Optimization of culture medium and growth conditions for production of L-arabinose isomerase and D-xylose isomerase by Lactobacillus bifermentans

Lactobacillus bifermentans was used to produce the intracellular enzymes L-arabinose isomerase and D-xylose isomerase. Various factors of cultivation (temperature, pH, or incubation period) and culture medium composition (mineral salts, carbon and nitrogen source) were studied to select the conditions that maximize production of these enzymes. Arabinose isomerase and xylose isomerase activities were 9.4 and 7.24 U/ml, respectively. They were highest at 9 h of cultivation in the optimized medium, 1.6 times higher than that in the basic MRS broth. The optimal medium composition and cultivation conditions were determined. On the other hand, the strain required for growth Tween 80 (1 g/l) and a source of inorganic nitrogen (e.g. ammonium citrate). The bacterium had no requirement for sodium acetate for both growth and production of isomerases. The production rate of enzymes was increased when metal ions were added and mainly manganese (2.5 mM).     

Integration and expression of theEscherichia coli xylose isomerase gene inSchizosaccharomyces pombe

Summary The xyclose isomerase gene inEscherichia coli was cloned complementarily into a Leu2-negativeSchizosaccharomyces pombe mutant (ATCC 38399). The subsequent integration of the plasmid into the chromosomal DNA of the host yeast was verified by using the dot blot and southern blot techniques. The expressed xylose isomerase showed activity on a nondenaturing polyacrylamide gel. The expression of xylose isomerase gene was influenced by the concentration of nutrients in the fermentation broth. The yeast possessed a xylose isomerase activity of 20 nmol/min/mg by growing in an enriched medium containing yeast extract-malt extract-peptone (YMP) andd-xylose. The conversion ofd-xylose tod-xylulose catalyzed by xylose isomerase in the transformed yeast cells makes it possible to fermentd-xylose with ethanol as a major product. When the fermentation broth contained YMP and 5% (w/v)d-xylose, the maximal ethanol yield and productivity reached 0.42 g/g and 0.19 g/l/h, respectively.     

Cloning of the glucose isomerase (D-xylose isomerase) and xylulose kinase genes of Streptomyces violaceoniger

Summary Xylose utilization mutants of Streptomyces violaceoniger were isolated lacking one or both of the enzymes, glucose isomerase (xylose isomerase) and xylulose kinase. Using pUT206 as a cloning vector, complementation of the glucose isomerase negative phenotype with fragments of the S. violaceoniger chromosome permitted isolation of two recombinant plasmids, designated pUT220 and pUT221, which contained 10.6 and 10.1 kb of chromosomal DNA, respectively. Both of these plasmids complemented all three different classes of xylose negative mutants and also provoked an increase of glucose isomerase and xylulose kinase activity in the mutant and wild-type strains. Plasmid pUT220 was chosen for detailed study by subcloning experiments. The putative glucose isomerase gene was localized to a 2.1 kb segment of the 10.6 kb chromosomal DNA fragment. The putative xylulose kinase gene resides nearby. Thus both genes seem to be clustered at a single chromosomal localization. This organization appears similar to that of the xylose utilization pathway in Escherichia coli, Salmonella typhimurium and Bacillus subtilis.     

Optimized culture conditions for the production of furanocoumarins by micropropagated shoots of Ruta graveolens

Ruta graveolens in vitro cultures are a potential source of secondary metabolites (furanocoumarins) of significant medical interest. Experiments led in our laboratory showed that micropropagated shoots were richer in furanocoumarins than any other plant material. In order to optimize the molecule production by such cultivation systems, several factors related to the culture medium were studied. Effects of medium composition on biomass growth and furanocoumarin content were analysed and optimal conditions were determined for phosphate (300 mg l⁻¹ of NaH₂PO₄), nitrate (2527 mg l⁻¹ of KNO₃), carbon source (10 g l⁻¹ of sucrose) and phytohormones (2,4-dichlorophenoxyacetic acid (2,4-D) 50 μM and benzylaminopurine (BAP) 50 μM). Ruta shoot growth and furanocoumarin production were compared for optimized and standard culture conditions. Specific medium gave better results in terms of growth (t_D equal to 6.9 days against 8.6 for standard conditions) but no significant differences appeared concerning metabolite concentrations. However, the present study opens the way to scale-up studies with bioreactor cultivation systems. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cultural characteristics and extraction of the fungal pigment phleichrome from the phytopathogenic fungus<Emphasis Type="Italic">Cladosporium phlei</Emphasis>

