Overproducing Mutant Research Articles

A photosystem II-associated carbonic anhydrase regulates the efficiency of photosynthetic oxygen evolution.

We show for the first time that Cah3, a carbonic anhydrase associated with the photosystem II (PSII) donor side in Chlamydomonas reinhardtii, regulates the water oxidation reaction. The mutant cia3, lacking Cah3 activity, has an impaired water splitting capacity, as shown for intact cells, thylakoids and PSII particles. To compensate this impairment, the mutant overproduces PSII reaction centres (1.6 times more than wild type). We present compelling evidence that the mutant has an average of two manganese atoms per PSII reaction centre. When bicarbonate is added to mutant thylakoids or PSII particles, the O2 evolution rates exceed those of the wild type by up to 50%. The donor side of PSII in the mutant also exhibits a much higher sensitivity to overexcitation than that of the wild type. We therefore conclude that Cah3 activity is necessary to stabilize the manganese cluster and maintain the water-oxidizing complex in a functionally active state. The possibility that two manganese atoms are enough for water oxidation if bicarbonate ions are available is discussed.

Activity of the enzymes involved in the synthesis of exopolysaccharide precursors in an overproducing mutant ropy strain ofStreptococcus thermophilus

The activities of some enzymes belonging to the Leloir pathway, phosphoglucomutase, UDP-glucose pyrophosphorylase, UDP-galactose 4-epimerase and galactose 1-P uridyl transferase, were studied in a wild ropy, a non-ropy and an overproducing mutant ropy strain of Streptococcus thermophilus. These activities were assayed over successive culture transfers along with exocellular polysaccharide (EPS) production. The overproducing mutant ropy strain showed increments in polysaccharide production over successive culture transfers, as opposed to reductions in production by the wild ropy strain. The observed variations among strains in the enzyme activities that were analysed in relation to EPS production suggest their involvement in the synthesis of sugar-nucleotide EPS precursors.

Activity of the enzymes involved in the synthesis of exopolysaccharide precursors in an overproducing mutant ropy strain of Streptococcus thermophilus

The activities of some enzymes belonging to the Leloir pathway, phosphoglucomutase, UDP-glucose pyrophosphorylase, UDP-galactose 4-epimerase and galactose 1-P uridyl transferase, were studied in a wild ropy, a non-ropy and an overproducing mutant ropy strain of Streptococcus thermophilus. These activities were assayed over successive culture transfers along with exocellular polysaccharide (EPS) production. The overproducing mutant ropy strain showed increments in polysaccharide production over successive culture transfers, as opposed to reductions in production by the wild ropy strain. The observed variations among strains in the enzyme activities that were analysed in relation to EPS production suggest their involvement in the synthesis of sugar-nucleotide EPS precursors.

Extractability of astaxanthin in a mixed culture of a carotenoid over-producing mutant of Xanthophyllomyces dendrorhous and Bacillus circulans in two-stage batch fermentation

The productivity and extractability of astaxanthin in a mixed culture of Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) NCHU-FS501, an astaxanthin over-producing mutant, and Bacillus circulans CCRC 11590 was evaluated in a 1.5 l fermentor using a two-stage batch fermentation technique. The first stage was for X. dendrorhous cultivation. The second stage was the mixed fermentation of the red yeast and B. circulans. The highest lytic enzyme activity of B. circulans was found at 24 h with yeast nitrogen base (YNB) as the nitrogen source and incubation at 30 °C in a pure culture. The induction of B. circulans lytic enzyme activity was influenced by the cell wall concentration of X. dendrorhous. The fastest induction was observed with addition of 2.5 g/l cell wall. The cultivation time in the first stage of fermentation significantly affected the extractability of carotenoid in the second stage of fermentation. Minimum extractability was found when X. dendrorhous was cultivated for 96 h in the first stage. Glucose concentration of 45 g/l in YNB supported high X. dendrorhous growth and astaxanthin production, with astaxanthin production of 9010 μg/l was observed during the first stage. Although the red yeast produced more astaxanthin with peptone as nitrogen source during the first stage, YNB supported the highest extractability of carotenoid and lytic activity of B. circulans in the second stage of fermentation. The optimal pH and temperature for pigment extraction in the mixed culture were pH 6.5 and 30–34 °C, respectively. Under these conditions, about 97% of the total pigment could be extracted after a 48 h incubation time. When the crude enzyme was recycled in the mixed culture, lytic enzyme activity of 20 units/ml was obtained after one incubation cycle (48 h), with 69% of total carotenoids extracted.

