Transcriptional effects of cadmium on iron homeostasis differ in calamine accessions of Noccaea caerulescens.

Calamine accessions of the zinc/cadmium/nickel hyperaccumulator, Noccaea caerulescens, exhibit striking variation in foliar cadmium accumulation in nature. The Ganges accession (GA) from Southern France displays foliar cadmium hyperaccumulation (>1000 µg g-1 DW), whereas the accession La Calamine (LC) from Belgium, with similar local soil metal composition, does not (<100 µg g-1 DW). All calamine accessions are cadmium hypertolerant. To find out the differences between LC and GA in their basic adaptation mechanisms, we bypassed the cadmium excluding phenotype of LC by exposing the plants to 50 µm cadmium in hydroponics, achieving equal cadmium accumulation in the shoots. The iron content increased in the roots of both accessions. GA exhibited significant decreases in manganese and zinc contents in the roots and shoots, approaching those in LC. Altogether 702 genes responded differently to cadmium exposure between the accessions, 157 and 545 in the roots and shoots, respectively. Cadmium-exposed LC showed a stress response and had decreased levels of a wide range of photosynthesis-related transcripts. GA showed less changes, mainly exhibiting an iron deficiency-like response. This included increased expression of genes encoding five iron deficiency-regulated bHLH transcription factors, ferric reduction oxidase FRO2, iron transporters IRT1 and OPT3, and nicotianamine synthase NAS1, and decreased expression of genes encoding ferritins and NEET (a NEET family iron-sulfur protein), which is possibly involved in iron transfer, distribution and/or management. The function of the IRT1 gene in the accessions was compared. We conclude that the major difference between the two accessions is in the way they cope with iron under cadmium exposure.

Expression of the GAF Sensor, Carbohydrate-Active Enzymes, Elicitins, and RXLRs Differs Markedly Between Two Phytophthora cactorum Isolates.

The phytopathogen Phytophthora cactorum infects economically important herbaceous and woody plant species. P. cactorum isolates differ in host specificity; for example, strawberry crown rot is often caused by a specialized pathotype. Here we compared the transcriptomes of two P. cactorum isolates that differ in their virulence to garden strawberry (Pc407: high virulence; Pc440: low virulence). De novo transcriptome assembly and clustering of contigs resulted in 19,372 gene clusters. Two days after inoculation of Fragaria vesca roots, 3,995 genes were differently expressed between the P. cactorum isolates. One of the genes that were highly expressed only in Pc407 encodes a GAF sensor protein potentially involved in membrane trafficking processes. Two days after inoculation, elicitins were highly expressed in Pc407 and lipid catabolism appeared to be more active than in Pc440. Of the carbohydrate-active enzymes, those that degrade pectin were often more highly expressed in Pc440, whereas members of glycosyl hydrolase family 1, potentially involved in the metabolism of glycosylated secondary metabolites, were more highly expressed in Pc407 at the time point studied. Differences were also observed among the RXLR effectors: Pc407 appears to rely on a smaller set of key RXLR effectors, whereas Pc440 expresses a greater number of RXLRs. This study is the first step toward improving understanding of the molecular basis of differences in the virulence of P. cactorum isolates. Identification of the key effectors is important, as it enables effector-assisted breeding strategies toward crown rot-resistant strawberry cultivars.

Gene expression differences between Noccaea caerulescens ecotypes help to identify candidate genes for metal phytoremediation.