The cultural characteristics of the fungusCladosporium phlei were assessed in order to develop an improved method for the production of the fungal pigment, phleichrome, which is an intermediate in the production of a photodynamic therapeutic agent. The growth ofC. phlei, as measured by the hyphal growth rate and increase in biomass, varies significantly depending on the culture media utilized (V8 juice-based medium proved optimal for both growth rate and biomass increase). How-ever, even on a V8 juice plate, the growth ofC. phlei occurred slowly and in a limited fashion, in that the colony covered only 75% of the agar surface after more than 4 weeks of cultivation at 20°C. Supplementations of glucose, fructose, galactose, and sucrose increased both hyphal expansion and mass production, whereas supplementations of other carbon sources, including glycerol and sorbitol, exerted no detectable effects. The effect of inorganic nitrogen supplementation was negligible, whereas organic nitrogen evidenced significant effects, with enhanced growth with malt extract and growth inhibition with yeast extract and tryptone. Sporulation was enhanced under conditions of continuous light, and a minimum of 10³ spores per mL of liquid media was found to be necessary for the optimal mass increase. A simple extraction procedure was established in order to isolate the deep red pigment which was subsequently identified as phleichrome via NMR analysis. WhenC. phlei was cultured on V8 medium containing 5% glucose and 2% malt extract, the quantity of mycelial mass was estimated as 20.6 g (dry weight) per liter of culture. The expected phleichrome yields from the mycelia and culture filtrates were estimated to be 43 and 2 mg/L, repectively. There was an equal contribution of the reported research by the first two authors.     

Cumene peroxide and Fe2+-ascorbate-induced lipid peroxidation and effect of phosphoglucose isomerase

Malondialdehyde (MDA) is one of cytotoxic aldehydes produced in cells as a result of lipid peroxidation and further MDA metabolism in cytoplasm is not known. In our experiments the liver fraction 10,000 g containing phosphoglucose isomerase and enzymes of the glyoxalase system was used and obtained experimental data shows that in this fraction there is an aggregate of reactions taking place both in membranes (lipid peroxidation) and outside membranes. MDA accumulation is relatively slow because MDA is a substrate of aldehyde isomerase (MDA ↔ methylglyoxal). The well known enzyme phosphoglucose isomerase acts as an aldehyde isomerase (Michaelis constant for this enzyme Km = 133 ± 8 μM). MDA conversion to methylglyoxal and further to neutral product D-lactate (with GSH as a cofactor) occurs in cytoplasm and D-lactate should be regarded as the end product of two different parametabolic reactions: lipid peroxidation or protein glycation.     

Production of polyhydroxyalkanoates by <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> UWD using swine wastewater: Effect of supplementing glucose,yeast extract,and inorganic salts

This study examined the effect of adding glucose, yeast extract, and inorganic salts to swine wastewater (SWW) in a batch culture on the production of a biodegradable plastic, polyhydroxyalkanoate (PHA). A bacterial strain, Azotobacter vinelandii UWD, was used to produce PHA without limiting the non-carbon nutrients. The addition of glucose (30 g/L) to the SWW medium increased the level of cell growth (4.4∼7.0 times) and PHA production (3.8∼8.5 times) depending upon the dilution of SWW. A 50% dilution of SWW was found to be optimal considering the dry cell weight (9.40 g/L), PHA content (58 wt%), and hydroxyvalerate (HV) mol fraction in the PHA (4.3 mol%). A 75% SWW medium was more advantageous for producing PHA with a higher HV fraction (7.1 mol%) at the expense of losing 22% of PHA production. The undiluted SWW medium produced less than one third of the PHA compared with the 50% SWW medium, but the HV fraction was the highest (10.8 mol%). Regarding the effect of the glucose concentration, at 20 g/L glucose, the dry cell weight and level of PHA production increased to 9.34 g/L (0.63 g PHA/g dry cell weight) and 5.90 g/L, respectively. At 50 g/L glucose, there was no significant increase in PHA production. For the glucose-supplemented (30 g/L) 50% SWW medium, the addition of a nitrogen source (1 g/L of yeast extract) did not increase the level of cell growth or PHA production because the C:N ratio (23:1) was already close to the optimal value (22:1). Better aeration increased the productivity of PHA. External nitrogen supplements (1 g/L of yeast extract) and other essential mineral salts was not necessary for bacterial growth because they were contained in the SWW. These results suggest that SWW is an excellent feedstock for producing larger amounts of the value-added material, PHA, if it is combined with carbohydrate-rich organic waste.     

