Effect of Biomass Pre-Treatment and Solvent Extraction on β-Carotene and Lycopene Recovery from Blakeslea trispora Cells

Abstract

The production of carotenoids from Blakeslea trispora cells in a synthetic medium has been reported, with the main products being β-carotene, lycopene, and γ-carotene. The effect of biomass pretreatment and solvent extraction on their selective recovery is reported here. Eight solvents of class II and III of the International Conference of Harmonization: ethanol, methanol, acetone, 2-propanol, pentane, hexane, ethyl acetate, and ethyl ether, and HPLC analysis were used for the evaluation of their selectivities towards the three main carotenoids with regard to different biomass pre-treatment. The average C_max values (maximum concentration of caronoids in a specific solvent) were estimated to 16 mg/L with the five out of eight solvents investigated, whereas methanol, pentane, and hexane gave lower values of 10, 11, and 9 mg/L, respectively. The highest carotenoid yield was obtained in the case of wet biomass, where 44–56% is recovered with one solvent and three extractions and the rest is recovered only after subsequent treatment with acetone; thus, four extractions of 2.5 h are needed. Two extractions of 54 min are enough to recover carotenoids from dehydrated biomass, with the disadvantage of a high degree of degradation. Our results showed that, for maximum carotenoid recovery, ethyl ether, 2-propanol, and ethanol could be successfully used with biomass without prior treatment, whereas fractions enriched in β-carotene or lycopene can be obtained by extraction with the proper solvent, thus avoiding degradation due to time-consuming processes.     

A Unique Pattern of Mycelial Elongation of Blakeslea trispora and Its Effect on Morphological Characteristics and β-Carotene Synthesis

The mycelial morphology of Blakeslea trispora was of crucial importance in the production of beta-carotene in submerged cultures of B. trispora. After the spores were inoculated, the time-course variation of mycelial morphology was closely examined under the microscope. With the addition of the non-ionic surfactant (Span 20: Sorbitan monolaurate, E493) to the culture medium, a unique pattern of mycelial elongation was observed: 1) slow formation of germ tubes from spores and 2) appearance of mycelia with very short length, which allowed a well-dispersed growth of B. trispora without significant pellet aggregation. Span 20 appears to act like a paramorphogen. Without Span 20, however, the fungal culture finally formed a big clump of mycelium owing to heavy cross-linking of long mycelia. But the short mycelium maintained in the course of cultivation seemed to be irrelevant to growth inhibition, because the final concentration of dry mycelium was much higher with Span 20 after 3-day cultivation. The 20-fold increase in specific yield of beta-carotene (mg beta-carotene produced per g mycelium) was achieved with this drastic change in the pattern of mycelial elongation. The reason for this result might be more effective mass transfer and/or enhanced sensitivity to environmental oxidative stress in the well-dispersed mycelial cultures of B. trispora.     

Metabolic Engineering for Production of β-Carotene and Lycopene in Saccharomyces cerevisiae

We have engineered a conventional yeast, Saccharomyces cerevisiae, to confer a novel biosynthetic pathway for the production of β-carotene and lycopene by introducing the bacterial carotenoid biosynthesis genes, which are individually surrounded by the promoters and terminators derived from S. cerevisiae. β-Carotene and lycopene accumulated in the cells of this yeast, which was considered to be a result of the carbon flow for the ergosterol biosynthetic pathway being partially directed to the pathway for the carotenoid production.     

Production of the Carotenoids Lycopene, β-Carotene,and Astaxanthin in the Food Yeast Candida utilis