Populations of Noccaea caerulescens show tremendous differences in their capacity to hyperaccumulate and hypertolerate metals. To explore the differences that could contribute to these traits, we undertook SOLiD high-throughput sequencing of the root transcriptomes of three phenotypically well-characterized N. caerulescens accessions, i.e., Ganges, La Calamine, and Monte Prinzera. Genes with possible contribution to zinc, cadmium, and nickel hyperaccumulation and hypertolerance were predicted. The most significant differences between the accessions were related to metal ion (di-, trivalent inorganic cation) transmembrane transporter activity, iron and calcium ion binding, (inorganic) anion transmembrane transporter activity, and antioxidant activity. Analysis of correlation between the expression profile of each gene and the metal-related characteristics of the accessions disclosed both previously characterized (HMA4, HMA3) and new candidate genes (e.g., for nickel IRT1, ZIP10, and PDF2.3) as possible contributors to the hyperaccumulation/tolerance phenotype. A number of unknown Noccaea-specific transcripts also showed correlation with Zn(2+), Cd(2+), or Ni(2+) hyperaccumulation/tolerance. This study shows that N. caerulescens populations have evolved great diversity in the expression of metal-related genes, facilitating adaptation to various metalliferous soils. The information will be helpful in the development of improved plants for metal phytoremediation.

Improved chromosome-level genome assembly of the Glanville fritillary butterfly (Melitaea cinxia) integrating Pacific Biosciences long reads and a high-density linkage map.

BACKGROUND: The Glanville fritillary (Melitaea cinxia) butterfly is a model system for metapopulation dynamics research in fragmented landscapes. Here, we provide a chromosome-level assembly of the butterfly's genome produced from Pacific Biosciences sequencing of a pool of males, combined with a linkage map from population crosses. RESULTS: The final assembly size of 484 Mb is an increase of 94 Mb on the previously published genome. Estimation of the completeness of the genome with BUSCO indicates that the genome contains 92-94% of the BUSCO genes in complete and single copies. We predicted 14,810 genes using the MAKER pipeline and manually curated 1,232 of these gene models. CONCLUSIONS: The genome and its annotated gene models are a valuable resource for future comparative genomics, molecular biology, transcriptome, and genetics studies on this species.

Characterization of the glyoxalase 1 gene TcGLX1 in the metal hyperaccumulator plant Thlaspi caerulescens.

Stress tolerance is currently one of the major research topics in plant biology because of the challenges posed by changing climate and increasing demand to grow crop plants in marginal soils. Increased Zn tolerance and accumulation has been reported in tobacco expressing the glyoxalase 1-encoding gene from Brassica juncea. Previous studies in our laboratory showed some Zn tolerance-correlated differences in the levels of glyoxalase 1-like protein among accessions of Zn hyperaccumulator Thlaspi caerulescens. We have now isolated the corresponding gene (named here TcGLX1), including ca. 570 bp of core and proximal promoter region. The predicted protein contains three glyoxalase 1 motifs and several putative sites for post-translational modification. In silico analysis predicted a number of cis-acting elements related to stress. The expression of TcGLX1 was not responsive to Zn. There was no correlation between the levels of TcGLX1 expression and the degrees of Zn tolerance or accumulation among T. caerulescens accessions nor was there co-segregation of TcGLX1 expression with Zn tolerance or Zn accumulation among F3 lines derived from crosses between plants from accessions with contrasting phenotypes for these properties. No phenotype was observed in an A. thaliana T-DNA insertion line for the closest A. thaliana homolog of TcGLX1, ATGLX1. These results suggest that glyoxalase 1 or at least the particular isoform studied here is not a major determinant of Zn tolerance in the Zn hyperaccumulator plant T. caerulescens. In addition, ATGLX1 is not essential for normal Zn tolerance in the non-tolerant, non-accumulator plant A. thaliana. Possible explanations for the apparent discrepancy between this and previous studies are discussed.

Terpenoid and lipid profiles vary in different Phytophthora cactorum - strawberry interactions.

