Metabolic engineering strategies for de novo biosynthesis of sterols and steroids in yeast

Steroidal compounds are of great interest in the pharmaceutical field, with steroidal drugs as the second largest category of medicine in the world. Advances in synthetic biology and metabolic engineering have enabled de novo biosynthesis of sterols and steroids in yeast, which is a green and safe production route for these valuable steroidal compounds. In this review, we summarize the metabolic engineering strategies developed and employed for improving the de novo biosynthesis of sterols and steroids in yeast based on the regulation mechanisms, and introduce the recent progresses in de novo synthesis of some typical sterols and steroids in yeast. The remaining challenges and future perspectives are also discussed.

Nanostructure of PMMA/MAM Blends Prepared by Out-of-Equilibrium (Extrusion) and Near-Equilibrium (Casting) Self-Assembly and Their Nanocellular or Microcellular Structure Obtained from CO2 Foaming

Blends of poly(methyl methacrylate) (PMMA) and a triblock copolymer poly(methyl methacrylate)-b-poly(butyl acrylate)-b-poly(methyl methacrylate) (MAM) have been obtained following both out-of-equilibrium (extrusion) and near-equilibrium (solvent casting) production routes. The self-assembly capability and the achievable nanostructures of these blends are analyzed by transmission electron microscopy (TEM) regarding their production route and potential for the achievement of nanocellular foams by CO2 gas dissolution foaming. The influence of the initial nanostructure of the solids on the obtained cellular structure of bulk and film samples is determined by high-resolution scanning electron microscopy (HRSEM) for diverse foaming conditions (saturation pressure, saturation temperature, and post-foaming stage), taking into account the required use of a foaming mold to achieve foams from films. Moreover, the influence of the nanostructuration on the presence of solid outer layers, typical of the selected foaming process, is addressed. Finally, consideration of a qualitative model and the obtained results in terms of nanostructuration, cellular structure, and foaming behavior, allow proposing a detailed cell nucleation, growth, and stabilization scheme for these materials, providing the first direct evidence of the cell nucleation happening inside the poly(butyl acrylate) phase in the PMMA/MAM blends.

A Method for Characterising the Influence of Casting Parameters on the Metallurgical Bonding of Copper and Steel Bimetals

Traditional casting technology offers two mayor drawbacks towards research activities. On the one hand, time and resources needed for every casting are rather high. The mould has to be able to withstand the high temperatures introduced by the melt and provide cooling for the cast part. Preparation and installation of measuring equipment therefore takes time. Additionally, due to the high mass of the mould when compared to the cast part, parameter variations are rather limited in their resulting effect on the temperature-time profile being one of the most prominent factors regarding cast quality. Especially when pouring by hand, variations in casting times and rates superimpose effects created intentionally. Therefore, a different process was advanced and evaluated, allowing to minimise some of the drawbacks mentioned before. The key idea is to drastically reduce casting size to the dimensions of one specimen and to apply a highly automated production route. As such, a mirror furnace was modified as to allow the processing of melt. Due to the specimens size, an adaption of mechanical testing equipment was performed and evaluated. As an example, copper-iron bimetal specimens were examined by light microscopy, micro hardness testing, nanoindentation as well as tensile and torsion testing. As the results were consistent, the newly introduced method can be applied successfully in casting research. This allows for highly reproducible results, reducing the uncertainty of temperature measurements of a specimen due to the distance between them. The possibility of separating influencing variables like maximum temperature and cooling rate allows an analysis of the casting process, which would require different moulds to do so in traditional casting methods. The next steps will be directed at a broader variety of metals processed and at a direct comparison between the new process route and traditional casting technology.