Purification, cloning, and DNA sequence analysis of a chitinase from an overproducing mutant of Streptomyces peucetius defective in daunorubicin biosynthesis

Extracellular chitinases of Streptomyces peucetius and a chitinase overproducing mutant, SPVI, were purified to homogeneity by ion exchange and gel filtration chromatography. The purified enzyme has a molecular mass of 42 kDa on SDS-PAGE, and the N-terminal amino acid sequence of the protein from the wild type showed homology to catalytic domains (Domain IV) of several other Streptomyces chitinases such as S. lividans 66, S. coelicolor A3(2), S. plicatus, and S. thermoviolaceus OPC-520. Purified SPVI chitinase cross-reacted to anti-chitinase antibodies of wild-type S. peucetius chitinase. A genomic library of SPVI constructed in E. coli using λ DASH II was probed with chiC of S. lividans 66 to screen for the chitinase gene. A 2.7 kb fragment containing the chitinase gene was subcloned from a λ DASH II clone, and sequenced. The deduced protein had a molecular mass of 68 kDa, and showed domain organization similar to that of S. lividans 66 chiC. The N-terminal amino acid sequence of the purified S. peucetius chitinase matched with the N-terminus of the catalytic domain, indicating the proteolytic processing of 68 kDa chitinase precursor protein to 42 kDa mature chitinase containing the catalytic domain only. A putative chiR sequence of a two-component regulatory system was found upstream of the chiC sequence.Key words: chitinase, chitinase purification, Streptomyces peucetius, daunorubicin, chiC.

Two different pathways are involved in the β‐oxidation of n‐alkanoic and n‐phenylalkanoic acids in Pseudomonas putida U: genetic studies and biotechnological applications

In Pseudomonas putida U, the degradation of n-alkanoic and n-phenylalkanoic acids is carried out by two sets of beta-oxidation enzymes (betaI and betaII). Whereas the first one (called betaI) is constitutive and catalyses the degradation of n-alkanoic and n-phenylalkanoic acids very efficiently, the other one (betaII), which is only expressed when some of the genes encoding betaI enzymes are mutated, catabolizes n-phenylalkanoates (n > 4) much more slowly. Genetic studies revealed that disruption or deletion of some of the betaI genes handicaps the growth of P. putida U in media containing n-alkanoic or n-phenylalkanoic acids with an acyl moiety longer than C4. However, all these mutants regained their ability to grow in media containing n-alkanoates as a result of the induction of betaII, but they were still unable to catabolize n-phenylalkanoates completely, as the betaI-FadBA enzymes are essential for the beta-oxidation of certain n-phenylalkanoyl-CoA derivatives when they reach a critical size. Owing to the existence of the betaII system, mutants lacking betaIfadB/A are able to synthesize new poly 3-OH-n-alkanoates (PHAs) and poly 3-OH-n-phenylalkanoates (PHPhAs) efficiently. However, they are unable to degrade these polymers, becoming bioplastic overproducer mutants. The genetic and biochemical importance of these results is reported and discussed.

Carrier-mediated transport of riboflavin in Ashbya gossypii.

