Enzymatic production of l-phenylalanine from acetamidocinnamic acid: breeding of an acetamidocinnamate amidohydrolase-hyperproducing mutant

To increase the productivity of l-phenylalanine from acetamidocinnamic acid, we screened bacteria containing high acetamidocinnamate amidohydrolase activity, and strain S-5 containing high activity was isolated from soil. The bacteria were identified as Corynebacterium sp. S-5.When strain S-5 was cultured in a medium containing acetamidocinnamic acid as the sole carbon source or enzyme inducer, the formation of acetamidocinnamate amidohydrolase was observed. This was controlled by catabolite repression. When the strain was cultured in a medium containing glucose and acetamidocinnamic acid as the sole nitrogen source, it showed low acetamidocinnamate amidohydrolase activity and an increased doubling time.To obtain acetamidocinnamate amidohydrolase-hyperproducing strain, we enriched cells growing faster than strain S-5 in a medium containing glucose and acetamidocinnamic acid by continuous culture of mutagenized cells. Mutant C-23 had 12-fold the enzyme production and 3-fold the growth rate of the wild-type strain in a medium containing glucose. Acetamidocinnamate amidohydrolase formation in the mutant did not require acetamidocinnamic acid as enzyme inducer and was resistant to catabolite repression.     

Over-production of hydantoinase and N-carbamoylamino acid amidohydrolase enzymes by regulatory mutants of Agrobacterium tumefaciens

While the hydantoin-hydrolysing enzymes from Agrobacterium strains are used as biocatalysts in the commercial production of D-p-hydroxyphenylglycine, they are now mostly produced in heterologous hosts such as Escherichia coli. This is due to the fact that the activity of these enzymes in the native strains is tightly regulated by growth conditions. Hydantoinase and N-carbamoylamino acid amidohydrolase (NCAAH) activities are induced when cells are grown in the presence of hydantoin or an hydantoin analogue, and in complete medium, enzyme activity can be detected only in early stationary growth phase. In this study, the ability of Agrobacterium tumefaciens RU-OR cells to produce active enzymes was found to be dependent upon the choice of nitrogen source and the presence of inducer, 2-thiouracil, in the growth medium. Growth with (NH4)2SO4 as the nitrogen source repressed the production of both enzymes (nitrogen repression) and also resulted in a rapid, but reversible loss of hydantoinase activity in induced cells (ammonia shock). Mutant strains with inducer-independent production of the enzymes and/or altered response to nitrogen control were isolated. Of greatest importance for industrial application was strain RU-ORPN1F9, in which hydantoinase and NCAAH enzyme activity was inducer-independent and no longer sensitive to nitrogen repression or ammonia shock. Such mutants offer the potential for native enzyme production levels equivalent to those achieved by current heterologous expression systems.     

Regulation of glutamate dehydrogenases in nit-2 and am mutants of Neurospora crassa.

The regulation of the glutamate dehydrogenases was investigated in wild-type Neurospora crassa and two classes of mutants altered in the assimilation of inorganic nitrogen, as either nitrate or ammonium. In the wild-type strain, a high nutrient carbon concentration increased the activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-glutamate dehydrogenase and decreased the activity of reduced nicotinamide adenine dinucleotide (NADH)-glutamate dehydrogenase. A high nutrient nitrogen concentration had the opposite effect, increasing NADH-glutamate dehydrogenase and decreasing NADPH-glutamate dehydrogenase. The nit-2 mutants, defective in many nitrogen-utilizing enzymes and transport systems, exhibited low enzyme activities after growth on a high sucrose concentration: NADPH-glutamate dehydrogenase activity was reduced 4-fold on NH(4)Cl medium, and NADH-glutamate dehydrogenase, 20-fold on urea medium. Unlike the other affected enzymes of nit-2, which are present only in basal levels, the NADH-glutamate dehydrogenase activity was found to be moderately enhanced when cells were grown on a low carbon concentration. This finding suggests that the control of this enzyme in nit-2 is hypersensitive to catabolite repression. The am mutants, which lack NADPH-glutamate dehydrogenase activity, possessed basal levels of NADH-glutamate dehydrogenase activity after growth on urea or l-aspartic acid media, like the wild-type strain, and possessed moderate levels (although three- to fourfold lower than the wild-type strain) on l-asparagine medium or l-aspartic acid medium containing NH(4)Cl. These regulatory patterns are identical to those of the nit-2 mutants. Thus, the two classes of mutants exhibit a common defect in NADH-glutamate dehydrogenase regulation. Double mutants of nit-2 and am had lower NADH-glutamate dehydrogenase activities than either parent. A carbon metabolite is proposed to be the repressor of NADH-glutamate dehydrogenase in N. crassa.     

