Conversion of glycerol to 1,3-dihydroxyacetone by glycerol dehydrogenase co-expressed with an NADH oxidase for cofactor regeneration

Objectives

To investigate the efficiency of a cofactor regeneration enzyme co-expressed with a glycerol dehydrogenase for the production of 1,3-dihydroxyacetone (DHA).

Results

In vitro biotransformation of glycerol was achieved with the cell-free extracts containing recombinant GlyDH (glycerol dehydrogenase from Escherichia coli), LDH (lactate dehydrogenase form Bacillus subtilis) or LpNox1 (NADH oxidase from Lactobacillus pentosus), giving DHA at 1.3 g l^?1 (GlyDH/LDH) and 2.2 g l^?1 (GlyDH/LpNox1) with total turnover number (TTN) of NAD⁺ recycling of 6039 and 11100, respectively. Whole cells of E. coli (GlyDH–LpNox1) co-expressing both GlyDH and LpNox1 were constructed and converted 10 g glycerol l^?1 to DHA at 0.2–0.5 g l^?1 in the presence of zero to 2 mM exogenous NAD⁺. The cell free extract of E. coli (GlyDH–LpNox) converted glycerol (2–50 g l^?1) to DHA from 0.5 to 4.0 g l^?1 (8–25 % conversion) without exogenous NAD⁺.

Conclusions

The disadvantage of the expensive consumption of NAD⁺ for the production of DHA has been overcome.

     

A relative quantitative positive/negative ion switching method for untargeted lipidomics via high resolution LC-MS/MS from any biological source

Introduction

Advances in high-resolution mass spectrometry have created renewed interest for studying global lipid biochemistry in disease and biological systems.

Objectives

Here, we present an untargeted 30 min. LC-MS/MS platform that utilizes positive/negative polarity switching to perform unbiased data dependent acquisitions (DDA) via higher energy collisional dissociation (HCD) fragmentation to profile more than 1000–1500 lipid ions mainly from methyl-tert-butyl ether (MTBE) or chloroform:methanol extractions.

Methods

The platform uses C₁₈ reversed-phase chromatography coupled to a hybrid QExactive Plus/HF Orbitrap mass spectrometer and the entire procedure takes?~10 h from lipid extraction to identification/quantification for a data set containing 12 samples (~4 h for a single sample). Lipids are identified by both accurate precursor ion mass and fragmentation features and quantified using LipidSearch and Elements software.

Results

Using this approach, we are able to profile intact lipid ions from up to 18 different main lipid classes and 66 subclasses. We show several studies from different biological sources, including cultured cancer cells, resected tissues from mice such as lung and breast tumors and biological fluids such as plasma and urine.

Conclusions

Using mouse embryonic fibroblasts, we showed that TSC2^?/? KD significantly abrogates lipid biosynthesis and that rapamycin can rescue triglyceride (TG) lipids and we show that SREBP^?/? shuts down lipid biosynthesis significantly via mTORC1 signaling pathways. We show that in mouse EGFR driven lung tumors, a large number of TGs and phosphatidylmethanol (PMe) lipids are elevated while some phospholipids (PLs) show some of the largest decrease in lipid levels from ~?2000 identified lipid ions. In addition, we identified more than 1500 unique lipid species from human blood plasma.

     

Asymmetric effects of litter removal and litter addition on the structure and function of soil microbial communities in a managed pine forest

Aims

The effect of different MeJA doses applied prior to or simultaneously with toxic Al on biochemical and physiological properties of Vaccinium corymbosum cultivars with contrasting Al resistance was studied.

Methods

Legacy (Al-resistant) and Bluegold (Al-sensitive) plants were treated with and without toxic Al under controlled conditions: a) without Al and MeJA, b) 100 μM Al, c) 100 μM Al + 5 μM MeJA, d) 100 μM Al + 10 μM MeJA and e) 100 μM Al + 50 μM MeJA. MeJA was applied to leaves 24 h prior to or simultaneously with Al in nutrient solution. After 48 h, Al-concentration, lipid peroxidation (LP), H₂O₂, antioxidant activity, total phenols, total flavonoids, phenolic compounds and superoxide dismutase activity (SOD) of plant organs were analyzed.

Results

Al-concentrations increased with Al-treatment in both cultivars, being Al, LP and H₂O₂ concentrations reduced with low simultaneous MeJA application. Higher MeJA doses induced more oxidative damage than the lowest. Legacy increased mainly non-enzymatic compounds, whereas Bluegold increased SOD activity to counteract Al³⁺.

