Increased production of low molecular weight recombinant proteins in Escherichia coli.

A general method for obtaining high-level production of low molecular weight proteins in Escherichia coli is described. This method is based on the use of a novel Met-Xaa-protein construction which is formed by insertion of a single amino acid residue (preferably Arginine or Lysine) between the N-terminal methionine and the protein of interest. The utility of this method is illustrated by examples for achieving high-level production of human insulin-like growth factor-1, human proinsulin, and their analogs. Furthermore, highly produced insulin-like growth factor-1 derivatives and human proinsulin analogs are converted to their natural sequences by removal of dipeptides with cathepsin C.     

Sclerotinia sclerotiorum growth and oxalic acid production on selected culture media

Abstract Two isolates of Sclerotinia sclerotiorum , the highly aggressive (B24) and the weakly aggressive (SS41), were grown on liquid media containing one of the following carbon sources: purified cell walls obtained from onion or sunflower, pectin, polygalacturonic acid, carboxymethylcellulose, xylan or arabinogalactan. Isolates were equally able to utilize these substrates for mycelial growth but differed in their ability to utilize them for oxalate production. B24 produces oxalic acid always to a substantial extent, SS41 only in traces. The poor ability to produce oxalic acid by SS41 seems to be due to a lower efficiency in the synthetic pathway.     

Contents of Free Adenine in Soybeans from Several Countries

Methionine auxotrophic mutants of Methylophilus methylotrophus AS1 expressing a mutant form of dapA (dapA24) encoding a dihydrodipicolinate synthase desensitized from feedback inhibition by L-lysine, and mutated lysE (lysE24) encoding the L-lysine exporter from Corynebacterium glutamicum 2256, produced higher amounts of L-lysine from methanol as sole carbon source than did other amino acid auxotrophic mutants. Especially, the M. methylotrophus 102 strain, carrying both dapA24 and lysE24, produced L-lysine in more than 1.5 times amounts higher than the parent. A single-base substitution was identified in this auxotroph in codon-329 of the open reading frame of metF, encoding 5,10-methylene-tetra-hydrofolate reductase. We constructed a metF disruptant mutant carrying both dapA24 and lysE24, and confirmed increases in L-lysine production. This is the first report to the effect that metF deficient increased L-lysine production in methylotroph.     

Decrease of the hydraulic conductivity of sand columns by Methanosarcina barkeri

The extent to which a methanogen can clog sand columns was examined: two permeameters packed with clean quartz sand were sterilized, saturated with water, inoculated with Methanosarcina barkeri and percolated under upward flow conditions. After approx. 5 months, the hydraulic conductivity of the sand had decreased to 3% and 25% of the highest values measured earlier. At that point, gas-filled regions in the sand were clearly visible through the transparent walls of the permeameters, and methane bubbles were continuously released from the columns into the effluent. Scanning electron microscopy observations and biomass assays indicated that cell mass accumulation did not contribute significantly to the observed decrease of the hydraulic conductivity. This decrease was therefore attributed to pore blocking due to the entrapment of methane bubbles.D. Sanchez de Lozada and P. Baveye are with the Department of Soil, Crop and Atmospheric Sciences, Bradfield Hall, Cornell University, Ithaca, NY 13853, USA; P. Vandevivere is with the College of Marine Studies, University of Delaware, Lewes, DE 19958, USA. S. Zinder is with the Department of Microbiology, Rice Hall, Cornell University, Ithaca, NY 14853, USA.     

Virus harvesting in perfusion culture: Choosing the right type of hollow fiber membrane

The use of bioreactors coupled to membrane-based perfusion systems enables very high cell and product concentrations in vaccine and viral vector manufacturing. Many virus particles, however, are not stable and either lose their infectivity or physically degrade resulting in significant product losses if not harvested continuously. Even hollow fiber membranes with a nominal pore size of 0.2 µm can retain much smaller virions within a bioreactor. Here, we report on a systematic study to characterize structural and physicochemical membrane properties with respect to filter fouling and harvesting of yellow fever virus (YFV; ~50 nm). In tangential flow filtration perfusion experiments, we observed that YFV retention was only marginally determined by nominal but by effective pore sizes depending on filter fouling. Evaluation of scanning electron microscope images indicated that filter fouling can be reduced significantly by choosing membranes with (i) a flat inner surface (low boundary layer thickness), (ii) a smooth material structure (reduced deposition), (iii) a high porosity (high transmembrane flux), (iv) a distinct pore size distribution (well-defined pore selectivity), and (v) an increased fiber wall thickness (larger effective surface area). Lowest filter fouling was observed with polysulfone (PS) membranes. While the use of a small-pore PS membrane (0.08 µm) allowed to fully retain YFV within the bioreactor, continuous product harvesting was achieved with the large-pore PS membrane (0.34 µm). Due to the low protein rejection of the latter, this membrane type could also be of interest for other applications, that is, recombinant protein production in perfusion cultures.     