Isolation and Characterization of a Bacillus cereus Mutant Strain Hyperproductive of Exo-β-Amylase

Starting with a strain of Bacillus cereus excreting about 40-fold more β-amylase than does the original wild-type strain, we isolated, after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine, a strain designated BQ10-S1 SpoIII which showed under optimal conditions a further 5.5-fold increase in β-amylase activity. The amylase production of this strain was observed to increase in the presence of 0.5% glucose or 1% maltose and, more markedly, in the presence of 2% soluble starch in the culture medium. The enzyme produced by this strain was immunologically identical to the wild-type enzyme, suggesting that either the copy number of the gene or the efficiency of enzyme synthesis from it, or both, are altered in this strain.     

Derepression of asparaginase II during exponential growth of Saccharomyces cerevisiae on ammonium ion

The biosynthesis of asparaginase II in Saccharomyces cerevisiae is sensitive to nitrogen catabolite repression. In cell cultures growing in complete ammonia medium, asparaginase II synthesis is repressed in the early exponential phase but becomes derepressed in the midexponential phase. When amino acids such as glutamine or asparagine replace ammonium ion in the growth medium, the enzyme remains repressed into the late exponential phase. The three nitrogen compounds permit a similar rate of cell growth and are assimilated at nearly the same rate. In the early exponential phase the internal amino acid pool is larger in cells growing with glutamine or asparagine than in cells growing with ammonium sulfate as the sole source of nitrogen.     

Biogenesis of mitochondria. The effects of catabolite repression on the in vitro phospholipid synthetic capacities of subcellular fractions of respiratory-competent and respiratory-deficient strains of Saccharomyces cerevisiae.

A respiratory-competent wild-type strain and a nuclear isogenic, mitochondrial DNA-less, petite mutant strain of Saccharomyces cerevisiae were grown under conditions of catabolite repression in batch cultures and under conditions of catabolite derepression in chemostat cultures. Subcellular fractions were isolated and the capacity of these fractions to incorporate sn-[2-³H]glycerol 3-phosphate into phospholipids was studied. Neither catabolite repression nor loss of mitochondrial DNA appreciably altered the total in vitro lipid synthesized by mitochondrial fractions during the incubation. Mitochondria isolated from catabolite-derepressed wild-type and petite cells had approximately the same specific activity in vitro for the synthesis of phosphatidylinositol. phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, and neutral lipids. Mitochondria isolated from the petite cells retained the capacity to synthesize phosphatidylglycerol and diphosphatidylglycerol, although the synthesis of these phospholipids was far less extensive than that by the mitochondria isolated from the wild-type cells. In both cases, mitochondria prepared from catabolite-repressed cells synthesized a greater proportion of phosphatidylserine than did mitochondria from catabolite-derepressed cells. The proportions of phospholipid species synthesized in vitro by the microsomal fractions studied were not grossly affected by catabolite repression or loss of mitochondrial DNA.     

