Regulation of biosynthesis of monensins on chemically defined medium

Addition ofL-valine andDL-isoleucine to the cultivation medium ofStreptomyces cinnamonensis was found to affect the ratio of synthesized monensins A and B. In the presence ofL-valine monensin A is synthesized predominantly, whereas in the presence ofDL-isoleucine the production of monensin B increases.     

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     

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     

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     

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.     

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.     

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.     

Effect of oleuropein and sodium chloride on viability and metabolism of Lactobacillus plantarum

 The effect of the addition of oleuropein (OLP) and NaCl on the growth and the DL-lactic acid production of Lactobacillus plantarum DSM 10492 has been investigated by using an unconventional medium. The growth of L. plantarum was not inhibited by the addition of increasing amounts of untreated OLP in the presence or absence of glucose. However, bacterial cells grew in quantity slightly with OLP alone. The increased addition of NaCl was associated with a delay in growth. Moreover, there was no growth with 8% NaCl. The addition of both NaCl and OLP resulted in growth inhibition, and the survival of cells decreased strongly. The main fermentation product was DL-lactic acid, but acetic acid was also detected after a prolonged incubation. L. plantarum produced DL-lactic acid in the presence of OLP alone but its formation decreased with increasing levels of OLP. On the other hand, heat-treated OLP had a bactericidal effect. Received: 16 October 1995/Received last revision: 5 February 1996/Accepted: 12 February 1996     

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.     

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.     

β-Carbon stereoselectivity of N-carbamoyl-d-α-amino acid amidohydrolase for α,β-diastereomeric amino acids

N-Carbamoyl-d-α-amino acid amidohydrolase (d-carbamoylase) was found to distinguish stereochemistry not only at the α-carbon but also at the β-carbon of N-carbamoyl-d-α-amino acids. The enzyme selectively acted on one of the four stereoisomers of N-carbamoyl-α,β-diastereomeric amino acids. This simultaneous recognition of two chiral centers by d-carbamoylase was useful for the fine stereoselective synthesis of α,β-diastereomeric amino acids such as threonine, isoleucine, 3,4-methylenedioxyphenylserine and β-methylphenylalanine. The stereoselectivity for the β-carbon was influenced by the pH of the reaction mixture and by the bulk of the substituent at the β-carbon. Received: 18 June 1999 / Received revision: 30 July 1999 / Accepted: 6 August 1999     

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.     

Immunolocalization of Lacrimal Gland PKC Isoforms. Effect of Phorbol Esters and Cholinergic Agonists on Their Cellular Distribution

In previous studies, we showed that lacrimal gland acini express three isoforms of protein kinase C (PKC): PKCα,-δ, and -ε. In the present study, we report the identification of two other PKC isoforms, namely PKCμ and -ι/λ. Using immunofluorescence techniques, we showed that these isoforms are differentially located. PKCα and -μ showed the most prominent membrane localization, whereas PKCδ, -ε and -ι/λ were mainly cytosolic. Using cell fractionation and western blotting techniques, we showed that the phorbol ester, phorbol 12, 13-dibutyrate (PdBu, 10⁻⁶ m), translocated all PKC isoforms, except PKCι/λ, from the soluble fraction into the particulate fraction. The effect was maximum at 5 min and persisted at 10 min. PKCε was the most responsive to PdBu reaching almost maximal translocation at a PdBu concentration as low as 10⁻⁹ m. The cholinergic agonist, carbachol (10⁻⁵ and 10⁻³ m), induced translocation which was transient for PKCδ, and -μ, but persisted for 10 min for PKCε. Carbachol did not translocate PKCα and, like PdBu, did not translocate PKCι/λ. We concluded that lacrimal gland PKC isoforms are differentially localized and that they translocate differentially in response to phorbol esters and cholinergic agonists. Received: 25 June 1996/Revised: 24 December 1996     

