The lead ion-catalyzed oligomerization of 5′-phosphorimidazolides of D-, L- or racemic DL-adenosine (D-ImpA, L-ImpA and DL-ImpA) gave oligoadenylates up to a pentamer. The oligomers resulting from racemic ImpA were comparable in yields and length to those from chiral D- or L-ImpA. A complex mixture of homochiral and heterochiral oligomers was formed in the reaction from racemic ImpA. Total dimer product from racemic ImpA by the lead ion catalyst showed homochiral selectivity. The reaction catalyzed by uranyl ion yielded oligoadenylates up to 15mer from chiral D- or L-ImpA in over 95% yield. A complex mixture of isomeric oligoadenylates was formed from racemic DL-ImpA in the presence of uranyl ion catalyst in comparable yields to those from D- or L-ImpA. The analysis of the dimer product from DL-ImpA showed that the homochiral 2′ –5′ linked dimer was selectively formed. D-ImpA polymerized effectively on a poly(U) template, which is exclusively composed of D-uridine, yielding oligoadenylates up to a pentamer. In contrast, L-ImpA or racemic DL-ImpA polymerized far less efficiently on the poly(U) template, demonstrating that chiral selection takes place in the poly(U) template-directed oligoadenylate formation.     

Synthesis of the amino acid conjugates of <Emphasis Type="Italic">epi</Emphasis>-jasmonic acid

The TES ether of the C6-hydroxy derivative of naturally occurring epi-jasmonic acid (epi-JA) was designed as epimerization-free equivalent of epi-JA. The TES ether was synthesized from (1R,4S)-4-hydroxycyclopent-2-enyl acetate in 13 steps. The acid part of the ether was activated with ClCO₂Bu ⁱ and subjected to condensation with l-amino acid at room temperature for 48 h. The TES group in the condensation product was removed in HCO₂H (0°C, 30 min) and the resulting hydroxyl group was oxidized with Jones reagent (acetone, 0°C, 30 min) to furnish the amino acid conjugate of epi-JA. The amino acids examined are l-isoleucine, l-leucine, l-alanine, l-valine, and d-allo-isoleucine, which afforded the conjugates in 48–68% yields with 89–96% diastereomeric purity over the trans isomers. Similarly, the possible three stereoisomers of epi-JA were condensed with l-isoleucine successfully, producing the corresponding stereoisomers in good yields.