Purification and kinetic behavior of glucose isomerase from Streptomyces lividans RSU26

Glucose isomerase (GI), an enzyme with deserved high potential in the world market. GI plays a major role in high Fructose Corn Syrup Production (HFCS). HFCS is used as a sweetener in food and pharmaceutical industries. Streptomyces are well-known producers of various industrially valuable enzymes, including Glucose isomerase. Currently, recombinant strains have been available for the production of various enzymes, but it has limitation in the large scale production. Therefore, identifying effective streptomyces strains have emerged. The current study, the novel S. lividans RSU26 was isolated from a marine source and optimized its potential to produce glucose isomerase at different physical and chemical conditions. The optimum pH and temperature for GI and biomass production were 7.5 and 35 °C, respectively at 96 h. Characterization study revealed that the approximate molar mass of GI was 43 kDa for monomeric and 170 kDa for tetrameric forms. Kinetic behavior exhibits Km, and Vmax values for the conversion of fructose to glucose conversion were 48.8 mM and 2.54 U mg⁻¹ at 50 °C and glucose to fructose were 29.4 mM and 2.38 U mg⁻¹ at 65 °C protein, respectively. Therefore, the present study suggested that the wild–type S. lividans RSU26 has strong potential to produce glucose isomerase for various industrial applications.     

Development of a selection system for the detection of L-ribose isomerase expressing mutants of <Emphasis Type="Italic">Escherichia coli</Emphasis>

L-Arabinose isomerase (E.C. 5.3.1.14) catalyzes the reversible isomerization between L-arabinose and L-ribulose and is highly selective towards L-arabinose. By using a directed evolution approach, enzyme variants with altered substrate specificity were created and screened in this research. More specifically, the screening was directed towards the identification of isomerase mutants with L-ribose isomerizing activity. Random mutagenesis was performed on the Escherichia coli L-arabinose isomerase gene (araA) by error-prone polymerase chain reaction to construct a mutant library. To enable screening of this library, a selection host was first constructed in which the mutant genes were transformed. In this selection host, the genes encoding for L-ribulokinase and L-ribulose-5-phosphate-4-epimerase were brought to constitutive expression and the gene encoding for the native L-arabinose isomerase was knocked out. L-Ribulokinase and L-ribulose-5-phosphate-4-epimerase are necessary to ensure the channeling of the formed product, L-ribulose, to the pentose phosphate pathway. Hence, the mutant clones could be screened on a minimal medium with L-ribose as the sole carbon source. Through the screening, two first-generation mutants were isolated, which expressed a small amount of L-ribose isomerase activity.     

Glucose-6-phosphate isomerase from the hyperthermophilic archaeon<Emphasis Type="Italic"> Methanococcus jannaschii:</Emphasis> characterization of the first archaeal member of the phosphoglucose isomerase superfamily

ORF MJ1605, previously annotated as pgi and coding for the putative glucose-6-phosphate isomerase (phosphoglucose isomerase, PGI) of the hyperthermophilic archaeon Methanococcus jannaschii, was cloned and functionally expressed in Escherichia coli. The purified 80-kDa protein consisted of a single subunit of 45 kDa, indicating a homodimeric (₂) structure. The K _m values for fructose 6-phosphate and glucose 6-phosphate were 0.04 mM and 1 mM, the corresponding V _max values were 20 U/mg and 9 U/mg, respectively (at 50 °C). The enzyme had a temperature optimum at 89 °C and showed significant thermostability up to 95 °C. The enzyme was inhibited by 6-phosphogluconate and erythrose-4-phosphate. RT-PCR experiments demonstrated in vivo expression of ORF MJ1618 during lithoautotrophic growth of M. jannaschii on H₂/CO₂. Phylogenetic analyses indicated that M. jannaschii PGI was obtained from bacteria, presumably from the hyperthermophile Thermotoga maritima.     

Medium optimization for the production of glucose isomerase from thermophilic Streptomyces thermonitrificans

A thermophilic strain of Streptomyces thermonitrificans produced a high activity of intracellular glucose isomerase (12 U/ml) when grown in a medium containing 1% (w/v) xylose, supplemented with 2% (w/v) sorbitol as the second carbon source, at 50°C for 16 h. Addition of Mg²⁺ enhanced enzyme production but the organism could grow and produce the enzyme in the absence of Co²⁺.The authors are with the Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, IndiaNCL Communication No. 5813     

Activity of NAD-dependent isocitrate dehydrogenase,isocitrate lyase,and malate dehydrogenase in <Emphasis Type="Italic">Mucor circinelloides</Emphasis> var. <Emphasis Type="Italic">lusitanicus</Emphasis> INMI under different modes of nitrogen supply

