Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis

Background

In the last years, the biotechnological production of platform chemicals for fuel components has become a major focus of interest. Although ligno-cellulosic material is considered as suitable feedstock, the almost inevitable pretreatment of this recalcitrant material may interfere with the subsequent fermentation steps. In this study, the fungus Ustilago maydis was used to produce itaconic acid as platform chemical for the synthesis of potential biofuels such as 3-methyltetrahydrofuran. No studies, however, have investigated how pretreatment of ligno-cellulosic biomass precisely influences the subsequent fermentation by U. maydis. Thus, this current study aims to first characterize U. maydis in shake flasks and then to evaluate the influence of three exemplary pretreatment methods on the cultivation and itaconic acid production of this fungus. Cellulose enzymatically hydrolysed in seawater and salt-assisted organic-acid catalysed cellulose were investigated as substrates. Lastly, hydrolysed hemicellulose from fractionated beech wood was applied as substrate.

Results

U. maydis was characterized on shake flask level regarding its itaconic acid production on glucose. Nitrogen limitation was shown to be a crucial condition for the production of itaconic acid. For itaconic acid concentrations above 25 g/L, a significant product inhibition was observed. Performing experiments that simulated influences of possible pretreatment methods, U. maydis was only slightly affected by high osmolarities up to 3.5 osmol/L as well as of 0.1 M oxalic acid. The production of itaconic acid was achieved on pretreated cellulose in seawater and on the hydrolysed hemicellulosic fraction of pretreated beech wood.

Conclusion

The fungus U. maydis is a promising producer of itaconic acid, since it grows as single cells (yeast-like) in submerged cultivations and it is extremely robust in high osmotic media and real seawater. Moreover, U. maydis can grow on the hemicellulosic fraction of pretreated beech wood. Thereby, this fungus combines important advantages of yeasts and filamentous fungi. Nevertheless, the biomass pretreatment does indeed affect the subsequent itaconic acid production. Although U. maydis is insusceptible to most possible impurities from pretreatment, high amounts of salts or residues of organic acids can slow microbial growth and decrease the production. Consequently, the pretreatment step needs to fit the prerequisites defined by the actual microorganisms applied for fermentation.     

Clinical features and predictors for disease natural progression in adults with Pompe disease: a nationwide prospective observational study

Background

Due partly to physicians’ unawareness, many adults with Pompe disease are diagnosed with great delay. Besides, it is not well known which factors influence the rate of disease progression, and thus disease outcome. We delineated the specific clinical features of Pompe disease in adults, and mapped out the distribution and severity of muscle weakness, and the sequence of involvement of the individual muscle groups. Furthermore, we defined the natural disease course and identified prognostic factors for disease progression.

Methods

We conducted a single-center, prospective, observational study. Muscle strength (manual muscle testing, and hand-held dynamometry), muscle function (quick motor function test), and pulmonary function (forced vital capacity in sitting and supine positions) were assessed every 3–6 months and analyzed using repeated-measures ANOVA.

Results

Between October 2004 and August 2009, 94 patients aged between 25 and 75 years were included in the study. Although skeletal muscle weakness was typically distributed in a limb-girdle pattern, many patients had unfamiliar features such as ptosis (23%), bulbar weakness (28%), and scapular winging (33%). During follow-up (average 1.6 years, range 0.5-4.2 years), skeletal muscle strength deteriorated significantly (mean declines of ?1.3% point/year for manual muscle testing and of ?2.6% points/year for hand-held dynamometry; both p<0.001). Longer disease duration (>15 years) and pulmonary involvement (forced vital capacity in sitting position <80%) at study entry predicted faster decline. On average, forced vital capacity in supine position deteriorated by 1.3% points per year (p=0.02). Decline in pulmonary function was consistent across subgroups. Ten percent of patients declined unexpectedly fast.

Conclusions

Recognizing patterns of common and less familiar characteristics in adults with Pompe disease facilitates timely diagnosis. Longer disease duration and reduced pulmonary function stand out as predictors of rapid disease progression, and aid in deciding whether to initiate enzyme replacement therapy, or when.