Conclusions

Low MeJA doses applied simultaneously with Al³⁺ increased Al-resistance in Legacy by increasing phenolic compounds, while Bluegold reduced oxidative damage through increment of SOD activity, suggesting a diminution of its Al-sensitivity. Higher MeJA doses could be potentially toxic. Studies are needed to determine the molecular mechanisms involved in the protective MeJA effect against Al-toxicity.

     

A novel malic enzyme gene, <Emphasis Type="Italic">Mime2</Emphasis>, from <Emphasis Type="Italic">Mortierella isabellina</Emphasis> M6-22 contributes to lipid accumulation

Objective

This study was aimed at cloning and characterizing a novel malic enzyme (ME) gene of Mortierella isabellina M6-22 and identifying its relation with lipid accumulation.

Methods

Mime2 was cloned from strain M6-22. Plasmid pET32aMIME2 was constructed to express ME of MIME2 in Escherichia coli BL21. After purification, the optimal pH and temperature of MIME2, as well as K_m and V_max for NADP⁺ were determined. The effects of EDTA or metal ions (Mn²⁺, Mg²⁺, Co²⁺, Cu²⁺, Ca²⁺, or Zn²⁺) on the enzymatic activity of MIME2 were evaluated. Besides, plasmid pRHMIME2 was created to express MIME2 in Rhodosporidium kratochvilovae YM25235, and its cell lipid content was measured by the acid-heating method. The optimal pH and temperature of MIME2 are 5.8 and 30 °C, respectively.

Results

The act ivity of MIME2 was significantly increased by Mg²⁺, Ca²⁺, or Mn²⁺ at 0.5 mM but inhibited by Cu²⁺ or Zn²⁺ (p?<?0.05). The optimal enzymatic activity of MIME2 is 177.46 U/mg, and the K_m and V_max for NADP⁺ are 0.703 mM and 156.25 μg/min, respectively. Besides, Mime2 transformation significantly increased the cell lipid content in strain YM25235 (3.15?±?0.24 vs. 2.17?±?0.31 g/L, p?<?0.01).

Conclusions

The novel ME gene Mime2 isolated from strain M6-22 contributes to lipid accumulation in strain YM25235.

     

Discovery of an acidic,thermostable and highly NADP<Superscript>+</Superscript> dependent formate dehydrogenase from <Emphasis Type="Italic">Lactobacillus buchneri</Emphasis> NRRL B-30929

Objectives

To identify a robust NADP⁺ dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929 (LbFDH) with unique biochemical properties.

Results

A new NADP⁺ dependent formate dehydrogenase gene (fdh) was cloned from genomic DNA of L. buchneri NRRL B-30929. The recombinant construct was expressed in Escherichia coli BL21(DE3) with 6?×?histidine at the C-terminus and the purified protein obtained as a single band of approx. 44 kDa on SDS-PAGE and 90 kDa on native-PAGE. The LbFDH was highly active at acidic conditions (pH 4.8–6.2). Its optimum temperature was 60 °C and 50 °C with NADP⁺ and NAD⁺, respectively and its T_m value was 78 °C. Its activity did not decrease after incubation in a solution containing 20% of DMSO and acetonitrile for 6 h. The K_M constants were 49.8, 0.12 and 1.68 mM for formate (with NADP⁺), NADP⁺ and NAD⁺, respectively.

Conclusions

An NADP⁺ dependent FDH from L. buchneri NRRL B-30929 was cloned, expressed and identified with its unusual characteristics. The LbFDH can be a promising candidate for NADPH regeneration through biocatalysis requiring acidic conditions and high temperatures.

     

Effect of candesartan monotherapy on lipid metabolism in patients with hypertension: a retrospective longitudinal survey using data from electronic medical records

Background

Studies focusing on the add-on effects of angiotensin II type 1 receptor blockers (ARBs) other than their antihypertensive effect are receiving attention. However, the effects of prolonged administration of ARBs on lipid metabolism in clinical cases are unclear. Our aims were to survey the changes in plasma lipid profile in patients with hypertension over a one-year period, and to examine the correlations between these values and the time after the start of ARB monotherapy with candesartan.

