Metabolic engineering of Lactobacillus plantarum for production of L-ribulose

L-Ribulose is a rare and expensive sugar that can be used as a precursor for the production of other rare sugars of high market value such as L-ribose. In this work we describe a production process for L-ribulose using L-arabinose, a common component of polymers of lignocellulosic materials, as the starting material. A ribulokinase-deficient mutant of the heterofermentative lactic acid bacterium Lactobacillus plantarum NCIMB8826 was constructed. Expression of araA, which encodes the critical enzyme L-arabinose isomerase, was repressed by high glucose concentrations in batch cultivations. A fed-batch cultivation strategy was therefore used to maximize L-arabinose isomerase production during growth. Resting cells of the ribulokinase-deficient mutant were used for the production of L-ribulose. The isomerization of L-arabinose to L-ribulose was very unfavorable for L-ribulose formation. However, high L-ribulose yields were obtained by complexing the produced L-ribulose with borate. The process for L-ribulose production in borate buffer by resting cells was optimized using central composite designs. The experiment design suggested that the process has an optimal operation point around an L-arabinose concentration of 100 g liter(-1), a borate concentration of 500 mM, and a temperature of 48 degrees C, where the statistical software predicted an initial L-ribulose production rate of 29.1 g liter(-1) h(-1), a best-achievable process productivity of 14.8 g liter(-1) h(-1), and a conversion of L-arabinose to L-ribulose of 0.70 mol mol(-1).     

Melanoma cell lines are susceptible to histone deacetylase inhibitor TSA provoked cell cycle arrest and apoptosis

Melanoma is the most aggressive of skin cancers because of its high resistance to currently available therapy. Although melanoma cells often retain wild-type p53 tumour suppressor protein and express it at high levels, the p53 mediated apoptosis pathway is suppressed. Histone deacetylase (HDAC) inhibitors are a promising group of compounds inducing differentiation, growth arrest and apoptosis in tumour cells in preclinical studies. We have studied the cellular effects of trichostatin A (TSA), a HDAC inhibitor, in a panel of melanoma cell lines and its mechanism of action in relation to p53. TSA stabilized wild-type p53, but p53 protein accumulation was overridden by simultaneous downregulation of p53 mRNA leading to a decrease in p53 protein. While growth arrest was induced in all cell lines studied and apoptosis in most (6/7), these cellular effects were independent of the p53 status of the cells. Inhibiting p53 function by a dominant negative p53 (p53(175His)) confirmed that the HDAC inhibitor induced apoptosis was independent of wild-type p53, even though TSA slightly activated p53 in a reporter assay. The results indicate that while the action of TSA is independent of p53, the activation of the apoptosis pathway by the HDAC inhibitors may provide therapeutic approaches for melanoma treatment.     

2,4-Dichlorophenoxyacetic acid and kinetin in anther culture of cultivated and wild oats and their interspecific crosses: plant regeneration from A. sativa L.

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin was studied in anther culture of oat Avena sativa L., wild oat A. sterilis L. and progeny of crosses between them. A high 2,4-D concentration (5–6 mg l^–1) increased embryo production in genotypes of both species and promoted plant regeneration in anther cultures of A. sterilis and A. sativa×A. sterilis progeny, while kinetin caused severe browning. However, a low concentration of kinetin was essential for initiation of regenerable embryos from anther culture of A. sativa cv. Kolbu: one green and one albino plant were produced. In addition, medium containing W₁₄ salts gave higher regenerant recovery compared with medium containing Murashge and Skoog salts, when cross progeny were tested. Received: 6 March 1998 / Revised: 30 April 1998 / Accepted: 16 November 1998     

The effect of explant material on somatic embryogenesis of Cyclamen persicum Mill

Somatic embryos of Cyclamen persicum Mill. could be produced through a callus phase from juvenile explant material including anthers, ovaries and zygotic embryos. The auxin 2,4-D (1.0–1.5 mg l^-1) and coconut milk (10% v/v) in MS medium were important factors for the induction of somatic embryogenesis. Somatic embryos germinated into plantlets in MS medium without growth regulators. The plants grew well in the greenhouse and flowered normally. The plants were phenotypically identical to the mother plants with a few exceptions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthylacetic acid - IAA 3-indoleacetic acid - BA 6-benzyladenine - ABA abscisic acid - CM coconut milk     

Optimization of a Bifidobacterium longum production process

Bifidobacteria are used as probiotics mainly in the dairy industry as cell suspensions or as freeze-dried additives. So far there have been no reports on a thorough investigation on factors influencing the production process or a statistical approach to the optimization thereof. A 2(8-4) fractional factorial design was used in determining the critical parameters influencing bioreactor cultivations of Bifidobacterium longum ATCC 15707. Glucose, yeast extract and l-cysteine concentrations were found critical for the cultivation of this strain. Glucose and yeast extract concentrations were further studied together with temperature in a three factor central composite design. The optimized cultivation conditions were temperature 40 degrees C, yeast extract concentration 35 gl(-1) and glucose concentration 20 gl(-1). Freeze-drying of frozen cell suspensions of B. longum was studied first in controlled temperatures and thereafter with temperature programming experiments. The results were statistically evaluated. A temperature program with a 2 h temperature gradient from -10 to 0 degrees C, a 10 h temperature gradient from 0 to +10 degrees C and a 12 h temperature hold at +10 degrees C was found best for the freeze-drying process. Temperature programming reduced drying times by over 50% and improved the product activity by over 160%.     