     

Suitability of Anabaena PCC7120 expressing mosquitocidal toxin genes from Bacillus thuringiensis subsp. israelensis for biotechnological application

We present evidence that Anabaena PCC7120 (A.7120) strains expressing mosquitocidal toxin genes from Bacillus thuringiensis subsp. israelensis (Bti) have a strong potential for biotechnological application. Characterization of two 4-year-old recombinant A.7120 clones constructed previously in our laboratory [clone 7 and clone 11, each carrying three Bti genes (cry4Aa, cry11Aa, and p20)] revealed three facts. First, the Bti genes were stable in A.7120 even in the absence of antibiotic selection when the genes were integrated in the chromosome (in clone 11); and the genes were also stable as plasmid-borne constructs (in clone 7), provided the cultures were maintained under continued selection. Second, clone 7 (kept under selection) and clone 11 (either kept or not kept under selection) continued to be mosquitocidal through 4 years of culture. Third, growth of the recombinant clones was comparable to the wild type under optimal growth conditions, indicating that growth was not compromised by the expression of toxin genes. These results clear the way for the development of mass production techniques for A.7120 strains expressing Bti toxin genes.     

Complete Sequence and Organization of pBtoxis, the Toxin-Coding Plasmid of Bacillus thuringiensis subsp. israelensis

The entire 127,923-bp sequence of the toxin-encoding plasmid pBtoxis from Bacillus thuringiensis subsp. israelensis is presented and analyzed. In addition to the four known Cry and two known Cyt toxins, a third Cyt-type sequence was found with an additional C-terminal domain previously unseen in such proteins. Many plasmid-encoded genes could be involved in several functions other than toxin production. The most striking of these are several genes potentially affecting host sporulation and germination and a set of genes for the production and export of a peptide antibiotic.     

Effect of Accessory Proteins P19 and P20 on Cytolytic Activity of Cyt1Aa from Bacillus thuringiensis subsp. israelensis in Escherichia coli

The gene coding for the accessory protein P19 of Bacillus thuringiensis subsp. israelensis was expressed in Escherichia coli and its product was characterized. To investigate its putative role in δ-endotoxin crystallization as a P20-like polypeptide, each of the two encoding genes, p20 and p19, was cloned for inducible expression coordinatively with cyt1Aa. The latter is known to kill its transgenic host. P20 but not P19 stabilized Cyt1Aa and protected the host cells from its lethal effect. Neither GroEL nor GroES, expressed in trans, affected Cyt1Aa as did P20. The function of P20 is thus more specific than that of the chaperones, but that of P19 remains enigmatic. The correct sequence of p19, confirmed in all five isolates of B. thuringiensis subsp. israelensis, does not explain the slow electrophoretic mobility of its 179 amino acids product. Received: 5 March 2001 / Accepted: 3 April 2001     

Neural computing in discovering RNA interactions.

High-order RNA structures are involved in regulating many biological processes; various algorithms have been designed to predict them. Experimental methods to probe such structures and to decipher the results are tedious. Artificial intelligence and the neural network approach can support the process of discovering RNA structures. Secondary structures of RNA molecules are probed by autoradiographing gels, separating end-labeled fragments generated by base-specific RNases. This process is performed in both conditions, denaturing (for sequencing purposes) and native. The resultant autoradiograms are scanned using line-detection techniques to identify the fragments by comparing the lines with those obtained by 'alkaline ladders'. The identified paired bases are treated by either one of two methods to find the foldings which are consistent with the RNases' 'cutting' rules. One exploits the maximum independent set algorithm; the other, the planarization algorithm. They require, respectively, n and n2 processing elements, where n is the number of base pairs. The state of the system usually converges to the near-optimum solution within about 500 iteration steps, where each processing element implements the McCulloch-Pitts binary neuron. Our simulator, based on the proposed algorithm, discovered a new structure in a sequence of 38 bases, which is more stable than that formerly proposed.