Genetic Modification of Carbon Catabolite Repression in Trichoderma reesei for Improved Protein Production

The cellulase and hemicellulase genes of the filamentous fungus Trichoderma reesei have been shown to be under carbon catabolite repression mediated by the regulatory gene cre1. In this study, strains were constructed in which the cre1 gene was either completely removed or replaced by a truncated mutant variant, cre1-1, found previously in the Rut-C30 mutant strain with enhanced enzyme production capability. The T. reesei transformants with either deletion or truncation of cre1 had clearly altered colony morphology compared with the parental strains, forming smaller colonies and fewer aerial hyphae and spores. Liquid cultures in a medium with glucose as a carbon source showed that the transformants were derepressed in cellulase and hemicellulase production. Interestingly, they also produced significantly elevated levels of these hydrolytic enzymes in fermentations carried out in a medium inducing the hydrolase genes. This suggests that cre1 acts as a modulator of cellulase and hemicellulase gene expression under both noninducing and inducing conditions. There was no phenotypic difference between the Δcre1 and cre1-1 mutant strains in any of the experiments done, indicating that the cre1-1 gene is practically a null allele. The results of this work indicate that cre1 is a valid target gene in strain engineering for improved enzyme production in T. reesei.The filamentous fungus Trichoderma reesei (Hypocrea jecorina) produces large amounts of extracellular enzymes. The majority of the secreted proteins are various plant polymer-degrading enzymes; the most abundant of these enzymes are the cellobiohydrolases and endoglucanases that act synergistically to break down cellulose. This fungus has been used as a production host for various industrial enzymes, including products tailored for textile, feed, food, and pulp and paper applications (6, 10). It has been reported that protein production levels in the industrial T. reesei process exceed 100 g/liter (7).The major cellulase and hemicellulase genes are regulated in a coordinate manner by the carbon source available (2, 9, 14). Cellulose and other plant materials and other substances (for example, lactose) induce the expression of cellulase and hemicellulase genes, while glucose acts as a repressing carbon source. Several genes coding for regulators of cellulase and hemicellulase expression have been isolated. These include CREI mediating carbon catabolite repression, the repressor ACEI, the activator ACEII, the CCAAT binding complex Hap2/3/5 (reviewed in references 2, 17, and 27) and the activator XYRI (29). The CREI protein has sequence similarity with other fungal proteins mediating glucose repression, such as Aspergillus nidulans CREA (8) and Saccharomyces cerevisiae MIG1 and RGR1 (22). In T. reesei, glucose repression has been shown to occur upon binding of CREI to specific sequences in the cbh1 promoter (13). A mutant cre1 gene (cre1-1) encoding a truncated form of CREI has been isolated from the hypercellulolytic T. reesei strain Rut-C30, which is capable of cellulase and hemicellulase production on glucose-containing media. Further evidence for the function of CREI in glucose repression was obtained by complementation of the cre1-1 mutation of Rut-C30 by the wild-type cre1 gene, which restored the glucose-repressed phenotype of the strain (15).In this paper, we wanted to address three questions. (i) What is the effect of cre1 mutations in the wild-type background? (ii) Is cre1-1 a null mutation? (iii) Can enzyme production be further improved by cre1 deletion in an industrial production strain improved greatly by mutagenesis and screening programs? Therefore, we introduced cre1-1 allele and cre1 deletion to the wild-type strain QM6a and the cre1 deletion into the industrial strain VTT-D-80133 and studied the effects of these mutations on enzyme production.     

Egg-laying tactic in Phyllomorpha laciniata in the presence of parasitoids

Female insects are expected to choose oviposition sites that have the best conditions for offspring development and survival. Natural enemies, such as predators and parasitoids, may have a strong influence on the selection of oviposition substrates by phytophagous insects. The golden egg bug, Phyllomorpha laciniata (Villers) (Heteroptera: Coreidae) has an unusual reproductive strategy. Females mainly use conspecifics, both males and other females, as egg-laying substrates, but occasionally they oviposit on plants as well. Survival of the eggs is higher when eggs are carried by conspecifics than when they are laid on plants, due to predation and parasitism. We investigated egg-laying behavior in the forced presence of the egg parasitoid Gryon bolivari (Giard) (Hymenoptera: Scelionidae). Specifically, we studied whether females provide egg protection by avoiding oviposition under the risk of egg parasitization. We expected a lower oviposition rate under parasitoid presence, and the eggs, if any, to be placed preferably on conspecifics and not on plants, thus ensuring higher survival of the progeny. The results show that P. laciniata 's egg-laying rate was lower when they were enclosed with parasitoids than when parasitoids were absent, especially when plants were the only substrate to oviposit on. Moreover, females showed strong preference for laying eggs on conspecifics rather than on plants. Egg-laying in P. laciniata appears to be not only influenced by the availability of conspecifics, but also by the presence of egg parasitoids. This indicates that females may be able to detect G. bolivari and avoid oviposition when parasitoids are present. We discuss the possibility of conspecifics as enemy-free space.     