Kinetic Study on Microwave-Assisted Oligomerization of 1-Decene over a HY Catalyst

A promising production route for a high-quality base stock for lubricants is the oligomerization of high molecular-weight olefins in a high energy efficiency system. Oligomerization of 1-decene (C10) was conducted in a microwave-assisted system over a HY zeolite catalyst at different reaction temperatures and times. Higher reaction temperature resulted in increasing formation of dimers and trimers. The oligomerization reaction yielded 80% conversion, 54.2% dimer product, 22.3% trimer product and 3.4% heavier product at 483 K for a reaction time of 3 h. The best fit kinetic model for the dimerization reaction was formulated from an assumption of no vacant reaction sites. For the trimerization reaction, a molecule of dimer (C20) formed on the active site, interacted with a molecule of 1-decene in the bulk solution to form a molecule of trimer (C30). Apparent activation energies for the dimerization and trimerization reactions were 70.8 ± 0.8 and 83.6 ± 0.9 kJ/mol, respectively. The C13-NMR spectrum indicated that the oligomer product contained a significant portion of highly branched hydrocarbons, causing a substantial reduction in the viscosity index compared to conventional poly-alpha olefin lubricant (PAO).

Processing and Characterization of Self-Reducing Briquettes Made of Jarosite and Blast Furnace Sludges

Jarosite sludge coming from the hydrometallurgical zinc production route is a hazardous material, which is currently neutralized and landfilled by the so-called Jarofix® process. The present study aims to assess the mechanical and metallurgical properties of briquettes made of jarosite powder with blast furnace sludges, acting as a reductant material, to recover the iron oxide in the form of pig iron and produce an inert slag, increasing the recovery of materials considered as wastes nowadays. Starch was used as a binder (0, 5, 10 wt%), and two different briquetting pressure levels were used (20 and 40 MPa). The results show that briquetting without a binder is not desirable, as the agglomerating forces provided by pressure only are not sufficient, as the briquettes are very fragile and not handy. The binder addition increased noticeably the briquettes resistance, however, only little distinction between the 5 and 10 wt% levels were seen. The briquetting pressure, on the other hand, showed a bigger role on the cold mechanical properties of the bound briquettes. The briquettes pressed at 40 MPa reached an average compressive strength higher than 12 MPa and good abrasion and drop resistance were seen, also showing that their production with starch as a binder is feasible. A special remark is done regarding the roasting treatment of the jarosite powder before the briquetting process, as an undesirable compound (thenardite) was formed within some briquettes due to a non-uniform heating of the powder, which hindered the briquettes mechanical properties. Metallurgical properties open the possibility to use such briquettes for iron production in cupola furnaces. Graphical Abstract

Characterization of Komagataeibacter Isolate Reveals New Prospects on Waste Stream Valorization for Bacterial Cellulose Production

Komagataeibacter spp. have been used for the bioconversion of industrial wastes and lignocellulosic hydrolysates to bacterial cellulose (BC). Recently studies have demonstrated the capacity of Komagataeibacter spp. in the biotransformation of inhibitors found in lignocellulosic hydrolysates, aromatic lignin-derived monomers (LDMs) and acetate. In general, detoxification and BC synthesis from lignocellulosic inhibitors requires a carbon flow from acetyl-coA towards tricarboxylic acid and gluconeogenesis, respectively. However, the related molecular aspects have not yet been identified in Komagataeibacter spp. In this study, we isolated a cellulose producing bacteria capable of synthesizing BC in a minimal medium containing crude glycerol, a by-product from biodiesel production process. The isolate, affiliated to Komagataeibacter genus, synthesized cellulose in minimal medium containing glucose (3.3±0.3 g/L), pure glycerol (2.2±0.1 g/L) and crude glycerol (2.1±0.1 g/L). Genome assembly and annotation identified four copies of bacterial cellulose synthase operon and genes for redirecting the carbon from central metabolic pathway to gluconeogenesis. According to the genome annotations, a BC production route from acetyl-CoA, a central metabolic intermediate, was hypothesized and was validated using acetate. We identified that when K. rhaeticus ENS9b was grown in minimal medium supplemented with acetate, BC production was not observed. However, in presence of readily utilizable substrate, such as spent yeast hydrolysate, acetate supplementation improved BC synthesis.