The filamentous hemiascomycete Ashbya gossypii is used for industrial riboflavin production. We examined riboflavin uptake and excretion at the plasma membrane using riboflavin auxotrophic and overproducing mutants. The riboflavin uptake system had low activity [Vmax = 20 +/- 4 nmol min(-1) g(-1) mycelial dry weight (dw)] and high affinity (KM = 40 +/- 12 microM). Inhibitor studies with the analogs FMN and FAD revealed high specificity of the uptake system. Excretion of riboflavin was not the consequence of non-specific permeability of the plasma membrane. Excretion rates in the mid-production phase were determined to be 2.5 nmol min(-1) g(-1) dw for wild-type cells and 66.7 nmol min(-1) g(-1) dw for an overproducing mutant, respectively. Inhibition of the reverse reaction, riboflavin uptake, led to an increase in apparent riboflavin efflux in the early production phase, indicating the presence of a separate excretion carrier. Riboflavin accumulation in A. gossypii vacuoles leading to product retention was found to be a secondary transport process. To address the question of whether a flux from the vacuoles back into the cytoplasm is present, we characterized efflux in hyphae in which the plasma membrane was permeabilized with digitonin. Efflux kinetics across the vacuolar membrane were unaffected by the lack of vacuolar H+ATPase activity and ATP, suggesting a passive mechanism. Based on the characterization of riboflavin transport processes in this study, the design of new production strains with improved riboflavin excretion may be possible.

Purification, cloning, and DNA sequence analysis of a chitinase from an overproducing mutant of <i>Streptomyces peucetius </i>defective in daunorubicin biosynthesis

Extracellular chitinases of Streptomyces peucetius and a chitinase overproducing mutant, SPVI, were purified to homogeneity by ion exchange and gel filtration chromatography. The purified enzyme has a molecular mass of 42 kDa on SDS-PAGE, and the N-terminal amino acid sequence of the protein from the wild type showed homology to catalytic domains (Domain IV) of several other Streptomyces chitinases such as S. lividans 66, S. coelicolor A3(2), S. plicatus, and S. thermoviolaceus OPC-520. Purified SPVI chitinase cross-reacted to anti-chitinase antibodies of wild-type S. peucetius chitinase. A genomic library of SPVI constructed in E. coli using lambda DASH II was probed with chiC of S. lividans 66 to screen for the chitinase gene. A 2.7 kb fragment containing the chitinase gene was subcloned from a lambda DASH II clone, and sequenced. The deduced protein had a molecular mass of 68 kDa, and showed domain organization similar to that of S. lividans 66 chiC. The N-terminal amino acid sequence of the purified S. peucetius chitinase matched with the N-terminus of the catalytic domain, indicating the proteolytic processing of 68 kDa chitinase precursor protein to 42 kDa mature chitinase containing the catalytic domain only. A putative chiR sequence of a two-component regulatory system was found upstream of the chiC sequence.

Pigment Diverse Mutants of Pseudomonas sp.: Inhibition of Fungal Growth and Stimulation of Growth of Cicer arietinum

A Pseudomonas strain MRS16 inhibited growth of different pathogenic fungi (Aspergillus sp., Fusarium oxysporum, Pythium aphanidermatum and Rhizoctonia solani) in vitro. Larger inhibition zones were obtained on nutrient agar and King's B media compared to potato dextrose agar and pigment production media. Mutants altered in production of fluorescent pigment were derived by nitrosoguanidine mutagenesis. The pigment overproducer mutant MRS16M-1 was more inhibitory whereas nonproducer mutant MRS16M-5 was less inhibitory than parent strain on nutrient agar medium. Addition of iron (100 µM FeCl3) in the medium decreased inhibition of fungal growth, suggesting the involvement of siderophores and other antifungal secondary metabolites. Seed bacterization of two cultivars of chickpea (Cicer arietinum cvs. H8618 and C235) differing in susceptibility to wilt caused initial root and shoot stunting at 5 d of growth followed by proliferation of secondary root growth at 10 d. Coinoculation of chickpea with Pseudomonas strain MRS16 or mutants and Rhizobium sp. Cicer strain Ca181 enhanced nodulation, nitrogen fixation and plant dry mass as compared to single inoculation with Rhizobium strain under sterile conditions.