Methylglyoxal-mediated growth inhibition in an Escherichia coli cAMP receptor protein mutant

Under certain growth conditions, some strains of Escherichia coli accumulate toxic levels of methylglyoxal. This report characterizes a strain which synthesizes a mutant cAMP receptor protein in an adenylate cyclase deletion background. When cultured in glucose 6-phosphate minimal medium, this strain (222) was prematurely growth arrested due to methylglyoxal production; growth inhibition did not occur when the strain was grown in glucose minimal medium. A comparison of a variety of enzyme and cofactor levels in the related strains 222 (mutant) and 225 (wild-type) grown on either glucose or glucose 6-phosphate medium was carried out. The only difference found that might explain an increase in methylglyoxal accumulation was an elevated level of phosphofructokinase in strain 222 grown on glucose 6-phosphate. Since this enzyme activity probably limits hexose phosphate metabolism, it is suggested that growth inhibition in strain 222 may be due to increased production of triose phosphate, some of which is converted to methylglyoxal.     

Growth of and trehalase activity in the thermophilic fungusThermomyces lanuginosus

The thermophilic fungus,Thermomyces lanuginosus, was grown in a glucose-asparagine liquid medium. Optimal mycelial growth occurred at 50°C. The conditions for sporulation were different from those required for vegetative growth. the former being favoured by lower nitrogen level and temperature. Trehalase (α, α-glu coside-l-glucohydrolase, EC 3.2.1.28) was one of the most active glycosidases at 50°C. Non-sporulating mycelium had higher levels of this enzyme than the sporulating mycelium. Trehalase was synthesized constitutively and its activity appears to be controlled by catabolite repression.     

Transductional construction of aspartase-hyperproducing strain of Escherichia coli B

Two aspartase-overproducing mutants of Escherichia coli B were characterized. Strain EAPc7 had a mutation enhancing aspartase formation in the region of aspartase gene. This mutation did not affect catabolite repression by aspartase. Strain EAPc244 showed a high cAMP content and an elevated adenylate cyclase activity. This mutation was closely linked to the ilv locus and caused the release of catabolite repression for various catabolite repression-sensitive enzymes, resulting in overproduction of adenylate cyclase. This mutation was transduced to an Ile⁻ strain derived from strain EAPc7 using the Ile⁺ selective marker. The constructed strain AT202, having the above 2 mutations, produced about 3-fold and 18-fold more aspartase than did the 2 parent strains and the wild-type strain, respectively, when cultured in the medium used for industrial production of aspartase. Strain AT202 maintained stably high aspartase activity after 30 cell generations. On the other hand, in E. coli K-12 harboring the aspA⁺ recombinant plasmid pYT471 (pBR322-aspA⁺), the activity decreased to the E. coli K-12 level. Hence, strain AT202 is more advantageous for industrial production of l-aspartic acid than cells harboring the aspA⁺-recombinant plasmid pYT471.     

Isolation and Characterization of a Catabolite Repression-Insensitive Mutant of a Methanol Yeast, Candida boidinii A5, Producing Alcohol Oxidase in Glucose-Containing Medium

Mutants exhibiting alcohol oxidase (EC 1.1.3.13) activity when grown on glucose in the presence of methanol were found among 2-deoxyglucose-resistant mutants derived from a methanol yeast, Candida boidinii A5. One of these mutants, strain ADU-15, showed the highest alcohol oxidase activity in glucose-containing medium. The growth characteristics and also the induction and degradation of alcohol oxidase were compared with the parent strain and mutant strain ADU-15. In the parent strain, initiation of alcohol oxidase synthesis was delayed by the addition of 0.5% glucose to the methanol medium, whereas it was not delayed in mutant strain ADU-15. This showed that alcohol oxidase underwent repression by glucose. On the other hand, degradation of alcohol oxidase after transfer of the cells from methanol to glucose medium (catabolite inactivation) was observed to proceed at similar rates in parent and mutant strains. The results of immunochemical titration experiments suggest that catabolite inactivation of alcohol oxidase is coupled with a quantitative change in the enzyme. Mutant strain ADU-15 was proved to be a catabolite repression-insensitive mutant and to produce alcohol oxidase in the presence of glucose. However, it was not an overproducer of alcohol oxidase and, in both the parent and mutant strains, alcohol oxidase was completely repressed by ethanol.     