Specialized metabolites are essential components in plant defence systems, serving as signalling molecules and chemical weapons against pathogens. The manipulation of plant defence metabolome or metabolites can thus be an important virulence strategy for pathogens. Because of their central role, metabolites can give valuable insights into plant-pathogen interactions. Here, we have conducted nontargeted metabolite profiling with UPLC-ESI-qTOF-MS to investigate the metabolic changes that have taken place in the crown tissue of Fragaria vesca L. (woodland strawberry) and Fragaria × ananassa (Weston) Duchesne ex Rozier (garden strawberry) during 48 h after Phytophthora cactorum challenge. Two P. cactorum isolates were compared: Pc407 is highly virulent to F. × ananassa and causes crown rot, whereas Pc440 is mildly virulent. In total, 45 metabolites differentially accumulated between the treatment groups were tentatively identified. Triterpenoids and various lipid compounds were highly represented. The levels of several triterpenoids increased upon inoculation, some of them showing distinct accumulation patterns in different interactions. Triterpenoids could either inhibit or stimulate P. cactorum growth and, therefore, triterpenoid profiles might have significant impact on disease progression. Of the lipid compounds, lysophospholipids, linoleic acid and linolenic acid were highly accumulated in the most compatible Pc407 - F. × ananassa interaction. As lysophospholipids promote cell death and have been linked to susceptibility, these compounds might be involved in the pathogenesis of crown rot disease. This metabolite analysis revealed potential factors contributing to the outcome of P. cactorum - strawberry interactions. The information is highly valuable, as it can help to find new breeding strategies and new solutions to control P. cactorum in strawberry.

Safety evaluation of the food enzyme α-amylase from the genetically modified Bacillus licheniformis strain DP-Dzb52.

The food enzyme α-amylase (1,4-α-d-glucan glucanohydrolase; EC 3.2.1.1) is produced with the genetically modified Bacillus licheniformis strain DP-Dzb52 by Danisco US Inc. The production strain contains multiple copies of an antimicrobial resistance gene. However, based on the absence of viable cells and DNA from the production organism in the food enzyme, this is not considered to be a risk. The α-amylase is intended to be used in starch processing for the production of glucose syrups, brewing processes and distilled alcohol production. Since residual amounts of the food enzyme are removed by the purification steps applied during the production of glucose syrups and distillation, no dietary exposure was calculated. Based on the maximum use levels recommended for the brewing processes and individual data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the enzyme-total organic solids (TOS) was estimated to be up to 0.145 TOS/kg body weight per day in European populations. The toxicity studies were carried out with another α-amylase from B. licheniformis strain DP-Dzb54, considered by the Panel as a suitable substitute. Toxicological tests indicated that there was no concern with respect to genotoxicity or systemic toxicity. A no observed adverse effect level was identified in rats which, compared with the dietary exposure, results in a margin of exposure of at least 750. A search for similarity of the amino acid sequence to known allergens was made and one match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions can be excluded in distilled alcohol production and is considered low when the enzyme is used in starch processing and brewing. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme endo-1,3(4)-ß-glucanase from the genetically modified Bacillus subtilis strain DP-Ezm28.

The food enzyme endo-1,3(4)-ß-glucanase (3(or 4)-ß-d-glucan 3(4)-glucanohydrolase; EC 3.2.1.6) is produced with a genetically modified Bacillus subtilis strain DP-Ezm28 by Danisco US Inc. The genetic modifications do not give rise to safety concerns. The production strain of the food enzyme contains multiple copies of a known antimicrobial resistance gene. However, based on the absence of viable cells and DNA from the production organism in the food enzyme, this is not considered to be a risk. The food enzyme is intended to be used in distilled alcohol production and brewing processes. Since residual amounts of total organic solids (TOS) are removed by distillation, dietary exposure was only calculated for brewing processes. Based on the maximum use levels recommended for brewing processes and individual data from the EFSA Comprehensive European Food Database, dietary exposure to the food enzyme-total organic solids was estimated to be up to 0.183 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,000 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 5464. Similarity of the amino acid sequence to those of known allergens was searched and two matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is considered low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Genetic and environmental influence on maize kernel proteome.