SIRT1 regulates nuclear number and domain size in skeletal muscle fibers

Skeletal muscle fibers are giant multinucleated cells wherein individual nuclei govern the protein synthesis in a finite volume of cytoplasm; this is termed the myonuclear domain (MND). The factors that control MND size remain to be defined. In the present study, we studied the contribution of the NAD⁺‐dependent deacetylase, sirtuin 1 (SIRT1), to the regulation of nuclear number and MND size. For this, we isolated myofibers from mice with tissue‐specific inactivation (mKO) or inducible overexpression (imOX) of SIRT1 and analyzed the 3D organisation of myonuclei. In imOX mice, the number of nuclei was increased whilst the average MND size was decreased as compared to littermate controls. Our findings were the opposite in mKO mice. Muscle stem cell (satellite cell) numbers were reduced in mKO muscles, a possible explanation for the lower density of myonuclei in these mice; however, no change was observed in imOX mice, suggesting that other factors might also be involved, such as the functional regulation of stem cells/muscle precursors. Interestingly, however, the changes in the MND volume did not impact the force‐generating capacity of muscle fibers. Taken together, our results demonstrate that SIRT1 is a key regulator of MND sizes, although the underlying molecular mechanisms and the cause‐effect relationship between MND and muscle function remain to be fully defined.     

Geostrategic Supply Risk and Economic Importance as Drivers for Implementation of Industrial Ecology Measures in a Nitrogen Fertilizer Production Company

Among other concerns, safeguarding the supply chains of raw materials is an important task for industrial companies. Therefore, not surprisingly, the number of scientific publications concerning the evaluation of resource criticality has increased in recent years. However, it was noticed that currently published methodologies are too complex to be applied by industrial companies on a daily basis. For this reason, the need to develop a methodology that would allow not only assessing resource criticality, but could also be integrated into widely applied methodological frameworks as an additional driver to improve resource efficiency was identified. Geostrategic supply risk and economic importance were chosen as key indicators to analyze and assess relative resource criticality. The developed methodology was field tested by applying it to a resource‐intensive nitrogen fertilizer production company. Five scenarios for resource efficiency improvements, consisting of cleaner production and industrial symbiosis measures, were investigated. If all the proposed measures were implemented, consumption of natural gas would decrease by 3.552 million cubic meters per year (0.3% of the total consumption). However, not all identified measures contribute to a reduction of the overall criticality of resources for the production company. Nevertheless, the integration of criticality assessments into the widely applied methodologies for development and implementation of resource efficiency innovations is a valuable addition and should be included in the analysis for sustainable innovations and development.     

Enhanced production of total flavones from Inonotus baumii by multiple strategies

As one kind of important secondary metabolites produced by Inonotus baumii, flavones can be applied in food, medicine, and other industries due to their biological activities such as antioxidant, anticancer, and antibacterial activity. To enhance total flavone production in submerged fermentation of I. baumii, three different strategies, optimization of fermentation parameters by statistical designs including Plackett–Burman design and response surface methodology, addition of precursors and elicitors, and two-phase culture, were used. The production of total flavones (PTF) reached 1532.83?mg/L when the optimized medium was used. All precursors and elicitors can increase the PTF. The maximum PTF (2184.06?mg/L, up to 1.57-fold) was obtained with the addition of both AgNO₃ and glutathione in fermentation media. Interestingly, when 0.5% (w/v) DM130 macroporous resin as adsorbent was added to fermentation broth on day 4 of culture, the highest production reached 2407.79?mg/L with this two-phase culture strategy. These methods can be further applied to large-scale industrial production and broaden the application of flavones.