Cell wall invertase in tobacco crown gall cells : enzyme properties and regulation by auxin

The cell wall invertase from an Agrobacterium tumefaciens-transformed Nicotiana tabacum cell line (SR1-C58) was purified. The heterogeneously glycosylated enzyme has the following properties: M_r 63,000, pH optimum at 4.7, K_{m sucrose} 0.6 millimolar (at pH 4.7), pl 9.5. Enzyme activity is inhibited by micromolar concentrations of HgCl₂ but is insensitive to H₂O₂, N-ethylmaleimide and dithiothreitol. Upon transfer of transformed cells from the stationary phase to fresh medium, a cycloheximide- and tunicamycin-sensitive de novo formation of cell wall invertase is demonstrated in the absence or presence of sucrose. While in an auxin mutant (lacking gene 1;SR1-3845) 1 micromolar 1-naphthaleneacetic acid led to a further increased activity, the wild-type transformed cell line (SR1-C58) responded with a decreased activity compared to the control. An analysis of cell wall invertase in and around tumors initiated with Agrobacterium tumefaciens (strain C58) on Nicotiana tabacum stem and Kalanchoë daigremontiana leaves revealed gradients of activity. The results indicate that the auxin-stimulated cell wall invertase is essential for the establishment of the tumor sink.     

Glycerol kinase, the pacemaker for the dissimilation of glycerol in Escherichia coli

The activity of glycerol kinase is rate-limiting in the metabolism of glycerol by cells of Escherichia coli. A mutant strain producing a glycerol kinase resistant to inhibition by fructose-1,6-diphosphate grows faster than its wild-type parent on glycerol as the sole source of carbon and energy. The amount of intracellular fructose-1,6-diphosphate was determined for wild-type cells growing exponentially on glycerol. The water content of such cells was also determined, allowing calculation of the intracellular concentration of fructose-1,6-diphosphate. This value, 1.7 mm, is adequate to exert substantial inhibition on the wild-type glycerol kinase. The desensitization of glycerol kinase to feedback inhibition also enhances the power of glycerol to exert catabolite repression, both on the enzymes of the glycerol system itself and on those of the lactose system. However, desensitization of glycerol kinase alone does not eliminate the phenomenon of diauxic growth in a glucose-glycerol medium. Biphasic growth in such a medium is abolished if the altered enzyme is produced constitutively. The constitutive production of the mutant kinase at high levels, however, renders the cells vulnerable to glycerol. Thus, when the cells have been grown on a carbon source with a low power for catabolite repression, e.g., succinate, sudden exposure to glycerol leads to overconsumption of the nutrient and cell death.     

Purification and regulation of glutamine synthetase in a collagenolytic Vibrio alginolyticus strain

Glutamine synthetase (EC 6.3.1.2) has been purified from a collagenolytic Vibrio alginolyticus strain. The apparent molecular weight of the glutamine synthetase subunit was approximately 62,000. This indicates a particle weight for the undissociated enzyme of 744,000, assuming the enzyme is the typical dodecamer. The glutamine synthetase enzyme had a sedimentation coefficient of 25.9 S and seems to be regulated by a denylylation and deadenylylation. The pH profiles assayed by the -glutamyltransferase method were similar for NH₄-shocked and unshocked cell extracts and isoactivity point was not obtained from these eurves. The optimum pH for purified and crude cell extracts was 7.9. Cell-free glutamine synthetase was inhibited by some amino acids and AMP. The transferase activity of glutamine synthetase from mid-exponential phase cells varied greatly depending on the sources of nitrogen or carbon in the growth medium. Glutamine synthetase level was regulated by nitrogen catabolite repression by (NH₄)₂SO₄ and glutamine, but cells grown, in the presence of proline, leucine, isoleucine, tryptophan, histidine, glutamic acid, glycine and arginine had enhanced levels of transferase activity. Glutamine synthetase was not subject to glucose, sucrose, fructose, glycerol or maltose catabolite repression and these sugars had the opposite effect and markedly enhanced glutamine synthetase activity.Abbreviations GS glutamine synthetase - SMM succinate minimal medium - ASMM ammonium/succinate minimal medium - GT -glutamyl transferase - SVP snake venom phosphodiesterase     