The growth and morphology as well as lipogenesis and activity of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle were studied in the fungus Mucor circinelloides var. lusitanicus INMI grown at various concentrations of urea (nitrogen source) added to the medium in different modes. It was shown that the maximum lipid content in the biomass was observed at a low (0.5 g/l) concentration of the nitrogen source, whereas the highest content of γ-linolenic acid in the lipids was detected at high (up to 4.0 g/l) concentrations of the nitrogen source. It was found that, when the feed-batch mode of nitrogen supply was used, the amount of γ-linolenic acid in total fatty acids was higher (up to 35%) than in the case of a single administration of the same amount of nitrogen source to the medium. The differences in the fatty acid composition and the unsaturation degree of the lipids from different subcellular fractions were demonstrated. The mycelium from the culture grown after a single administration of the nitrogen source was deformed to a great extent. The activities of the TCA cycle enzymes, NAD-dependent isocitrate dehydrogenase (IDH), and malate dehydrogenase (MDH) were lower than in the case of the feed-batch mode of urea addition, whereas the activity of isocitrate lyase (ICL), the key enzyme of the glyoxylate cycle, was higher. The coupling of the cell metabolism and the lipid composition of fungal cells and the process of cell differentiation in fungi depending on the conditions of nitrogen supply is discussed.     

Optimization of Nutritional Factors for Exopolysaccharide Production by Submerged Cultivation of the Medicinal Mushroom Oudemansiella radicata

Summary Effects of nutritional factors on exopolysaccharide production by submerged cultivation of the medicinal mushroom Oudemansiella radicata were investigated in shake flasks. Sucrose and peptone were optimal carbon and nitrogen sources for cell growth and exopolysaccharide production. The exopolysaccharide production was increased with an increase in initial sucrose concentration within the range of 10–40 g l⁻¹ and initial peptone concentration within the range of 1–3 g l⁻¹. To enhance further exopolysaccharide production, the effect of carbon/nitrogen ratios was studied using central composite design (CCD) and response surface analysis. The maximum exopolysaccharide production of 2.67 ± 0.15 g l⁻¹ was achieved in medium with optimized carbon and nitrogen sources, i.e. 39.3 g sucrose l⁻¹ and 3.16 g peptone l⁻¹ in the same cultivation conditions. The information obtained is helpful for the hyperproduction of exopolysaccharide by submerged cultivation of O. radicata on a large scale.     

Enhanced lovastatin production by solid state fermentation ofMonascus ruber

The purpose of this study was to optimize the solid state cultivation ofMonascus ruber on sterile rice. A single-level-multiple-factor and a single-factor-multiple-level experimental design were employed to determine the optimal medium constituents and to optimize carbon and nitrogen source concentrations for lovastatin production. Simultaneous quantitative analyses of the β-hydroxyacid form and β-hydroxylactone for of lovastatin were performed by the high performance liquid chromatography (HPLC) method with a UV photodiode-array (PDA) detector. The total lovastatin yield (4≈6 mg/g, average of five repeats) was achieved by adding soybean powder, glycerol, sodium nitrate, and acetic acid at optimal composition of the medium increased by almost 2 times the yield observed prior to optimization. The experimental results also indicated that the β-hydroxylactone form of lovastatin (LFL) and the β-hydroxyacid form of lovastatin (AFL) simultaneously existed in solid state cultures ofMonascus ruber, while the latter was the dominant form in the middle-late stage of continued fermentation. These results indicate that optimized culture conditions can be used for industrial production of lovastatin to obtain high yields.     

Study of docosahexaenoic acid production by the heterotrophic microalga <Emphasis Type="Italic">Crypthecodinium cohnii</Emphasis> CCMP 316 using carob pulp as a promising carbon source

In this work, carob pulp syrup was used as carbon source in C. cohnii fermentations for docosahexaenoic acid production. In preliminary experiments different carob pulp dilutions supplemented with sea salt were tested. The highest biomass productivity (4 mg/lh) and specific growth rate (0.04/h) were observed at the highest carob pulp dilution (1:10.5 (v/v), corresponding to 8.8 g/l glucose). Ammonium chloride and yeast extract were tested as nitrogen sources using different carob pulp syrup dilutions, supplemented with sea salt as growth medium. The best results were observed for yeast extract as nitrogen source. A C. cohnii fed-batch fermentation was carried out using diluted carob pulp syrup (1:10.5 v/v) supplemented with yeast extract and sea salt. The biomass productivity was 420 mg/lh, and the specific growth rate 0.05/h. Under these conditions the DHA concentration and DHA production volumetric rate attained 1.9 g/l and 18.5 mg/lh respectively after 100.4 h. The easy, clean and safe handling of carob pulp syrup makes this feedstock a promising carbon source for large-scale DHA production from C. cohnii. In this way, this carob industry by-product could be usefully disposed of through microbial production of a high value fermentation product.     