The food-grade yeast Candida utilis has been engineered to confer a novel biosynthetic pathway for the production of carotenoids such as lycopene, β-carotene, and astaxanthin. The exogenous carotenoid biosynthesis genes were derived from the epiphytic bacterium Erwinia uredovora and the marine bacterium Agrobacterium aurantiacum. The carotenoid biosynthesis genes were individually modified based on the codon usage of the C. utilis glyceraldehyde 3-phosphate dehydrogenase gene and expressed in C. utilis under the control of the constitutive promoters and terminators derived from C. utilis. The resultant yeast strains accumulated lycopene, β-carotene, and astaxanthin in the cells at 1.1, 0.4, and 0.4 mg per g (dry weight) of cells, respectively. This was considered to be a result of the carbon flow into ergosterol biosynthesis being partially redirected to the nonendogenous pathway for carotenoid production.Carotenoids are yellow, orange, and red pigments which are widely distributed in nature (3). Industrially, carotenoid pigments such as β-carotene are utilized as food or feed supplements. β-Carotene is also a precursor of vitamin A in mammals (11). Recently, carotenoids have attracted greater attention, due to their beneficial effect on human health: e.g., the functions of lycopene and astaxanthin include strong quenching of singlet oxygen (12), involvement in cancer prevention (2), and enhancement of immune responses (6). Astaxanthin has also been exploited for industrial use, principally as an agent for pigmenting cultured fish and shellfish.The genes responsible for the synthesis of carotenoids such as lycopene, β-carotene, and astaxanthin have been isolated from the epiphytic Erwinia species or the marine bacteria Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1, and their functions have been elucidated (13, 14). The first substrate of the encoded enzymes for carotenoid synthesis is farnesyl pyrophosphate (diphosphate) (FPP), which is the common precursor for the biosynthesis of numerous isoprenoid compounds such as sterols, hopanols, dolicols, and quinones. The ubiquitous nature of FPP among yeasts has been utilized in the microbial production of lycopene and β-carotene by the yeast Saccharomyces cerevisiae carrying the Erwinia uredovora carotenogenic genes (19). However, the amount of carotenoids produced in these hosts was only 0.1 mg of lycopene and 0.1 mg of β-carotene per g (dry weight) of cells, respectively.The edible yeast Candida utilis is generally recognized as a safe substance by the Food and Drug Administration. Large-scale production of the yeast cells has been developed with cheap biomass-derived sugars as the carbon source for the production of single-cell protein and several chemicals such as glutathione and RNA (1, 4). This yeast was also found to accumulate a large amount of ergosterol in the cell during stationary phase (6 to 13 mg/g [dry weight] of cells) (17). Thus, C. utilis has the potential to produce a large amount of carotenoids by redirecting the carbon flux for the ergosterol biosynthesis into the nonendogenous pathway for carotenoid synthesis via FPP. Previously, a C. utilis strain was made to produce lycopene (0.8 mg/g [dry weight]) by expressing the three nonmodified genes crtE, crtB, and crtI derived from E. uredovora (15).In this paper, the de novo biosynthesis of lycopene, β-carotene, and astaxanthin has been performed in C. utilis by using six carotenogenic genes, which were synthesized according to the codon usage of the C. utilis glyceraldehyde-3-phosphate dehydrogenase (GAP) gene, which is expressed at high levels. By this approach, increased carotenoid production in C. utilis was achieved.     

Spectroscopy analysis for simultaneous determination of lycopene and β-carotene in fungal biomass of Blakeslea trispora

Blakeslea trispora is a good alternative source for producing such carotenoids as lycopene and β-carotene. The objective of this research was to elaborate a method for the simultaneous determination of lycopene and β-carotene in Blakeslea trispora products using a usual UV-vis spectrophotometer. The standard solutions of the mixture of different concentrations of β-carotene and lycopene were measured with the UV-vis method and correlation formula for the extinction coefficients of 1% standard solution of lycopene in the solvent (hexane) and the ratios of the optical densities at the character peaks of 470 and 502 nm was elaborated. This gives a possibility to calculate the concentrations of lycopene and β-carotene in the mixture. The prediction quality of the UV-vis method was sufficient and the obtained results were very close to the ones, being measured with the HPLC technique. The proposed method can be used for both routine industrial work and academic research, providing the rapid analysis for simultaneous measurements of lycopene and β-carotene.     