New aspects of genes and enzymes for β-lactam antibiotic biosynthesis

Penicillins, cephalosporins and cephamycins are peptide antibiotics synthesized by condensation of l-α-aminoadipic acid, l-cysteine and l-valine to form the tripeptide δ(l-α-aminoadipyl)-l-cysteinyl-d-valine (Aad-Cys-Val) by a non-ribosomal peptide synthetase. The genes pcbAB and pcbC, common to all penicillin and cephalosporin producers, that encode the Aad-Cys-Val synthetase¹ and isopenicillin N (IPN) synthase¹ respectively, have been cloned and the encoded enzymes studied in detail. The IPN synthase has been crystallized and its active center identified, providing evidence for the molecular mechanism of cyclization of the tripeptide Aad-Cys-Val to isopenicillin N. The late genes of the penicillin and cephalosporin pathways have also been characterized although some of the molecular mechanisms catalyzed by the encoded enzymes (e.g. IPN acyltransferase) are still obscure. In cephamycin-producing organisms, biosynthesis of the α-aminoadipic acid precursor proceeds in two steps catalyzed by lysine 6-aminotransferase and piperideine-6-carboxylic acid dehydrogenase. The gene lat for the first of these enzymes is located in the cephamycin gene cluster, providing an interesting example of association of genes encoding enzymes for the formation of a precursor with genes involved in assembly of the antibiotics. Novel enzymes involved in methoxylation at C-7 and carbamoylation at C-3′ of the cephem nucleus were isolated from Nocardia lactamdurans and Streptomyces clavuligerus. The methoxylation system is encoded by two linked genes cmcI-cmcJ and their products (proteins P₇ and P₈) form a complex that is required for hydroxylation at C-7 and for the subsequent methylation of the 7-hydroxycephem derivative to form the methoxyl group. Carbamoylation at the C-3′-hydroxyl group of the cephem nucleus is catalyzed by a specific carbamoyltransferase encoded by the gene cmcH. Finally, genes for a β-lactamase (bla), a penicillin-binding protein (pbp) and a transmembrane protein (cmcT) that appears to be involved in cephamycin exportation, are clustered together with the biosynthetic genes in the cephamycin clusters of S. clavuligerus and N. lactamdurans. Availability of the cloned genes allows metabolic engineering of the β-lactam biosynthetic pathways such as a channelling precursors and directed removal of bottlenecks in the β-lactam biosynthetic pathways. Several new β-lactam antibiotics have been discovered in gram-positive and gram-negative bacteria that will provide new genes for combinatorial synthesis of new molecules. Received: 2 December 1997 / Received revision: 20 February 1998 / Accepted: 24 February 1998     

Preferential production of rapamycin vs prolylrapamycin by Streptomyces hygroscopicus

A trace of prolylrapamycin is often produced in rapamycin fermentations carried out by strains of Streptomyces hygroscopicus. Prolylrapamycin was produced as the major rapamycin when L-proline was added to the fermentation medium. Addition of proline plus thiazolidine-2-carboxylic acid (T2CA), a sulfur-containing proline analog, prevented rapamycin production and stimulated prolylrapamycin production, thereby resulting in an even greater selective production of prolylrapamycin. T2CA addition inhibited rapamycin production even in the presence of L-lysine which is converted into pipecolic acid intracellularly and normally stimulates rapamycin formation. Addition of the rapamycin precursor, DL-pipecolic acid, surprisingly failed to stimulate rapamycin production. However, when DL-pipecolic acid was added with L-proline, it reduced the formation of prolylrapamycin and stimulated rapamycin production; this was evident especially in the presence of T2CA. The evidence suggests that T2CA suppresses rapamycin production by inhibiting intracellular conversion of L-lysine into pipecolate. Furthermore, the data suggest that uptake of pipecolate into the cell is stimulated or induced by growth in the presence of L-proline and/or T2CA. Received 24 December 1997/ Accepted in revised form 12 May 1998     

Cloning and expression of the α–L-arabinofuranosidase gene (ABF2) of Aspergillus niger in Saccharomyces cerevisiae

 First-strand cDNA was prepared from mRNA of Aspergillus niger MRC11624 induced on oat spelts xylan. Using the cDNA as a template, the α-L-arabinofuranosidase gene (abf B) was amplified with the polymerase chain reaction technique. The abf B DNA fragment was inserted between the yeast phosphoglycerate kinase I gene promoter (PGK1 _P ) and terminator (PGK1 _T ) sequences on a multicopy episomal plasmid. The resulting construct PGK1 _P -abf B-PGK1 _T was designated ABF2. The ABF2 gene was expressed successfully in Saccharomyces cerevisiae and functional α-L-arabinofuranosidase was secreted from the yeast cells. The ABF2 nucleotide sequence was determined and verified to encode a 449-amino-acid protein (Abf 2) that is 94% identical to the α-L-arabinofuranosidase B of A. niger N400. Maximum α-L-arabinofuranosidase activities of 0.020 U/ml and 1.40 U/ml were obtained with autoselective recombinant S. cerevisiae strains when grown for 48 h in synthetic and complex medium respectively. Received: 29 January 1996/Received revision: 3 May 1996/Accepted: 9 May 1996     