Comparative targeted compositional analysis is currently an important element in the safety assessment of genetically modified plants. Profiling methods have been suggested as nontargeted tools to improve the detection of possible unintended effects. In this study, the capability of 2-dimensional electrophoresis to detect significant differences among seven conventional maize (Zea mays) cultivars grown in six different locations in Germany during two consecutive seasons was evaluated. Besides maize genotype, both geographic location and season had a significant effect on protein profiles. Differences as high as 55- and 53-fold in the quantity of specific proteins were recorded, the median observed difference being around 6- and 5-fold between the genotypes and growing locations, respectively. Understanding the variation in the quantity of individual proteins should help to put the variation of endogenous proteins and the novel proteins in the genetically modified plants in perspective. This together with the targeted analyses the profiling methods, including proteomics, could also help to get a deeper insight into the unintended alterations that might have occurred during the genetic modification process.

Elevation of night-time temperature increases terpenoid emissions from Betula pendula and Populus tremula.

Volatile organic compounds (VOCs) are expected to have an important role in plant adaptation to high temperatures. The impacts of increasing night-time temperature on daytime terpenoid emissions and related gene expression in silver birch (Betula pendula) and European aspen (Populus tremula) clones were studied. The plants were grown under five different night-time temperatures (6, 10, 14, 18, and 22 degrees C) while daytime temperature was kept at a constant 22 degrees C. VOC emissions were collected during the daytime and analysed by gas chromatography-mass spectrometry (GC-MS). In birch, emissions per leaf area of the C11 homoterpene 4,8-dimethy1-nona-1,3,7-triene (DMNT) and several sesquiterpenes were consistently increased with increasing night-time temperature. Total sesquiterpene (SQT) emissions showed an increase at higher temperatures. In aspen, emissions of DMNT and beta-ocimene increased from 6 degrees C to 14 degrees C, while several other monoterpenes and the SQTs (Z,E)-alpha-farnesene and (E,E)-alpha-farnesene increased up to 18 degrees C. Total monoterpene and sesquiterpene emission peaked at 18 degrees C, whereas isoprene emissions decreased at 22 degrees C. Leaf area increased across the temperature range of 6-22 degrees C by 32% in birch and by 59% in aspen. Specific leaf area (SLA) was also increased in both species. The genetic regulation of VOC emissions seems to be very complex, as indicated by several inverse relationships between emission profiles and expression of several regulatory genes (DXR, DXS, and IPP). The study indicates that increasing night temperature may strongly affect the quantity and quality of daytime VOC emissions of northern deciduous trees.

Proteomics of Thlaspi caerulescens accessions and an inter-accession cross segregating for zinc accumulation.

Metal hyperaccumulator plants have previously been characterized by transcriptomics, but reports on other profiling techniques are scarce. Protein profiles of Thlaspi caerulescens accessions La Calamine (LC) and Lellingen (LE) and lines derived from an LCxLE cross were examined here to determine the co-segregation of protein expression with the level of zinc (Zn) hyperaccumulation. Although hydrophobic proteins such as membrane transporters are not disclosed, this approach has the potential to reveal other proteins important for the Zn hyperaccumulation trait. Plants were exposed to metals. Proteins were separated using two-dimensional electrophoresis and those showing differences among accessions, lines or metal exposures were subjected to mass-spectrometric analysis for identification. Crossing decreased the number of different proteins in the lines compared with the parents, more so in the shoots than in the roots, but the frequencies of Zn-responsive proteins were about the same in the accessions and the selection lines. This supports the finding that the Zn accumulation traits are mainly determined by the root and that Zn accumulation itself is not the reason for the co-segregation. This study demonstrates that crossing accessions with contrasting Zn accumulation traits is a potent tool to investigate the mechanisms behind metal hyperaccumulation. Four tentatively identified root proteins showed co-segregation with high or low Zn accumulation: manganese superoxide dismutase, glutathione S-transferase, S-formyl glutathione hydrolase, and translation elongation factor 5A-2. However, these proteins may not be the direct determinants of Zn accumulation. The role of these and other tentatively identified proteins in Zn accumulation and tolerance is discussed.