The Contribution of Polystyrene Nanospheres towards the Crystallization of Proteins

Background

Protein crystallization is a slow process of trial and error and limits the amount of solved protein structures. Search of a universal heterogeneous nucleant is an effort to facilitate crystallizability of proteins.

Methodology

The effect of polystyrene nanospheres on protein crystallization were tested with three commercial proteins: lysozyme, xylanase, xylose isomerase, and with five research target proteins: hydrophobins HFBI and HFBII, laccase, sarcosine dimethylglycine N-methyltransferase (SDMT), and anti-testosterone Fab fragment 5F2. The use of nanospheres both in screening and as an additive for known crystallization conditions was studied. In screening, the addition of an aqueous solution of nanosphere to the crystallization drop had a significant positive effect on crystallization success in comparison to the control screen. As an additive in hydrophobin crystallization, the nanospheres altered the crystal packing, most likely due to the amphiphilic nature of hydrophobins. In the case of laccase, nanospheres could be used as an alternative for streak-seeding, which insofar had remained the only technique to produce high-diffracting crystals. With methyltransferase SDMT the nanospheres, used also as an additive, produced fewer, larger crystals in less time. Nanospheres, combined with the streak-seeding method, produced single 5F2 Fab crystals in shorter equilibration times.

Conclusions

All in all, the use of nanospheres in protein crystallization proved to be beneficial, both when screening new crystallization conditions to promote nucleation and when used as an additive to produce better quality crystals, faster. The polystyrene nanospheres are easy to use, commercially available and close to being inert, as even with amphiphilic proteins only the crystal packing is altered and the nanospheres do not interfere with the structure and function of the protein.     

Genome-wide Association Study in a High-Risk Isolate for Multiple Sclerosis Reveals Associated Variants in STAT3 Gene

Genetic risk for multiple sclerosis (MS) is thought to involve both common and rare risk alleles. Recent GWAS and subsequent meta-analysis have established the critical role of the HLA locus and identified new common variants associated to MS. These variants have small odds ratios (ORs) and explain only a fraction of the genetic risk. To expose potentially rare, high-impact alleles, we conducted a GWAS of 68 distantly related cases and 136 controls from a high-risk internal isolate of Finland with increased prevalence and familial occurrence of MS. The top 27 loci with p < 10⁻⁴ were tested in 711 cases and 1029 controls from Finland, and the top two findings were validated in 3859 cases and 9110 controls from more heterogeneous populations. SNP (rs744166) within the STAT3 gene was associated to MS (p = 2.75 × 10⁻¹⁰, OR 0.87, confidence interval 0.83–0.91). The protective haplotype for MS in STAT3 is a risk allele for Crohn disease, implying that STAT3 represents a shared risk locus for at least two autoimmune diseases. This study also demonstrates the potential of special isolated populations in search for variants contributing to complex traits.     

Effects of ageing on motor unit activation patterns and reflex sensitivity in dynamic movements

Both contraction type and ageing may cause changes in H-reflex excitability. H reflex is partly affected by presynaptic inhibition that may also be an important factor in the control of MU activation. The purpose of the study was to examine age related changes in H-reflex excitability and motor unit activation patterns in dynamic and in isometric contractions. Ten younger (YOUNG) and 13 elderly (OLD) males performed isometric (ISO), concentric (CON) and eccentric (ECC) plantarflexions with submaximal activation levels (20% and 40% of maximal soleus surface EMG). Intramuscular EMG data was analyzed utilizing an intramuscular spike amplitude frequency histogram method. Average H/M ratio was always lowest in ECC (n.s.). Mean spike amplitude increased with activation level (P < .05), whereas no significant differences were found between contraction types. Both H-reflex excitability, which may be due to an increase in presynaptic inhibition, and mean spike frequency were higher in YOUNG compared to OLD. In OLD the mean spike frequency was significantly smaller in CON compared to ISO. Lack of difference in mean spike amplitude and frequency across contraction types in YOUNG would imply a similar activation strategy, whereas the lower frequency in dynamic contractions in OLD could be related to synergist muscle behavior.     