Investigation of poly ( β-L-malic acid) production by strains of Aureobasidium pullulans

 Eight strains of the genus Aureobasidium obtained from culture collections were tested for their capability to produce poly(β-L-malic acid) (PMA). Four of the tested strains showed positive results. The most productive strain, A. pullulans CBS 591.75, was used to study the production of PMA in stirred-tank reactors. It was found that PMA was mainly produced in the late exponential phase, and the production related positively to glucose consumption. At the beginning of the fermentation the pH increased from 4.0 to about 7.0; subsequently the pH decreased and remained stable at around 3.0–3.5 for several days. Temperatures higher than 25^°C were detrimental to PMA production and cell growth. PMA production and cell growth at 20^°C and 25^°C exhibited no significant differences. PMA production and cell growth were studied under pH-controlled fermentation (at pH 2.0, 4.0, 5.5). The highest PMA production occurred at pH 4.0. PMA production was reduced at pH 2.0 although quite reasonable cell growth occurred at this pH value. Under optimized conditions 9.8 g PMA/l was produced during 9 days of fermentation in the stirred-tank reactors with an overall yield of 0.11 g PMA/g glucose. A procedure for the isolation of PMA and its separation from the other components of the fermentation broth was developed. The isolated PMA was characterized by ¹H and ¹³C-NMR spectroscopy as well as by infrared absorption spectroscopy. Gel-permeation chromatography revealed a relative molecular mass of approximately 3000–5000 by comparison with polyethylene glycol standards. Received: 13 February 1996/Received revision: 25 April 1996/Accepted: 1 May 1996     

Kinetics of Thermoascus aurantiacus solid-state fermentation on sugar-beet pulp – polysaccharide alteration and production of related enzymatic activities

 The fungal solubilization of cell wall components of sugar-beet pulp, during solid-state fermentation of Thermoascus aurantiacus, is reported here. The extracellular fungal enzyme activities related to the substrate degradation were also studied. In 120 h, more than 60% of the main sugar-beet pulp polysaccharides, i.e. pectins, arabinose- and glucose-containing polysaccharides, were rapidly brought into solution by the fungus. The slow accumulation of monosaccharides compared to the fast degradation of the polysaccharides suggested that most of the released sugars were consumed by the fungus. The analysis of the enzymes present in the water extracts of the solid-state cultures proved that the fungus was able to synthesize a complete enzymatic system required for the hydrolysis of the main sugar-beet pulp polysaccharides. The highest enzyme activities measured were β-glucosidase and α-L-arabinofuranosidase. Received: 22 September 1995/Received revision: 15 January 1996/Accepted: 22 January 1996     

Low-specificity l-threonine aldolase of Pseudomonas sp. NCIMB 10558: purification, characterization and its application to β-hydroxy-α-amino acid synthesis

Low-specificity l-threonine aldolase, catalyzing the reversible cleavage/condensation reaction between l-threonine/l-allo-threonine and glycine plus acetaldehyde, was purified to homogeneity from Pseudomonas sp. NCIMB 10558. The enzyme has an apparent molecular mass of approximately 145 kDa and consists of four identical subunits with a molecular mass of 38 kDa. The enzyme, requiring pyridoxal- 5′-phosphate as a coenzyme, is strictly l-specific at the α position, whereas it can not distinguish between threo and erythro forms at the β position. Besides the reversible cleavage/condensation of threonine, the enzyme also catalyzes the reversible interconversion between glycine plus various aldehydes and l-β-hydroxy-α-amino acids, including l-β-(3,4-dihydroxyphenyl)serine, l-β-(3,4-met‐hylenedioxyphenyl)serine and l-β-phenylserine, providing a new route for the industrial production of these important amino acids. Received: 10 November 1997 / Received revision: 7 January 1998 / Accepted 30 January 1998