Gene Conversion and Functional Divergence in the β-Globin Gene Family

Different models of gene family evolution have been proposed to explain the mechanism whereby gene copies created by gene duplications are maintained and diverge in function. Ohta proposed a model which predicts a burst of nonsynonymous substitutions following gene duplication and the preservation of duplicates through positive selection. An alternative model, the duplication–degeneration–complementation (DDC) model, does not explicitly require the action of positive Darwinian selection for the maintenance of duplicated gene copies, although purifying selection is assumed to continue to act on both copies. A potential outcome of the DDC model is heterogeneity in purifying selection among the gene copies, due to partitioning of subfunctions which complement each other. By using the d_N/d_S () rate ratio to measure selection pressure, we can distinguish between these two very different evolutionary scenarios. In this study we investigated these scenarios in the -globin family of genes, a textbook example of evolution by gene duplication. We assembled a comprehensive dataset of 72 vertebrate -globin sequences. The estimated phylogeny suggested multiple gene duplication and gene conversion events. By using different programs to detect recombination, we confirmed several cases of gene conversion and detected two new cases. We tested evolutionary scenarios derived from Ohtas model and the DDC model by examining selective pressures along lineages in a phylogeny of -globin genes in eutherian mammals. We did not find significant evidence for an increase in the ratio following major duplication events in this family. However, one exception to this pattern was the duplication of -globin in simian primates, after which a few sites were identified to be under positive selection. Overall, our results suggest that following gene duplications, paralogous copies of -globin genes evolved under a nonepisodic process of functional divergence.[Reviewing Editor: Martin Kreitman]     

Mutations in Nicastrin Protein Differentially Affect Amyloid β-Peptide Production and Notch Protein Processing

The γ-secretase complex is responsible for intramembrane processing of over 60 substrates and is involved in Notch signaling as well as in the generation of the amyloid β-peptide (Aβ). Aggregated forms of Aβ have a pathogenic role in Alzheimer disease and, thus, reducing the Aβ levels by inhibiting γ-secretase is a possible treatment strategy for Alzheimer disease. Regrettably, clinical trials have shown that inhibition of γ-secretase results in Notch-related side effects. Therefore, it is of great importance to find ways to inhibit amyloid precursor protein (APP) processing without disturbing vital signaling pathways such as Notch. Nicastrin (Nct) is part of the γ-secretase complex and has been proposed to be involved in substrate recognition and selection. We have investigated how the four evenly spaced and conserved cysteine residues in the Nct ectodomain affect APP and Notch processing. We mutated these cysteines to serines and analyzed them in cells lacking endogenous Nct. We found that two mutants, C213S (C2) and C230S (C3), differentially affected APP and Notch processing. Both the formation of Aβ and the intracellular domain of amyloid precursor protein (AICD) were reduced, whereas the production of Notch intracellular domain (NICD) was maintained on a high level, although C230S (C3) showed impaired complex assembly. Our data demonstrate that single residues in a γ-secretase component besides presenilin are able to differentially affect APP and Notch processing.     

Effect of the aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor: mathematical modeling

The effect of aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor was investigated. B. trispora formed hyphae, zygophores and zygospores during the fermentation. The zygospores were the morphological form responsible for β-carotene production. Both aeration and agitation significantly affected β-carotene concentration, productivity, biomass and the volumetric mass transfer coefficient (K_La). The highest β-carotene concentration (1.5 kg m⁻³) and the highest productivity (0.08 kg m⁻³ per day) were obtained at low impeller speed (150 rpm) and high aeration rate (1.5 vvm). Also, maximum productivity (0.08 kg m⁻³ per day) and biomass dry weight (26.4 kg m⁻³) were achieved at high agitation speed (500 rpm) and moderate aeration rate (1.0 vvm). Conversely, the highest value of K_La (0.33 s⁻¹) was observed at high agitation speed (500 rpm) and high aeration rate (1.5 vvm). The experiments were arranged according to a central composite statistical design. Response surface methodology was used to describe the effect of impeller speed and aeration rate on the most important fermentation parameters. In all cases, the fit of the model was found to be good. All fermentation parameters (except biomass concentration) were strongly affected by the interactions among the operation variables. β-Carotene concentration and productivity were significantly influenced by the aeration, agitation, and by the positive or negative quadratic effect of the aeration rate. Biomass concentration was principally related to the aeration rate, agitation speed, and the positive or negative quadratic effect of the impeller speed and aeration rate, respectively. Finally, the volumetric mass transfer coefficient was characterized by the significant effect of the agitation speed, while the aeration rate had a small effect on K_La.     