Isolation and characterization of a dihydropyrimidine dehydrogenase mutant of Pseudomonas chlororaphis

A dihydropyrimidine dehydrogenase mutant of Pseudomonas chlororaphis ATCC 17414 was isolated and characterized in this study. Initially, reductive catabolism of uracil was confirmed to be active in ATCC 17414 cells. Following chemical mutagenesis and d-cycloserine counterselection, a mutant strain unable to utilize uracil as a nitrogen source was identified. It was also unable to utilize thymine as a nitrogen source but could use either dihydrouracil or dihydrothymine as a sole source of nitrogen. Subsequently, it was determined that the mutant strain was deficient for the initial enzyme in the reductive pathway dihydropyrimidine dehydrogenase. The lack of dehydrogenase activity did not seem to have an adverse effect upon the activity of the second reductive pathway enzyme dihydropyrimidinase activity. It was shown that both dihydropyrimidine dehydrogenase and dihydropyrimidinase levels were affected by the nitrogen source present in the growth medium. Dihydropyrimidine dehydrogenase and dihydropyrimidinase activities were elevated after growth on uracil, thymine, dihydrouracil or dihydrothymine as a source of nitrogen.     

AiiA Quorum-Sensing Quenching Controls Proteolytic Activity and Biofilm Formation by Enterobacter cloacae

The aim of this work was to evaluate a quorum-quenching approach to identify functions regulated by quorum sensing in Enterobacter cloacae. We employed an aiiA transconjugant strain of E. cloacae that synthesizes a lactonase enzyme that hydrolyzes N-acyl homoserine lactone signaling molecules to compare bacterial phenotypes in the presence and absence of quorum signals. The aiiA-expressing strain displayed increased proteolytic activity and intensity of a milk-clotting reaction when compared to the wild-type strain. Although both strains growing on polystyrene plates in rich media and a minimal medium of salts formed biofilms, the wild-type strain exhibited a higher number of adhered cells. On the surface of stainless steel coupons that were submerged in culture media, the number of adhered cells of the wild type contained up to one log more cells compared with the aiiA transconjugant. However, after 48?h of incubation, there was no significant difference between the strains. The results demonstrated that the quorum-sensing system negatively regulates proteolytic activity and is likely involved in the early steps of biofilm formation by E. cloacae 067.     

Improved α-amylase production by Aspergillus oryzae after a double deletion of genes involved in carbon catabolite repression

In filamentous fungi, the expression of secretory glycoside hydrolase encoding genes, such as those for amylases, cellulases, and xylanases, is generally repressed in the presence of glucose. CreA and CreB have been observed to be regulating factors for carbon catabolite repression. In this study, we generated single and double deletion creA and/or creB mutants in Aspergillus oryzae. The α-amylase activities of each strain were compared under various culture conditions. For the wild-type strain, mRNA levels of α-amylase were markedly decreased in the later stage of submerged culture under inducing conditions, whereas this reduced expression was not observed for single creA and double creA/creB deletion mutants. In addition, α-amylase activity of the wild-type strain was reduced in submerged culture containing high concentrations of inducing sugars, whereas all constructed mutants showed higher α-amylase activities. In particular, the α-amylase activity of the double deletion mutant in a medium containing 5 % starch was >10-fold higher than that of the wild-type strain under the same culture conditions. In solid-state cultures using wheat bran as a substrate, the α-amylase activities of single creA and double deletion mutants were >2-fold higher than that of the wild-type strain. These results suggested that deleting both creA and creB resulted in dramatic improvements in the production of secretory glycoside hydrolases in filamentous fungi.