Screening of Phytase Overproducing Strains in Aspergillus spp. by UV Mutagenesis

Phytases (myo-ino sitol hexakisphosphate phosphohydrolase; EC 3.1.3.8) are enzymes which catalyze the hydrolisys of phytate into myo-inositol and inorganic phosphates. Phytases are found in plants and a variety of microorganisms. Aspergillus species were treated with 254 nm of UV irradiation for the screening of phytase overproducing mutant strains. At 15 minute irradiation, the survivals of population were less than 5%, and UV irradiation time was decided at 20 minute for the isolation of mutant strains. Four UV mutant strains in A. oryzae (YUV-47, -169 , -341, -511) and six in A. ficuum (FUV-17 , -36, -69 , -193 , -317 , -419) were isolated on PSM media containing ammonium phosphate. The specific enzyme activities of A. ficuum mutants are 110 to 140% high er than that of wild type.

Plant growth-promoting bacteria that decrease heavy metal toxicity in plants.

Kluyvera ascorbata SUD165 and a siderophore-overproducing mutant of this bacterium, K. ascorbata SUD165/26, were used to inoculate tomato, canola, and Indian mustard seeds which were then grown in soil for 25-42 days in the presence of either nickel, lead, or zinc. The parameters that were monitored included plant wet and dry weight, protein and chlorophyll content in the plant leaves, and concentration of heavy metal in the plant roots and shoots. As indicated by a decrease in the measured values of these parameters, in all instances, plant growth was inhibited by the presence of the added metal. Both bacterial strains were effective, although not always to a statistically significant extent, at relieving a portion of the growth inhibition caused by the metals. In most cases, the siderophore overproducing mutant K. ascorbata 165/26 exerted a more pronounced effect on plant growth than did the wild-type bacterium K. ascorbata SUD165. The data suggest that the ability of these bacteria to protect plants against the inhibitory effects of high concentrations of nickel, lead, and zinc is related to the bacteria providing the plants with sufficient iron.

Deletion of the pyc gene blocks clavulanic acid biosynthesis except in glycerol-containing medium: evidence for two different genes in formation of the C3 unit.

The beta-lactamase inhibitor clavulanic acid is formed by condensation of a pyruvate-derived C3 unit with a molecule of arginine. A gene (pyc, for pyruvate converting) located upstream of the bls gene in the clavulanic acid gene cluster of Streptomyces clavuligerus encodes a 582-amino-acid protein with domains recognizing pyruvate and thiamine pyrophosphate that shows 29.9% identity to acetohydroxyacid synthases. Amplification of the pyc gene resulted in an earlier onset and higher production of clavulanic acid. Replacement of the pyc gene with the aph gene did not cause isoleucine-valine auxotrophy in the mutant. The pyc replacement mutant did not produce clavulanic acid in starch-asparagine (SA) or in Trypticase soy broth (TSB) complex medium, suggesting that the pyc gene product is involved in the conversion of pyruvate into the C3 unit of clavulanic acid. However, the beta-lactamase inhibitor was still formed at the same level as in the wild-type strain in defined medium containing D-glycerol, glutamic acid, and proline (GSPG medium) as confirmed by high-pressure liquid chromatography and paper chromatography. The production of clavulanic acid by the replacement mutant was dependent on addition of glycerol to the medium, and glycerol-free GSPG medium did not support clavulanic acid biosynthesis, suggesting that an alternative gene product catalyzes the incorporation of glycerol into clavulanic acid in the absence of the Pyc protein. The pyc replacement mutant overproduces cephamycin.

Identification of growth phenotypes in Aspergillus niger pectinase over-producing mutants using image analysis procedures

Relative growth rates of four strains of Aspergillus niger were estimated by image analysis of colonies grown with mineral salts and 10 g pectin/l. Water activity (aw) levels were 0.96 and 0.99 and 2-deoxy-D-glucose (2DG) was added up to 0.1 g/l. These techniques proved useful to select mould strains for pectinase production in culture media with different water activities. © Rapid Science Ltd. 1998

Non-inducible, mainly cell-associated beta-lactamase from Nocardia asteroides strain 108.