Production of γ-linolenic acid by the fungus Mucor rouxii in fed-batch and continuous culture

Summary The production of -linolenic acid (GLA) and lipid was studied in Mucor rouxii CBS 416.77. In a fed-batch culture productivities of 39.4 mg/l per hour for GLA and 99 mg/l per hour for the total amount of lipid were determined at 18 h of cultivation. At this point the highest value of GLA in lipid (39.7%, w/w) was also reached. Production of GLA was also studied in a series of continuous cultures. It was observed that, in addition to growth rate, the nitrogen concentration of the input medium was of great importance for high productivities. The highest productivity values for GLA (37 mg/l per hour) and for lipid (95 mg/l per hour) were reached at a dilution rate of 0.10 h^-1 with a concentration of 4.5g/l NH₄Cl in the input medium.     

Carotene Hydroxylase Activity Determines the Levels of Both α-Carotene and Total Carotenoids in Orange Carrots

The typically intense carotenoid accumulation in cultivated orange-rooted carrots (Daucus carota) is determined by a high protein abundance of the rate-limiting enzyme for carotenoid biosynthesis, phytoene synthase (PSY), as compared with white-rooted cultivars. However, in contrast to other carotenoid accumulating systems, orange carrots are characterized by unusually high levels of α-carotene in addition to β-carotene. We found similarly increased α-carotene levels in leaves of orange carrots compared with white-rooted cultivars. This has also been observed in the Arabidopsis thaliana lut5 mutant carrying a defective carotene hydroxylase CYP97A3 gene. In fact, overexpression of CYP97A3 in orange carrots restored leaf carotenoid patterns almost to those found in white-rooted cultivars and strongly reduced α-carotene levels in the roots. Unexpectedly, this was accompanied by a 30 to 50% reduction in total root carotenoids and correlated with reduced PSY protein levels while PSY expression was unchanged. This suggests a negative feedback emerging from carotenoid metabolites determining PSY protein levels and, thus, total carotenoid flux. Furthermore, we identified a deficient CYP97A3 allele containing a frame-shift insertion in orange carrots. Association mapping analysis using a large carrot population revealed a significant association of this polymorphism with both α-carotene content and the α-/β-carotene ratio and explained a large proportion of the observed variation in carrots.     

Characterization of the β-Carotene Hydroxylase Gene DSM2 Conferring Drought and Oxidative Stress Resistance by Increasing Xanthophylls and Abscisic Acid Synthesis in Rice