Methods

We carried out candesartan monotherapy in patients with mild to moderate hypertension and examined the longitudinal changes in plasma lipid profile. Data from 405 patients for triglyceride (TG), 440 for total cholesterol (TC), 313 for high density lipoprotein cholesterol (HDL-C) and 304 for low density lipoprotein cholesterol (LDL-C) were obtained from the electronic medical records (EMRs) in the Clinical Data Warehouse (CDW) of Nihon University School of Medicine (NUSM). The inverse probability of treatment weighting (IPTW) method (calculated from the inverse of the propensity score) was used to balance the covariates and reduce bias in each treatment duration. Linear mixed effects models were used to analyse the relationship between these longitudinal data of blood examinations and covariates of patient sex, age, diagnosis of diabetes mellitus (DM) and duration of candesartan monotherapy.

Results

Plasma HDL-C level was associated with sex, duration of treatment, and interaction of sex and treatment duration, but not with age or diagnosis of DM. HDL-C level was significantly decreased during the 6~9 months period (p = 0.0218) compared with baseline. TG and TC levels were associated with sex, but not with age, diagnosis of DM or treatment duration. LDL-C level was not associated with any covariate. Analysis of the subjects divided by sex revealed a decrease in HDL-C in female subjects (during the 6~9 months period: p = 0.0054), but not in male subjects.

Conclusions

Our study revealed that administration of candesartan slightly decreased HDL-C in female subjects. However, TG, TC and LDL-C levels were not influenced by candesartan monotherapy. Candesartan may be safely used for patients with hypertension with respect to lipid metabolism, because the effect of candesartan on lipids may be small.

     

A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD<Superscript>+</Superscript> regeneration

Objectives

To find an efficient and cheap system for NAD⁺ regeneration

Results

A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K _m value for NADH of 10 μM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD⁺ regeneration was established. The system is attractive in that the O₂ consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD⁺ was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C

Conclusion

The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD⁺ regeneration.

     

Response of Degarelix treatment in human prostate cancer monitored by HR-MAS <Superscript>1</Superscript>H NMR spectroscopy

Introduction

The androgen receptor (AR) is the master regulator of prostate cancer cell metabolism. Degarelix is a novel gonadotrophin-releasing hormone blocker, used to decrease serum androgen levels in order to treat advanced human prostate cancer. Little is known of the rapid metabolic response of the human prostate cancer tissue samples to the decreased androgen levels.

Objectives

To investigate the metabolic responses in benign and cancerous tissue samples from patients after treatment with Degarelix by using HRMAS ¹H NMR spectroscopy.

Methods

Using non-destructive HR-MAS ¹H NMR spectroscopy we analysed the metabolic changes induced by decreased AR signalling in human prostate cancer tissue samples. Absolute concentrations of the metabolites alanine, lactate, glutamine, glutamate, citrate, choline compounds [t-choline = choline + phosphocholine (PC) + glycerophosphocholine (GPC)], creatine compounds [t-creatine = creatine (Cr) + phosphocreatine (PCr)], taurine, myo-inositol and polyamines were measured in benign prostate tissue samples (n = 10), in prostate cancer specimens from untreated patients (n = 7) and prostate cancer specimens from patients treated with Degarelix (n = 6).

Results

Lactate, alanine and t-choline concentrations were significantly elevated in high-grade prostate cancer samples when compared to benign samples in untreated patients. Decreased androgen levels resulted in significant decreases of lactate and t-choline concentrations in human prostate cancer biopsies.

Conclusions

The reduced concentrations of lactate and t-choline metabolites due to Degarelix could in principle be monitored by in vivo ¹H MRS, which suggests that it would be possible to monitor the effects of physical or chemical castration in patients by that non-invasive method.

     

Genetic susceptibility to dyslipidemia and incidence of cardiovascular disease depending on a diet quality index in the Malmö Diet and Cancer cohort

Background

By taking diet quality into account, we may clarify the relationship between genetically elevated triglycerides (TG) and low-density lipoprotein-cholesterol (LDL-C), and better understand the inconsistent results regarding genetically elevated high-density lipoprotein-cholesterol (HDL-C), and cardiovascular disease (CVD) risk.

Methods

We included 24,799 participants (62 % women, age 44–74 years) from the Malmö Diet and Cancer cohort. During a mean follow-up time of 15 years, 3068 incident CVD cases (1814 coronary and 1254 ischemic stroke) were identified. Genetic risk scores (GRSs) were constructed by combining 80 validated genetic variants associated with higher TG and LDL-C or lower HDL-C. The participants’ dietary intake, assessed by a modified diet history method, was ranked according to a diet quality index that included six dietary components: saturated fat, polyunsaturated fat, fish, fiber, fruit and vegetables, and sucrose.