Plant selection principle based on xylose isomerase

Summary The xylose isomerase genes (xylA) from Thermoanaerobacterium thermosulfurogenes and Streptomyces rubiginosus were introduced and expressed in three plant species (potato, tobacco and tomato) and transgenic plants were selected on xylose-containing medium. The xylose isomerase genes were transferred to explants of the target plant by Agrobacterium-mediated transformation. The xylose isomerase genes were expressed under the control of the enhanced cauliflower mosaic virus 35S promoter and the Ω′ translation enhancer sequence from tobacco mosaic virus. In potato and tomato, xylose isomerase selection was more efficient than the established kanamycin selection. The level of enzyme activity in the regenerated transgenic plants selected on xylose was 5–25-fold higher than the enzyme activity in control plants selected on kanamycin. The xylose isomerase system enables transgenic cells to utilize xylose as a carbohydrate source. In contrast to antibiotic or herbicide resistance-based system where transgenic cells survive on a selective medium but nontransgenic cells are killed, the xylose system is an example of a positive selection system where transgenic cells proliferate while non-transgenic cells are starved but still survive. The results show that a new selection method, is established. The xylose system is devoid of the disadvantages of antibiotic or herbicide selection, and depends on an enzyme which is already being widely utilized in specific food processes and that is generally recognized as safe for use in the starch industry.     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

Conditions of the biosynthesis of an extracellular subtilisin-like proteinase by <Emphasis Type="Italic">Bacillus pumilus</Emphasis> KMM 62

The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46–48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46–48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The involvement of different regulatory mechanisms of the synthesis of this proteinase is assumed in the early and late stationary phases of growth.     

The use of the phosphomannose isomerase gene as alternative selectable marker for Agrobacterium-mediated transformation of flax (Linum usitatissimum)

In order to meet the future requirement of using non-antibiotic resistance genes for the production of transgenic plants, we have adapted the selectable marker system PMI/mannose to be used in Agrobacterium-mediated transformation of flax (Linum usitatissimum L.) cv. Barbara. The Escherichia coli pmi gene encodes a phosphomannose isomerase (E.C. 5.1.3.8) that converts mannose-6-phosphate, an inhibitor of glycolysis, into fructose-6-phosphate (glycolysis intermediate). Its expression in transformed cells allows them to grow on mannose-selective medium. The Agrobacterium tumefaciens strain GV3101 (pGV2260) harbouring the binary vector pNOV2819 that carries the pmi gene under the control of the Cestrum yellow leaf curling virus constitutive promoter was used for transformation experiments. Transgenic flax plants able to root on mannose-containing medium were obtained from hypocotyl-derived calli that had been selected on a combination of 20 g L⁻¹ sucrose and 10 g L⁻¹ mannose. Their transgenic state was confirmed by PCR and Southern blotting. Transgene expression was detected by RT-PCR in leaves, stems and roots of in vitro grown primary transformants. The mean transformation efficiency of 3.6%, that reached 6.4% in one experiment was comparable to that obtained when using the nptII selectable marker on the same cultivar. The ability of T1 seeds to germinate on mannose-containing medium confirmed the Mendelian inheritance of the pmi gene in the progeny of primary transformants. These results indicate that the PMI/mannose selection system can be successfully used for the recovery of flax transgenic plants under safe conditions for human health and the environment.     

Activities of the enzymes of formaldehyde catabolism in recombinant strains of <Emphasis Type="Italic">Hansenula polymorpha</Emphasis>

Activities of the enzymes of formaldehyde (FA) catabolism in recombinant strains of the methylotrophic yeast Hansenula polymorpha overproducing NAD⁺- and glutathione-dependent formaldehyde dehydrogenase (FADH) were studied under different cultivation conditions and at elevated FA content. Southern dot-blot analysis confirmed the presence of six to eight copies of the target FLD1 gene in stable recombinant clones of H. polymorpha. Under certain cultivation conditions, the transformants resistant to elevated FA concentrations were shown to produce FADH and other bioanalytically important enzymes: formate dehydrogenase, alcohol dehydrogenase, alcohol oxidase, and formaldehyde reductase. The optimal cultivation conditions for recombinants were determined, resulting in maximum synthesis of FADH: methanol as a carbon source, methylamine as a nitrogen source, FA as an inducer, temperature of 37°C, and cells in the early exponential phase of growth.