Safety evaluation of the food enzyme triacylglycerol lipase from the genetically modified Ogataea polymorpha strain DP-Jzk33.

The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase EC 3.1.1.3) is produced with the genetically modified Ogataea polymorpha strain DP-Jzk33 by Danisco US Inc. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and recombinant DNA. It is intended to be used in baking and cereal-based processes. Based on the maximum use levels recommended for baking and cereal-based processes and individual data from the EFSA Comprehensive European Food Database, dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.520 mg TOS/kg body weight (bw) per day. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 669 mg TOS/kg bw per day, the highest dose tested. Comparison with the estimated dietary exposure results in a margin of exposure of at least 1,287. A search was made of the similarity of the amino acid sequence of the lipase to those of known allergens and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood of such reactions to occur is likely to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Stilbene synthase gene transfer caused alterations in the phenylpropanoid metabolism of transgenic strawberry (Fragaria x ananassa).

The gene encoding stilbene synthase is frequently used to modify plant secondary metabolism with the aim of producing the self-defence phytoalexin resveratrol. In this study, strawberry (Fragaria x ananassa) was transformed with the NS-Vitis3 gene encoding stilbene synthase from frost grape (Vitis riparia) under the control of the cauliflower mosaic virus 35S and the floral filament-specific fil1 promoters. Changes in leaf metabolites were investigated with UPLC-qTOF-MS (ultra performance liquid chromatography-quadrupole time of flight mass spectrometry) profiling, and increased accumulation of cinnamate, coumarate, and ferulate derivatives concomitantly with a decrease in the levels of flavonols was observed, while the anticipated resveratrol or its derivatives were not detected. The changed metabolite profile suggested that chalcone synthase was down-regulated by the genetic modification; this was verified by decreased chalcone synthase transcript levels. Changes in the levels of phenolic compounds led to increased susceptibility of the transgenic strawberry to grey mould fungus.

NMR and UPLC-qTOF-MS/MS characterisation of novel phenylethanol derivatives of phenylpropanoid glucosides from the leaves of strawberry (Fragaria x ananassa cv. Jonsok).

INTRODUCTION: Strawberry (Fragaria x ananassa) is rich in polyphenols, particularly anthocyanins, flavonols, condensed tannins and ellagic tannins. In addition to the fruits, the leaves of strawberry also contain a wide range of phenolic compound classes, but have not been investigated to the same extent as the fruit. OBJECTIVE: To characterise a metabolite group present in the leaves of strawberry, that was not amenable for identification based on earlier information available in the literature. METHODOLOGY: Methanolic extracts of strawberry leaves were analysed by UPLC-qTOF-MS/MS and iterative quantum mechanical NMR spectral analysis. RESULTS: The structures of phenylethanol derivatives of phenylpropanoid glucosides Eutigoside A ( F4) and its two isomeric forms 2-(4-hydroxyphenyl)ethyl-[6-O-(Z)-coumaroyl]-beta-D-glucopyranoside (F6) and 4-(2-hydroxyethyl)phenyl-[6-O-(E)-coumaroyl]-beta-D-glucopyranoside (F1) were resolved by NMR and UPLC-qTOF-MS/MS. In addition, two other derivatives of phenylpropanoid glucosides similar to Eutigoside A but possessing different phenolic acid moieties, namely Grayanoside A ( F5) and 2-(4-hydroxyphenyl)ethyl-[6-O-(E)-caffeoyl]-beta-D-glucopyranoside (F14), were similarly identified. Also, accurate characteristic coupling constants for the subunits are reported and their usefulness in structural analysis is highlighted. CONCLUSION: Chemical analysis of the leaves of strawberry (Fragaria x ananassa cv. Jonsok) resulted in the identification of a compound class, phenylethanol derivatives of phenylpropanoid glycosides, not previously found in strawberry.