Control of ��-amylase production by Bacillus subtilis

This study proposes two adaptive control algorithms for the fed-batch production of α-amylase. The first one uses online information from hardware measuring glucose. Online information of both biomass and glucose concentrations measured with different frequency is used in the second algorithm. Hardware measuring variables are inputs for software sensors of glucose concentration and (specific) glucose consumption rate. Either of the algorithms do not require any kinetic coefficients. This is a benefit, because the kinetic coefficients can vary during cultivation and between cultivations, leading to low process reproducibility and the non-stationary state of the bioprocess. The results of simulation investigations show good performance of the proposed control schemes.     

Modeling and simulation of Streptomyces peucetius var. caesius N47 cultivation and epsilon-rhodomycinone production with kinetic equations and neural networks

This study focuses on comparing different kinetic growth models and the use of neural networks in the batch cultivation of Streptomyces peucetius var. caesius producing epsilon-rhodomycinone. Contois, Monod and Teissier microbial growth models were used as well as the logistic growth modeling approach, which was found best in the simulations of growth and glucose consumption in the batch growth phase. The lag phase was included in the kinetic model with a CO2 trigger and a delay factor. Substrate consumption and product formation were included as Luedeking-Piret and logistic type equations, respectively. Biomass formation was modeled successfully with a 6-8-2 network, and the network was capable of biomass prediction with an R2-value of 0.983. Epsilon-rhodomycinone production was successfully modeled with a recursive 8-3-1 network capable of epsilon-rhodomycinone prediction with an R2-value of 0.903. The predictive power of the neural networks was superior to the kinetic models, which could not be used in predictive modeling of arbitrary batch cultivations.     

Biotechnological production of <Emphasis Type="SmallCaps">l</Emphasis>-ribose from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

l-Ribose is a rare and expensive sugar that can be used as a precursor for the production of l-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing l-ribose from the readily available raw material l-arabinose. This was achieved by introducing l-ribose isomerase activity into l-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for l-ribose production by resting cells was investigated. The initial l-ribose production rates at 39°C and pH 8 were 0.46 ± 0.01 g g⁻¹ h⁻¹ (1.84 ± 0.03 g l⁻¹ h⁻¹) and 0.27 ± 0.01 g g⁻¹ h⁻¹ (1.91 ± 0.1 g l⁻¹ h⁻¹) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates having both l-arabinose isomerase and l-ribose isomerase activity were successfully used for converting l-arabinose to l-ribose.     

Regeneration of anther-derived plants ofAvena sterilis

Anther culture response of accessions of several hexaploid Avena species was studied with respect to requirement for auxin. The highest callus induction frequency was achieved withA. sativa L. cv. Stout (17.3%). The three most responsive genotypes, two of A. sativa and one of A. nuda L., had two-fold higher anther culture responses on growth regulator -free medium than on a medium containing 2,4-d. The A. sterilis L. accession CAV 2648 was the only genotype which consistently produced white, embryogenic structures (on solid MS+ 10% sucrose) and did so irrespective of the presence of 2,4-d. When transferred onto medium with lower sucrose concentration and an auxin transport inhibitor (TIBA), three green (two diploid [2t n=6t x=42] and one haploid [1t n=3t x=21]) and two albino plantlets were regenerated. The diploid regenerants set seed in the greenhouse. This first report of plants recovered from anther culture in the wild oat A. sterilis may provide an avenue to understand better and possibly overcome problems associated with androgenesis in cultivated oat. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of cold and heat pretreatments on anther culture response of Avena sativa and A. sterilis

The effect of stress pretreatments on embryo induction in anther cultures of selected genotypes of Avena sativa and A. sterilis was tested. A heat pretreatment of isolated anthers at +32°C for 5 days was best for the A. sativa line WW 18019 and for A. sterilis line CAV 2648. Genotype dependency may exist since in ‘Stout’ heat pretreatment did not increase embryo production. For A. sterilis 13 green and three albino regenerants were produced, of which five plants (haploids) survived transfer to the greenhouse. For A. sativa, 30 various differentiation media/treatment combinations were used in an attempt to regenerate plants from embryos, with no success. Seven day cold treatment of cut tillers increased slightly the response level in ‘Stout’ and was routinely used in subsequent experiments. Maltose proved to be better then sucrose as a carbon source for the genotypes tested. Fourteen percent maltose promoted the highest induction in A. sterilis, but the quality of embryos was improved in the presence of 10% maltose for both species. Sub-optimal carbohydrate levels did not enhance embryo induction in oats. This revised version was published online in June 2006 with corrections to the Cover Date.