Results

The GRS_LDL-C (P?=?5?×?10^?6) and GRS_HDL-C (P?=?0.02) but not GRS_TG (P?=?0.08) were significantly associated with CVD risk. No significant interaction between the GRSs and diet quality was observed on CVD risk (P?>?0.39). A high compared to a low diet quality attenuated the association between GRS_LDL-C and the risk of incident ischemic stroke (P interaction?=?0.01).

Conclusion

We found some evidence of an interaction between diet quality and GRS_LDL-C on ischemic stroke.

     

Enhanced succinic acid production in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> with increasing the available NADH supply and glucose consumption rate by decreasing H<Superscript>+</Superscript>-ATPase activity

Objectives

To enhance succinic acid production in Corynebacterium glutamicum by increasing the supply of NADH and the rate of glucose consumption by decreasing H⁺-ATPase activity.

Results

A mutant of C. glutamicum NC-3-1 with decreased H⁺-ATPase activity was constructed. This increased the rate of glycolysis and the supply of NADH. Fermentation of C. glutamicum NC-3-1 gave 39 % higher succinic acid production (113 and 81 g/l), a 29 % higher succinic acid yield (0.94 and 0.73 g succinic acid/g glucose) and decreased by-products formation compared to that of C. glutamicum NC-3 in 5 l bioreactor.

Conclusion

The point mutation in C. glutamicum NC-3-1 increased the rate of glycolysis and resulted in higher succinic acid production, higher succinic acid yield and significantly decreased formation of by-products.

     

Plasma metabolomics in calves with acute bronchopneumonia

Background

Bovine respiratory disease is one of the main health issues in dairy calves. Inflammatory lung diseases are highly complex with respect to pathogenesis and relationships between inflammation, clinical disease and response to treatment. Metabolomics may offer the potential to identify biomarkers that define calf bronchopneumonia in terms of combined clinical, physiological and patho-biological abnormalities. While metabolomic studies are often encountered in childhood pneumonia, there is no knowledge related to the same approach to calf pneumonia.

Objective

The aim of this first study was to reveal the new potential biomarkers for acute calf bronchopneumonia by single proton (¹H) Nuclear magnetic resonance (NMR) based quantitative metabolomics.

Methods

Fifty dairy calves with acute bronchopneumonia presented for treatment to the teaching hospital, and ten healthy dairy calves belonging the teaching farm were used. Laboratory (hematological: complete blood count and blood gas analysis, and biochemical analysis related to health profile) were performed. NMR spectra of the all samples (50 diseased + 10 healthy water soluble extracts, 50 diseased + 10 healthy lipid extracts) were acquired using a standard Nuclear Overhauser Effect Spectroscopy pulse sequence.

Results

NMR based metabolomics analysis showed that calves suffering from bronchopneumonia and healthy calves have two different and distinguishable metabolic fingerprints using both water soluble and lipid extracts. Alterations in metabolites, increases in 2-methyl glutarate, phenylalanine, phosphatidylcholine, and decreases in ethanol, dimethylsulfone, propionate, acetate, allantoin, free cholesterol, cholesterol (–C18), were meaningful for pathogenic mechanisms of calf bronchopneumonia.

Conclusion

The NMR based metabolomics may contribute to better understanding bronchopneumonia in calves.

     

Selective oxidation of UDP-glucose to UDP-glucuronic acid using permeabilized <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> expressing human UDP-glucose 6-dehydrogenase

Objectives

To use permeabilized cells of the fission yeast, Schizosaccharomyces pombe, that expresses human UDP-glucose 6-dehydrogenase (UGDH, EC 1.1.1.22), for the production of UDP-glucuronic acid from UDP-glucose.

Results

In cell extracts no activity was detected. Therefore, cells were permeabilized with 0.3 % (v/v) Triton X-100. After washing away all low molecular weight metabolites, the permeabilized cells were directly used as whole cell biocatalyst. Substrates were 5 mM UDP-glucose and 10 mM NAD⁺. Divalent cations were not added to the reaction medium as they promoted UDP-glucose hydrolysis. With this reaction system 5 mM UDP-glucose were converted into 5 mM UDP-glucuronic acid within 3 h.

Conclusions

Recombinant permeabilized cells of S. pombe can be used to synthesize UDP-glucuronic acid with 100 % yield and selectivity.