Safety evaluation of the food enzyme endo-1,4-ß-xylanase from a genetically modified Bacillus licheniformis (strain NZYM-CE).

The food enzyme endo-1,4-ß-xylanase (4-ß-d-xylan xylanohydrolase; EC 3.2.1.8) is produced with a genetically modified Bacillus licheniformis (strain NZYM-CE) by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and recombinant DNA. This xylanase is intended to be used in baking and cereal-based processes. Based on the maximum use levels recommended for the respective food processes and individual data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.012 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a genotoxic concern. The systemic toxicity was assessed by a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level (NOAEL) of at least 1,020 mg TOS/kg bw per day, the highest dose tested. When the NOAEL value is compared to the estimated dietary exposure, this results in a margin of exposure (MoE) of at least 85,000. Similarity of the amino acid sequence to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is considered to be low. Overall, the Panel concluded that based on the data provided and the derived MoE, this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety of concentrated l-lysine (base), l-lysine monohydrochloride and l-lysine sulfate produced using different strains of Corynebacterium glutamicum for all animal species based on a dossier submitted by FEFANA asbl.

The EFSA FEEDAP Panel previously (2016) could not conclude on the safety of certain concentrated liquid l-lysine (base), l-lysine monohydrochloride (HCl) and l-lysine sulfate products manufactured using different strains of Corynebacterium glutamicum. New information on the safety of these products was provided by the applicant. The recipient strain C. glutamicum KCTC 12307BP qualifies for qualified presumption of safety (QPS) approach for safety assessment, the genetic modification does not introduce any safety concern and no introduced antibiotic resistance genes remain in its genome. Even if uncertainty remains concerning the absence/presence of the production strain and/or its recombinant DNA in the final products, these would not raise safety concerns. The liquid l-lysine (base) and l-lysine HCl produced by C. glutamicum KCTC 12307BP or C. glutamicum KCCM 11117P; and l-lysine HCl produced by C. glutamicum NRRL B-50547 are considered safe for the target species, consumers and the environment. Regarding the safety for the user, concentrated liquid l-lysine (base) and l-lysine HCl produced by C. glutamicum KCTC 12307BP, C. glutamicum NRRL B-50547 or C. glutamicum KCCM 11117P are not irritant to skin or eyes and they are not skin sensitisers. l-Lysine HCl is not hazardous by inhalation. The use of C. glutamicum DSM 24990 in the production of l-lysine sulfate is considered safe for the target species, consumers, users or the environment. No negative effects are to be expected for the target species within the proposed inclusion levels of 0.5-30 g lysine sulfate/kg complete feed provided that the total S intake complies with the recommendations of established scientific bodies. The use of C. glutamicum KCCM 10227 in the production of l-lysine sulfate is considered safe for the target species, consumers, users and the environment with regard to antimicrobial resistance. No negative effects are to be expected for the target species within common inclusion levels provided that the total S intake complies with the recommendations of established scientific bodies.

Safety and efficacy of l-lysine monohydrochloride and concentrated liquid l-lysine (base) produced by fermentation using Corynebacterium glutamicum strain NRRL B-50775 for all animal species based on a dossier submitted by ADM.