The beta-lactamase of the soil-borne strain 108 (parental strain) of Nocardia asteroides is a non-inducible enzyme mainly associated with the cells; it can be efficiently extracted by ultrasonication and SDS treatment. Crude enzyme preparations showed penicillinase and cephalosporinase activity. The kinetics of beta-lactamase production and in-vitro susceptibility to combinations of beta-lactam antibiotics plus beta-lactamase inhibitors have been studied in two stable overproducer mutants (A14 and B1) obtained by mutagenization of the parental strain with nitrosoguanidine. The cell-associated enzyme increased with bacterial growth in parental and mutant strains and was particularly abundant in stationary phase cells. The beta-lactamase inhibitors sulbactam and clavulanic acid decreased MIC values of penicillins more efficiently in the parental strain than in mutants, thus indicating some involvement of the enzyme in the resistance of N. asteroides strain 108 to beta-lactam antibiotics.

A RAPID BIOASSAY TO DETECT MYCOTOXINS USING A MELANIN PRECURSOR OVERPRODUCER MUTANT OF THE CILIATETETRAHYMENA THERMOPHILA

Four different mycotoxins (patulin, T-2 toxin, diacetoxyscirpenol and roquefortine) were used to study growth inhibitory effects on a melanin precursor overproducer mutant of the ciliateTetrahymena thermophilaThis strain is especially sensitive to diacetoxyscirpenol and T-2 toxin. The secretion capacity of melanin precursors into the culture medium by this mutant and its biosensor capacity are very useful characteristics to elaborate a rapid bioassay to detect some specific mycotoxins.

Purification and analysis of an extremely halophilic β-galactosidase from Haloferax alicantei

As a first step in the development of a reporter system for gene expression in halophilic archaea, a β-galactosidase was purified 140-fold from Haloferax alicantei (previously phenon K, strain Aa2.2). An overproducing mutant was first isolated by UV mutagenesis and screening on agar plates containing X-Gal substrate. Cytoplasmic extracts of the mutant contained 25-fold higher enzyme levels than the parent. Purification of the active enzyme was greatly facilitated by the ability of sorbitol to stabilise enzyme activity in the absence of salt, which allowed conventional purification methods (e.g., ion-exchange chromatography) to be utilised. The enzyme was optimally active at 4 M NaCl and was estimated to be 180±20 kDa in size, consisting of two monomers (each 78±3 kDa). It cleaves several different β-galactoside substrates such as ONP-Gal, X-Gal and lactulose, but not lactose, and also has β- d-fucosidase activity. No β-glucosidase, β-arabinosidase or β-xylosidase activity could be detected. The amino-acid sequence at the N-terminus and of four proteolytic products has been determined.

A dinucleotide mutation in dihydrodipicolinate synthase of Nicotiana sylvestris leads to lysine overproduction

By applying a mutagenesis/selection procedure to obtain resistance to a lysine analog, S-(2-aminoethyl)L-cysteine (AEC), a lysine overproducing mutant in Nicotiana sylvestris was isolated. Amino acid analyses performed throughout plant development and of different organs of the N. sylvestris RAEC-1 mutant, revealed a developmental-dependent accumulation of free lysine. Lysine biosynthesis in the RAEC-1 mutant was enhanced due to a lysine feedback-desensitized dihydrodipicolinate synthase (DHDPS). Several molecular approaches were undertaken to identify the nucleotide change in the dhdps-r1 gene, the mutated gene coding for the lysine-desensitized enzyme. The enzyme was purified from wild-type plants for amino end microsequencing and 10 amino acids were identified. Using dicotyledon dhdps probes, a genomic fragment was cloned from an enriched library of DNA from the homozygote RAEC-1 mutant plant. A dhdps cDNA, putatively full-length, was isolated from a tobacco cDNA library. Nucleotide sequence analyses confirmed the presence of the previously identified amino end preceded by a chloroplast transit peptide sequence. Nucleotide sequence comparisons, enzymatic and immunological analyses revealed that the tobacco cDNA corresponds to a normal type of DHDPS, lysine feedback-regulated, and the genomic fragment to the mutated DHDPS, insensitive to lysine inhibition. Functional complementation of a DHDPS-deficient Escherichia coli strain was used as an expression system. Reconstruction between the cDNA and genomic fragment led to the production of a cDNA producing an insensitive form of DHDPS. Amino acid sequence comparisons pointed out, at position 104 from the first amino acid of the mature protein, the substitution of Asn to Ileu which corresponds to a dinucleotide mutation. This change is unique to the dhdps-r1 gene when compared with the wild-type sequence. The identification of the nucleotide and amino acid change of the lysine-desensitized DHDPS from RAEC-1 plant opens new perspectives for the improvement of the nutritional value of crops and possibly to develop a new plant selectable marker.