     

Investigation on left ventricular multi-directional deformation in patients of hypertension with different LVEF

Background

This study is aimed at investigating myocardial multi-directional systolic deformation in hypertensive with different left ventricular ejection fraction (LVEF), and exploring its contribution to LVEF.

Methods

One hundred and twenty-three patients with primary hypertension (HT) were divided into group A (LVEF ≥ 55%), group B (45% ≤ LVEF < 50%, or 50% ≤ LVEF < 55% + LVEDVI ≥ 97 ml/m²), and group C (LVEF < 45%). Two-dimensional strain echocardiography (2DSE) including LV longitudinal strain (SL), radial strain (SR) and circumferential strain (SC) were measured.

Results

SL decreased gradually from group A, B to C (all p < 0.05) while SR and SC were reduced only in group B and C (all p < 0.05). All strain measurements correlated to LVEF, with the strongest correlation in SC (r = ?0.82, p < 0.01) and the second in SL (r = ?0.76). The diastolic E/e increased from group A, B to C.

Conclusions

Left ventricular multi-directional deformation correlated well to LVEF in hypertension and particularly SC, indicating that it was SC, not SL or SR, that makes the prominent contribution to left ventricular pump function.

     

A simple method to determine evaporation and compensate for liquid losses in small-scale cell culture systems

Objectives

Establish a method to indirectly measure evaporation in microwell-based cell culture systems and show that the proposed method allows compensating for liquid losses in fed-batch processes.

Results

A correlation between evaporation and the concentration of Na⁺ was found (R²?=?0.95) when using the 24-well-based miniature bioreactor system (micro-Matrix) for a batch culture with GS-CHO. Based on these results, a method was developed to counteract evaporation with periodic water additions based on measurements of the Na⁺ concentration. Implementation of this method resulted in a reduction of the relative liquid loss after 15 days of a fed-batch cultivation from 36.7?±?6.7% without volume corrections to 6.9?±?6.5% with volume corrections.

Conclusion

A procedure was established to indirectly measure evaporation through a correlation with the level of Na⁺ ions in solution and deriving a simple formula to account for liquid losses.

     

Embedded rock fragments affect alpine steppe plant growth,soil carbon and nitrogen in the northern Tibetan Plateau

Background and Aims

Rock fragments within topsoil have important effects on soil properties and plant growth. This study mainly aimed to investigate the relationships between rock fragments, soil carbon (C) and nitrogen (N) densities and vegetation biomass in an alpine steppe.

Methods

Rock fragments, plant and soil samples were collected from four topographic positions (top, upper, lower, and bottom) on a hillslope.

Results

Volumetric rock fragment content within the 0–30 cm soil profile varied from 17.8 to 30.5%, the upper position value was significantly greater (P < 0.05) than those at other positions. The highest aboveground biomass was observed at the lower position (921 kg ha^?1), while the highest belowground biomass within the 0–30 cm profile was found at the upper position (4460 kg ha^?1). More fine earth and plant litter input accompanied by lower C and N losses induced by rainfall erosion resulted in higher soil organic C and total N densities (28.6 Mg C ha^?1 and 2.87 Mg N ha^?1) at the lower position.

Conclusions

Rock fragments may promote root growth but limit aboveground biomass production, and can therefore change the biomass distribution pattern. Our findings provide more evidence for scientifically assessing alpine steppe productivity.

     

In-vivo quantification of the revascularization of a human acellular dermis seeded with EPCs and MSCs in co-culture with fibroblasts and pericytes in the dorsal chamber model in pre-irradiated tissue

Introduction

Transplantation of a cell-seeded graft may improve wound healing after radiotherapy. However, the survival of the seeded cells depends on a rapid vascularization of the graft. Co-culturing of adult stem cells may be a promising strategy to accelerate the vessel formation inside the graft. Thus, we compared the in vivo angiogenic potency of mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC) using dorsal skinfold chambers and intravital microscopy.

Materials and methods

Cells were isolated from rat bone marrow and adipose tissue and characterized by immunostaining and flow cytometry. Forty-eight rats received a dorsal skinfold chamber and were divided into 2 main groups, irradiated and non-irradiated. Each of these 2 groups were further subdivided into 4 groups: unseeded matrices, matrices + fibroblasts + pericytes, matrices + fibroblasts + pericytes + MSCs and matrices + fibroblasts + pericytes + EPCs. Vessel densities were quantified semi-automatically using FIJI.