The European Commission asked EFSA for an opinion on the safety for the target animals, consumer, user and the environment and on the efficacy of a l-lysine monohydrochloride (HCl, minimum 98.5%) and of a concentrated liquid l-lysine (base, minimum 50%) produced by a genetically modified strain of Corynebacterium glutamicum (NRRL B-50775). They are intended to be used in feed or water for drinking for all animal species and categories. Neither the production strain C. glutamicum NRRL B-50775 nor its recombinant DNA was detected in the final product. Therefore, the product does not pose any safety concern associated with the genetic modification of the production strain. l-Lysine HCl and concentrated liquid l-lysine (base) produced by C. glutamicum NRRL B-50775 are considered safe for the target species, for the consumer and for the environment. l-Lysine HCl produced by C. glutamicum NRRL B-50775 is considered not irritant to skin or eyes and not a skin sensitiser. In the absence of data, the FEEDAP Panel cannot conclude on the potential toxicity by inhalation of l-lysine HCl produced by C. glutamicum NRRL B-50775. Concentrated liquid l-lysine (base) produced by C. glutamicum NRRL B-50775, due to its high pH (11) it is anticipated to be corrosive to skin and eyes and poses a risk by inhalation. l-Lysine HCl and concentrated liquid l-lysine (base) produced by C. glutamicum NRRL B-50775 are considered as efficacious sources of the essential amino acid l-lysine for non-ruminant animal species. For the supplemental l-lysine to be as efficacious in ruminants as in non-ruminant species, it would require protection against degradation in the rumen.

Safety evaluation of the food enzyme pullulanase from a genetically modified Bacillus licheniformis (strain DP-Dzp39).

The food enzyme pullulanase (pullulan 6-α-glucanohydrolase; EC 3.2.1.41) is produced with a genetically modified Bacillus licheniformis (strain DP-Dzp39) by Danisco US Inc. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its recombinant DNA. This pullulanase is intended to be used in brewing processes, starch processing for glucose syrups production and distilled alcohol production. Residual amounts of total organic solids (TOS) are removed by distillation and by the purification steps applied during the production of glucose syrups, consequently, dietary exposure was not calculated for these food processes. For brewery products, based on the maximum use level recommended for the brewing processes and individual data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the food enzyme-TOS was estimated to be up to 0.053 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests with the food enzyme did not raise concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no-observed-adverse-effect level at the highest dose of 500 mg TOS/kg bw per day that, compared to the estimated dietary exposure, results in sufficiently high margin of exposure (at least 9,400). The amino acid sequence of the food enzyme did not match those of known allergens. The Panel considered that, under the intended condition of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is considered low. Based on the data provided, the Panel concluded that this food enzyme does not raise safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme glucose oxidase from Aspergillus niger (strain ZGL).

The food enzyme glucose oxidase (ß-d-glucose:oxygen 1-oxidoreductase; EC 1.1.3.4) is produced with a genetically modified Aspergillus niger strain ZGL by DSM Food Specialties B.V.. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and recombinant DNA. The glucose oxidase is intended to be used in baking processes. Based on the maximum use levels, dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.004 mg TOS/kg body weight (bw) per day. The toxicity studies were carried out with an asparaginase from A. niger (strain ASP). The Panel considered this enzyme as a suitable substitute to be used in the toxicological studies, because they derive from the same recipient strain, the location of the inserts are comparable, no partial inserts were present and the production methods are essentially the same. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level (NOAEL) at the highest dose of 1,038 and 1,194 mg TOS/kg bw per day (for males and females, respectively) that, compared with the estimated dietary exposure, results in a sufficiently high margin of exposure (MoE) (of at least 260,000). Similarity of the amino acid sequence to those of known allergens was searched and one match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood to occur is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme triacylglycerol lipase from Aspergillus niger (strain LFS).

The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is produced with a genetically modified Aspergillus niger strain LFS by DSM Food Specialties B.V.. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and recombinant DNA. The triacylglycerol lipase food enzyme is intended to be used in baking processes. Based on the maximum use levels, dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.020 mg TOS/kg body weight (bw) per day. The toxicity studies were carried out with an asparaginase from A. niger (strain ASP). The Panel considered this enzyme as a suitable substitute to be used in the toxicological studies, because they derive from the same recipient strain, the location of the inserts are comparable, no partial inserts were present and the production methods are essentially the same. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level (NOAEL) at the highest dose of 1,038 and 1,194 mg TOS/kg bw per day (for males and females, respectively) that, compared with the estimated dietary exposure, results in a sufficiently high margin of exposure (MoE) (of at least 51,900). Similarity of the amino acid sequence to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood to occur is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.