Antigenic properties and immunoelectron microscopic localization of Mycobacterium fortuitum beta-lactamase.

Mycobacterium fortuitum is a fast-growing Mycobacterium species which produces a beta-lactamase involved in the intrinsic resistance of the microorganism to beta-lactam antibiotics. An anti-beta-lactamase serum against the purified enzyme was raised in rabbits. Antibody binding was specific for native beta-lactamase, and enzyme activity was partially inhibited by the serum; furthermore, cross-reactions with denatured class A beta-lactamases were observed. This serum was used as a probe in immunogold labeling for the localization of the cell-bound beta-lactamase in both the low-level producer ATCC 19542 (parental strain) and the overproducer mutant D316. By the combination of preembedding immunogold labeling and replica technique, it was shown that the beta-lactamase was uniformly distributed on the whole external cell surface, where it appeared to be associated with a Tween 80-removable capsule-like material. Compared with the parental strain, a much higher level of expression of surface enzyme was observed in strain D316. Surface labeling was more intense in the stationary phase of growth than in exponentially growing cells. The data obtained are interpreted in the context of the intrinsic resistance of M. fortuitum to beta-lactam antibiotics.

Enhanced Saline Stress Resistance in Threonine and Methionine Overproducing Mutant Cell Line from Protoplast Culture of Rice ( Oryza sativa L.)

Summary This study was designed to examine whether saline stress resistance was enhanced in mutant in which free amino acids were overproduced. We selected a mutant cell line (RLTA-2) that overproduced threonine and methionine, and a NaCl resistant cell line (RNaCI-1) through protoplast culture treated with ethylmethane-sulfonate (EMS) with addition of inhibitory concentrations of 1 mM each of lysine plus threonine (1 mM LT) and 200 mM NaCl, respectively. Concentrations of threonine, methionine and the total free amino acids changed largely, being 13.8, 6.2 and 1.6 times higher in RLTA-2 than in the wild type, respectively. Proline concentration increased slightly in RLTA-2 when it was subcultured on the medium supplemented with 200 mM NaCl (RLTA-2-200), i.e. ca. 1.4 times in RLTA-2, while a remarkable increase of proline concentration was observed in RNaCI-1, i.e. 11.5 times as compared with the wild type. Furthermore, calcium accumulation was 3 to 5 times in RLTA-2, but not in RNaCl-1, regardless of NaCl concentration. The selected RLTA-2 calli grew better than the wild type under high concentration of NaCl, but the growth rate of RLTA-2 and RNaCl-1 calli was almost similar above 150 mM NaCl concentration. Almost all RLTA-2 calli survived on N6 medium supplemented with 200 mM NaCl. The capacity to maintain the low level of K + and the high level of Ca ++ in the RLTA-2 cell line under the high level of Na + was highly correlated with free amino acid level as compatible cytoplasmic solute and nitrogen source in cytoplasm. The present results indicate the enhancement of saline resistance in the mutant cell line in which free amino acids are overproduced. The relationship of free amino acid accumulation to enhanced saline resistance is discussed.

Protein kinase activity in cell free extracts of Streptomyces avermitilis; comparing wild type and overproducing mutant

Protein kinase activity was assayed in cell-free extracts prepared from mycelia of a wild strain (MA) and an overproducing mutant (RX 2) of Streptomyces avermitilis. At least 10 different polypeptides ranging in Mr from 10 000 to 120 000 were found to be phosphorylated on analysis of 32P labeled cell lysates by gel electrophoresis and phosphorimage. The protein profile and the level of phosphorylation varied depending on the culture stage of the mycelia.