Drought is a major limiting factor for crop production. To identify critical genes for drought resistance in rice (Oryza sativa), we screened T-DNA mutants and identified a drought-hypersensitive mutant, dsm2. The mutant phenotype was caused by a T-DNA insertion in a gene encoding a putative β-carotene hydroxylase (BCH). BCH is predicted for the biosynthesis of zeaxanthin, a carotenoid precursor of abscisic acid (ABA). The amounts of zeaxanthin and ABA were significantly reduced in two allelic dsm2 mutants after drought stress compared with the wild type. Under drought stress conditions, the mutant leaves lost water faster than the wild type and the photosynthesis rate, biomass, and grain yield were significantly reduced, whereas malondialdehyde level and stomata aperture were increased in the mutant. The mutant is also hypersensitive to oxidative stresses. The mutant had significantly lower maximal efficiency of photosystem II photochemistry and nonphotochemical quenching capacity than the wild type, indicating photoinhibition in photosystem II and decreased capacity for eliminating excess energy by thermal dissipation. Overexpression of DSM2 in rice resulted in significantly increased resistance to drought and oxidative stresses and increases of the xanthophylls and nonphotochemical quenching. Some stress-related ABA-responsive genes were up-regulated in the overexpression line. DSM2 is a chloroplast protein, and the response of DSM2 to environmental stimuli is distinctive from the other two BCH members in rice. We conclude that the DSM2 gene significantly contributes to control of the xanthophyll cycle and ABA synthesis, both of which play critical roles in the establishment of drought resistance in rice.Abiotic stresses such as drought, salinity, and adverse temperatures are major limiting factors for plant growth and reproduction. To respond to environmental cues, plants have evolved a variety of biochemical and physiological mechanisms to adapt to adverse conditions during their growth and development (Boyer, 1982). Abscisic acid (ABA) has been recognized as a stress hormone that coordinates the complex networks of stress responses. Under drought or salt stress conditions, plant endogenous ABA level can rise to about 40-fold, triggering the closure of stomata and accumulating reactive oxygen species (ROS), dehydrins, and late embryogenesis abundant proteins for osmotic adjustment (Verslues et al., 2006). The endogenous ABA level is determined by ABA biosynthesis, catabolism, and release of ABA from ABA-Glc conjugates (Nambara and Marion-Poll, 2005; Lee et al., 2006). Therefore, identification of all the components affecting active ABA content is essential for a complete understanding of the action of the hormone.Numerous ABA biosynthetic genes have been identified through mutant analysis, such as maize (Zea mays) viviparous mutants vp2, vp5, vp7, vp9, vp14, w3, y3, and y9 (Schwartz et al., 1997; Hable et al., 1998; Singh et al., 2003); rice (Oryza sativa) preharvest-sprouting mutants psh1, psh2, psh3, and psh4 (Fang et al., 2008); sunflower (Helianthus annuus) nondormant mutant nd-1 (Conti et al., 2004); Arabidopsis (Arabidopsis thaliana) ABA- and nonphotochemical quenching (NPQ)-deficient mutants aba1, aba2, aba3, aba4, npq1, npq2, b1, b2, and nced3 (Havaux et al., 2000; Xiong et al., 2001; Tian et al., 2003; Barrero et al., 2005; Kim and DellaPenna, 2006; North et al., 2007); and tomato (Solanum lycopersicum) white-flower mutant wf (Galpaz et al., 2006; Supplemental Fig. S1). The mutants unable to biosynthesize carotenoid precursors for endogenous ABA synthesis often produced preharvest-sprouting seeds and wilted or white leaves (Gubler et al., 2005; Nambara and Marion-Poll, 2005; Finch-Savage and Leubner-Metzger, 2006).ABA biosynthesis initiates with the synthesis of a C5 building block, isopentenyl pyrophosphate, and its isomer dimethylallyl pyrophosphate through a plastid methylerythritol phosphate pathway (Eisenreich et al., 2001; Hunter, 2007). The three isopentenyl pyrophosphate molecules are then added to dimethylallyl pyrophosphate by geranylgeranyl diphosphate synthase to produce C20 geranylgeranyl diphosphate. Two geranylgeranyl diphosphates are condensed by a committing enzyme, phytoene synthase, to produce colorless C40 carotenoid phytoene, which is then desaturated and isomerized into red-colored lycopene by phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), and Z-ISO and CRTISO isomerases in plants (Isaacson et al., 2002; Park et al., 2002). Subsequently, several cyclization and hydroxylation reactions take place to yield α-carotene and β-carotene (Li et al., 1996; Hable et al., 1998; Park et al., 2002; Miki and Shimamoto, 2004; Fang et al., 2008). Heme-type cytochrome P450-type CYP97 and non-heme-type β-carotene hydroxylase (BCH) are primarily responsible for the hydroxylation of α-carotene and β-carotene to produce lutein and zeaxanthin, respectively. Zeaxanthin, an important component of the xanthophyll cycle, is epoxidated by zeaxanthin epoxidase to produce violaxanthin, and this reaction can be reversed by violaxanthin deepoxidase to increase the xanthophyll cycle for plants to adapt to high-light stress (Johnson et al., 2008). Neoxanthin synthase converts violaxanthin into neoxanthin (North et al., 2007). In chloroplast, 9-cis-epoxycarotenoid dioxygenase (NCED) cleaves violaxanthin and neoxanthin to produce xanthoxin, the direct substrate for ABA synthesis via ABA aldehyde (Schwartz et al., 1997, 2003; Xiong and Zhu, 2003). Increasing evidence suggest that the endogenous ABA level is fine-tuned by differential regulation of the multiple steps of ABA biosynthesis (Seo and Koshiba, 2002; Nambara and Marion-Poll, 2005; Destefano-Beltrán et al., 2006; Thompson et al., 2007; Rodríguez-Gacio et al., 2009; Supplemental Fig. S1).The xanthophyll cycle (light-dependent reversible conversion between violaxanthin and zeaxanthin) is involved in photoprotection in PSII by regulating the nonradiative dissipation of excess absorbed light energy as heat (Gilmore et al., 1994). Mutants with defects in the xanthophyll cycle exhibit a weak photoprotective ability and produce ROS such as hydrogen peroxide (H₂O₂) when the absorption of light energy exceeds that consumed for photosynthesis (Niyogi, 1999). Under dehydration stress, electrons at a high energy state can easily form ROS, which are toxic to proteins, DNA, and lipids (Mittler, 2002; Apel and Hirt, 2004). However, plants have evolved a variety of biochemical and physiological mechanisms to scavenge ROS, thus maintaining a balance between ROS production and scavenging (Mittler et al., 2004).An association between the xanthophyll cycle and stress tolerance has been reported in plants. In Arabidopsis, overexpression of a bacterial BCH gene caused a specific 2-fold increase in the size of the xanthophyll cycle and enhanced photooxidative tolerance (Davison et al., 2002). Constitutive overexpression of a bacterial BCH gene, crtZ, in tobacco (Nicotiana tabacum) led to increased zeaxanthin synthesis and enhanced UV light tolerance (Götz et al., 2002). In Arabidopsis, zeaxanthin synthesis can be catalyzed by both heme-type CYP97 hydroxylases LUT1 and LUT5 and non-heme-type hydroxylases BCH1 and BCH2, and these two types exhibit some overlapping activities (Tian et al., 2003, 2004; Kim and DellaPenna, 2006). In contrast to the intensive molecular and genetic studies of BCH in Arabidopsis, the counterpart in economically important crops such as rice has not been identified.In this study, we characterized the rice drought-sensitive mutant dsm2, impaired in the gene DSM2 encoding a BCH. Our results demonstrate that DSM2 acts as a putative enzyme catalyzing the biosynthesis of zeaxanthin, one of the precursors of ABA that participates in the process of NPQ. Decreases of NPQ, maximal efficiency of PSII photochemistry (F_v/F_m), xanthophylls, and ABA in the dsm2 mutant suggest that the drought hypersensitivity of dsm2 is due to the combination of impairments in the xanthophyll cycle and ABA synthesis under drought stress conditions. DSM2 overexpression lines, possessing high F_v/F_m and NPQ, showed significantly improved drought resistance at both seedling and reproductive stages. Furthermore, our results imply that DSM2 may be the major member of the BCH family in rice for controlling zeaxanthin synthesis in response to dehydration stresses.     

Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin β-Aryl Ether Cleavage Pathway from Sphingobium sp. SYK-6

There has been great progress in the development of technology for the conversion of lignocellulosic biomass to sugars and subsequent fermentation to fuels. However, plant lignin remains an untapped source of materials for production of fuels or high value chemicals. Biological cleavage of lignin has been well characterized in fungi, in which enzymes that create free radical intermediates are used to degrade this material. In contrast, a catabolic pathway for the stereospecific cleavage of β-aryl ether units that are found in lignin has been identified in Sphingobium sp. SYK-6 bacteria. β-Aryl ether units are typically abundant in lignin, corresponding to 50–70% of all of the intermonomer linkages. Consequently, a comprehensive understanding of enzymatic β-aryl ether (β-ether) cleavage is important for future efforts to biologically process lignin and its breakdown products. The crystal structures and biochemical characterization of the NAD-dependent dehydrogenases (LigD, LigO, and LigL) and the glutathione-dependent lyase LigG provide new insights into the early and late enzymes in the β-ether degradation pathway. We present detailed information on the cofactor and substrate binding sites and on the catalytic mechanisms of these enzymes, comparing them with other known members of their respective families. Information on the Lig enzymes provides new insight into their catalysis mechanisms and can inform future strategies for using aromatic oligomers derived from plant lignin as a source of valuable aromatic compounds for biofuels and other bioproducts.     