Admission neopterin and interleukin 12 concentrations in identifying infections in adult cancer patients

Differential diagnosis between infections and neoplastic fever is a common diagnostic problem. The utility of admission serum concentrations of neopterin and interleukin 12 (IL-12) was prospectively evaluated in this respect. The infection group (n=56) had a higher median neopterin value (12.8 nmol/l vs 4.0 nmol/l, P<0.001) and neopterin-to-IL-12 ratio (1.74 vs 0.11, P<0.001) than the non-infection group (n=36); the median IL-12 values were higher in the latter group (10.6 pg/ml vs 71.6 pg/ml, P=0.007). According to the area under the operating characteristics curves (AUC), especially neopterin (0.90), but also the neopterin-to-IL-12 ratio (0.79), was good at identifying bacteremia. However, in differentiating infections in general from neoplastic fever (n=10), the neopterin-to-IL-12 ratio was less powerful (0.64), though still better than neopterin (0.58) and clearly better than IL-12 (0.42). The present results show that the neopterin-to-IL-12 ratio, which reflects simultaneously both the ongoing infection and the tumour load, may have promising clinical implications for differential diagnosis between infections and neoplastic fever.     

Acid-base chemistry of vitamin B6 compounds in methanol

The enzymatic reactions involving pyridoxal 5-phosphate (PLP) can be simulated in solutions; thus, this system forms a favorable model for understanding the requirements of the enzymatic catalysis. We have studied in methanol protonic equilibria of the imines formed between PLP or salicylaldehyde (SA) and various amino acids, using UV and NMR spectroscopy. A glass electrode and an operationalpH* scale were used to control acidity. The first protonation of the phosphate of PLP imines can be detected by UV spectroscopy withpK* at 10.8, proved by [³¹P]-NMR. The second protonation of phosphate (pK* at 4.8) is accompanied by increased hydrolysis of the imines. The imines of aspartate deviate from the imines of nondicarboxylic amino acids indicating that the -carboxyl of aspartate is internally hydrogen-bonded. PLP-2-aminobutanol Schiff base does not show with [¹H]-NMR atpH* 7 separate peaks for ketoenamine-enolimine tautomers even at -90°C, SA-phenylalanine shows an unidentified absorption at 350–380 nm. This was tentatively assigned a trans structure.     

Difluoromethylornithine irreversibly inactivates ornithine decarboxylase of Pseudomonas aeruginosa, but does not inhibit the enzymes of Escherichia coli.

DL-alpha-Difluoromethylornithine, an enzyme-activated irreversible inhibitor of eukaryotic ornithine decarboxylase and consequently of putrescine biosynthesis, inhibited ornithine decarboxylase in enzyme extracts from Pseudomonas aeruginosa in a time-dependent manner t1/2 1 min, and also effectively blocked the enzyme activity in situ in the cell. Difluoromethylornithine, however, had no effect on the activity of ornithine decarboxylase assayed in enzyme extracts from either Escherichia coli or Klebsiella pneumoniae. However, the presence of the inhibitor in cell cultures did partially lower ornithine decarboxylase activity intracellularly in E. coli. Any decrease in the intracellular ornithine decarboxylase activity observed in E. coli and Pseudomonas was accompanied by a concomitant increase in arginine decarboxylase activity, arguing for a co-ordinated control of putrescine biosynthesis in these cells.     

Sexual selection for large male size in a polyandrous butterfly: the effect of body size on male versus female reproductive success in Pieris napi

Sexual selection theory predicts that the larger sex shouldbe that for which fitness increases at the faster rate withsize. In butterflies, as in most invertebrates, females areusually the larger sex, but previous comparative analysis hasshown that relative male size increases with female polyandryamong butterflies. In agreement with this pattern, males arelarger than females in the strongly polyandrous green-veinedwhite butterfly, Pieris napi L., and in this article we assessthe size dependence of reproductive success in both sexes. Inan experiment where virgin males and females were released inthe field, we found no strong association between size and malemating success. However, laboratory experiments showed thatthere was a strong correlation between size and the ejaculatethat the male delivered to the female at mating and that largeejaculates delayed female remating for a longer time comparedto small ejaculates. Moreover, female P. napi utilize male-derivednutrients received at mating to increase their fecundity. Hence,large males sire more offspring both by way of donating morenutrients to female egg production and by way of delaying femaleremating (given that the last male to mate with the female willfather most of the offspring). Laboratory experiments showedthat the association between size and fecundity was low, ornonexistent, among P. napi females allowed to mate only once.However, weak size dependence was found for polyandrous females.We hypothesize that size dependence of female fecundity maybe especially weak among polyandrous butterflies because a fundamentalsource of variation in fecundity relates to their ability tofind nutrient giving males, an ability which may be unrelatedto female size. According to this hypothesis there is a causalassociation between weak size dependence of female fecundityand polyandry, and a strong size dependence of male reproductivesuccess that may underlie the comparative pattern of positivecorrelation between relative male size and polyandry.