Results

Fibroblasts + pericytes ? seeded matrices showed a significantly higher vascular density in all groups with an exception of non-irradiated rats at day 12 compared to unseeded matrices. Co-seeding of MSCs increased vessel densities in both, irradiated and non-irradiated groups. Co-seeding with EPCs did not result in an increase of vascularization in none of the groups.

Discussion

We demonstrated that the pre-radiation treatment led to a significant decreased vascularization of the implanted grafts. The augmentation of the matrices with fibroblasts and pericytes in co-culture increased the vascularization compared to the non-seeded matrices. A further significant enhancement of vessel ingrowth into the matrices could be achieved by the co-seeding with MSCs in both, irradiated and non-irradiated groups.

     

Characterization of a new multifunctional beta-glucosidase from <Emphasis Type="Italic">Musca domestica</Emphasis>

Objective

To engineer Pichia pastoris for heterologous production of cellulase from Musca domestica and explore its potential for industrial applications.

Results

A new beta-glucosidase gene (bg), encoding 562 amino acids, was cloned from M. domestica by using rapid amplification of cDNA ends. The gene bg was linked to pPICZαA and expressed in P. pastoris with a yield of 500 mg l^?1. The enzyme has the maximum activity with 27.6 U mg^?1 towards cellulose. The beta-glucosidase has stable activity from 20 to 70 °C and can tolerate one-mole glucose. It has the maximum activities for salicin (25.9 ± 1.8 U mg^?1), cellobiose (40.1 ± 2.3 U mg^?1) and cellulose (27.6 ± 3.5 U mg^?1). The wide-range substrate activities of the beta-glucosidase were further verified by matrix-assisted laser desorption/ionization mass spectra. Structural analysis shows that the beta-glucosidase belongs to glycoside hydrolase family Ι and possesses O-glycosylation sites.

Conclusions

Thus, a multifunctional beta-glucosidase was expressed from M. domestica and provides a potential tool for industrial application of cellulose.

     

Intensive ground vegetation growth mitigates the carbon loss after forest disturbance

Aims

Slow or failed tree regeneration after forest disturbance is increasingly observed in the central European Alps, potentially amplifying the carbon (C) loss from disturbance. We aimed at quantifying C dynamics of a poorly regenerating disturbance site with a special focus on the role of non-woody ground vegetation.

Methods

Soil CO₂ efflux, fine root biomass, ground vegetation biomass, tree increment and litter input were assessed in (i) an undisturbed section of a ~ 110 years old Norway spruce stand, (ii) in a disturbed section which was clear-cut six years ago (no tree regeneration), and (iii) in a disturbed section which was clear-cut three years ago (no tree regeneration).

Results

Total soil CO₂ efflux was similar across all stand sections (8.5 ± 0.2 to 8.9 ± 0.3 t C ha^?1 yr.^?1). The undisturbed forest served as atmospheric C sink (2.1 t C ha^?1 yr.^?1), whereas both clearings were C sources to the atmosphere. The source strength three years after disturbance (?5.5 t C ha^?1 yr.^?1) was almost twice as high as six years after disturbance (?2.9 t C ha^?1 yr.^?1), with declining heterotrophic soil respiration and the high productivity of dense graminoid ground vegetation mitigating C loss.

Conclusions

C loss after disturbance decreases with time and ground vegetation growth. Dense non-woody ground vegetation cover can hamper tree regeneration but simultaneously decrease the ecosystem C loss. The role of ground vegetation should be more explicitly taken into account in forest C budgets assessing disturbance effects.

     

Expression of lycopene biosynthesis genes fused in line with Shine-Dalgarno sequences improves the stress-tolerance of <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Objectives

Lycopene biosynthetic genes from Deinococcus radiodurans were co-expressed in Lactococcus lactis to produce lycopene and improve its tolerance to stress.

Results

Lycopene-related genes from D. radiodurans, DR1395 (crtE), DR0862 (crtB), and DR0861 (crtI), were fused in line with S hine-Dalgarno (SD) sequences and co-expressed in L. lactis. The recombinant strain produced 0.36 mg lycopene g^-1 dry cell wt after 48 h fermentation. The survival rate to UV irradiation of the recombinant strain was higher than that of the non-transformed strain.

Conclusion

The L. lactis with co-expressed genes responsible for lycopene biosynthesis from D. radiodurans produced lycopene and exhibited increased resistance to UV stress, suggesting that the recombinant strain has important application potential in food industry.