Alzheimer’s Disease Risk Polymorphisms Regulate Gene Expression in the ZCWPW1 and the CELF1 Loci

Late onset Alzheimer’s disease (LOAD) is a genetically complex and clinically heterogeneous disease. Recent large-scale genome wide association studies (GWAS) have identified more than twenty loci that modify risk for AD. Despite the identification of these loci, little progress has been made in identifying the functional variants that explain the association with AD risk. Thus, we sought to determine whether the novel LOAD GWAS single nucleotide polymorphisms (SNPs) alter expression of LOAD GWAS genes and whether expression of these genes is altered in AD brains. The majority of LOAD GWAS SNPs occur in gene dense regions under large linkage disequilibrium (LD) blocks, making it unclear which gene(s) are modified by the SNP. Thus, we tested for brain expression quantitative trait loci (eQTLs) between LOAD GWAS SNPs and SNPs in high LD with the LOAD GWAS SNPs in all of the genes within the GWAS loci. We found a significant eQTL between rs1476679 and PILRB and GATS, which occurs within the ZCWPW1 locus. PILRB and GATS expression levels, within the ZCWPW1 locus, were also associated with AD status. Rs7120548 was associated with MTCH2 expression, which occurs within the CELF1 locus. Additionally, expression of several genes within the CELF1 locus, including MTCH2, were highly correlated with one another and were associated with AD status. We further demonstrate that PILRB, as well as other genes within the GWAS loci, are most highly expressed in microglia. These findings together with the function of PILRB as a DAP12 receptor supports the critical role of microglia and neuroinflammation in AD risk.     

The Effect of “Ferrodex”, Vitamin A and “Rovimix β-Carotene” on the Iron and Copper Levels in Blood Plasma of Calves

In 8 Versuchen wurde der Gärungsverlauf bei der Silierung von nitratarmem Grünfutter von Welschem Weidelgras, Knaulgras und Gras‐Leguminosen‐Gemenge geprüft. Aus den Ergebnissen geht hervor, daß bei Fehlen von Nitrat im Grünfutter bereits zu Gärbeginn Buttersäure entsteht, parallel zur Milchsäuregärung, auch in leicht vergärbarem Grünfutter. Diese frühzeitige Buttersäurebildung ist mit dem Fehlen von Nitrat als natürlicher Clostridieninhibitor zu erklären. Die Clostridienentwicklung verläuft zu Gärbeginn demnach wesentlich schneller als bisher angenommen wurde. Offensichtlich dienen leicht lösliche Kohlenhydrate als Substrat für die Buttersäurebildung. In nitratarmem Grünfutter werden deshalb Clostridien als Nahrungskonkurrenten für die Milchsäurebakterien wirksam. Aminosäuren werden zu Gärbeginn nicht abgebaut. Trotz z.T. hoher Buttersäuregehalte sind die Ammoniakgehalte gering. Höhere Homologe der Buttersäure fehlen. Die Milchsäuregärung erreicht trotz hoher Zuckergehalte im Grünfutter meist nur ein begrenztes Ausmaß. Es werden Unterschiede in der Vergärbarkeit der Kohlen‐hydratfraktion zwischen den Gräsern angenommen.     

Gene organization and plasticity of the β-lactam genes in different filamentous fungi

The genes pcbAB, pcbC and penDE encoding enzymes that catalyze the three steps of the penicillin biosynthesis have been cloned from Penicillium chrysogenum and Aspergillus nidulans. They are located in a cluster in Penicillium chrysogenum, Penicillium notatum, Aspergillus nidulans and Penicillium nalgiovense. The three genes are clustered in chromosome I (10.4 Mb) of P. chrysogenum, in chromosome II of P. notatum (9.6 Mb) and in chromosome VI (3.0 Mb) of A. nidulans. The cluster of the penicillin biosynthetic genes is amplified in strains with high level of antibiotic production. About five to six copies of the cluster are present in the AS-P-78 strain and 11 to 14 copies in the E1 strain (an industrial isolate), whereas only one copy is present in the wild type (NRRL 1951) strain and in the low producer Wis 54-1255 strain. The amplified region in strains AS-P-78 and E1 is arranged in tandem repeats of 106.5 or 57.6-kb units, respectively. In Acremonium chrysogenum the genes involved in cephalosporin biosynthesis are separated in at least two clusters. The pcbAB and pcbC genes are linked in the so-called early cluster of genes involved in the cephalosporin biosynthesis. The late cluster, which includes the cefEF and cefG genes, is involved in the last steps of cephalosporin biosynthesis. The early cluster was located in chromosome VII (4.6 Mb) in the C10 strain and the late cluster in chromosome I (2.2 Mb). Both clusters are present in a single copy in the A. chrysogenum genome, in the wild-type and in the high cephalosporin-producing C10 strains.