Simulation of the Refining Process of Ultra-Low Carbon (ULC) Steel

The standard production route for mild steels for automotive purposes is still based on conventional continuous casting (CC) and hot strip rolling (HSR). The current trend towards the "zero-carbon car" will demand the abating of material emissions in the future. Thin slab casting and direct rolling (e.g., Arvedi endless strip production (ESP)) is an approach to reduce CO2 emissions by 50% compared to CC and HSR. One of the main limitations in applying ESP for the production of ultra-low carbon/interstitial free (ULC/IF) steels is clogging. Clogging is the blockage of the submerged entry nozzle due to the build-up of oxide layers or an oxide network. The high clogging sensitivity of IF steels results most probably from the FeTi addition, and hence, a general change of the deoxidation practice might be an option to overcome these problems. In the present work, the thorough refining process of ULC steel was simulated by addressing the different deoxidation routes and the influence of titanium (Ti) alloying on steel cleanness. The developed ladle furnace (LF) and the Ruhrstahl Heraeus (RH) refining models were applied to perform the simulation. Before the simulations, the models are briefly described and validated by the published industrial data.

Progress in Vinyl Acetate

Vinyl acetate is an organic chemical raw material of great industrial value. It is widely used in synthetic film, adhesive, coating and textile slurry. The production route of vinyl acetate is introduced, and the advantages and disadvantages of synthesis methods of vinyl acetate method, acetylene method, EDA cracking method, base synthesis method and Halcon method are compared. The method of carbide acetylene has high economic efficiency, low energy consumption and good environmental protection, proposing the development direction of ethylene acetate production technology.

68Ga-Radiolabeling and Pharmacological Characterization of a Kit-Based Formulation of the Gastrin-Releasing Peptide Receptor (GRP-R) Antagonist RM2 for Convenient Preparation of [68Ga]Ga-RM2

Background: [68Ga]Ga-RM2 is a potent Gastrin-Releasing Peptide-receptor (GRP-R) antagonist for imaging prostate cancer and breast cancer, currently under clinical evaluation in several specialized centers around the world. Targeted radionuclide therapy of GRP-R-expressing tumors is also being investigated. We here report the characteristics of a kit-based formulation of RM2 that should ease the development of GRP-R imaging and make it available to more institutions and patients. Methods: Stability of the investigated kits over one year was determined using LC/MS/MS and UV-HPLC. Direct 68Ga-radiolabeling was optimized with respect to buffer (pH), temperature, reaction time and shaking time. Conventionally prepared [68Ga]Ga-RM2 using an automated synthesizer was used as a comparator. Finally, the [68Ga]Ga-RM2 product was assessed with regards to hydrophilicity, affinity, internalization, membrane bound fraction, calcium mobilization assay and efflux, which is a valuable addition to the in vivo literature. Results: The kit-based formulation, kept between 2 °C and 8 °C, was stable for over one year. Using acetate buffer pH 3.0 in 2.5–5.1 mL total volume, heating at 100 °C during 10 min and cooling down for 5 min, the [68Ga]Ga-RM2 produced by kit complies with the requirements of the European Pharmacopoeia. Compared with the module production route, the [68Ga]Ga-RM2 produced by kit was faster, displayed higher yields, higher volumetric activity and was devoid of ethanol. In in vitro evaluations, the [68Ga]Ga-RM2 displayed sub-nanomolar affinity (Kd = 0.25 ± 0.19 nM), receptor specific and time dependent membrane-bound fraction of 42.0 ± 5.1% at 60 min and GRP-R mediated internalization of 24.4 ± 4.3% at 30 min. The [natGa]Ga-RM2 was ineffective in stimulating intracellular calcium mobilization. Finally, the efflux of the internalized activity was 64.3 ± 6.5% at 5 min. Conclusion: The kit-based formulation of RM2 is suitable to disseminate GRP-R imaging and therapy to distant hospitals without complex radiochemistry equipment.

Arthrospira platensis as a Feasible Feedstock for Bioethanol Production

In recent decades and to deal with the scarcity of fossil fuels, many studies have been developed in order to set up a sustainable biofuel production sector. This new sector must be efficient (high productivity), economically profitable (low production costs and therefore acceptable fuel prices), and ethical (low carbon balance, no competition with food resources). The production of bioethanol is based on the fermentation of reserve sugars, accumulated in the form of starch in microalgae and glycogen in cyanobacteria. The advantage of this bioenergy production route lies in the fact that the post-crop fermentation process is at the industrial stage since it has already been tested for many years for the production of bioethanol from agricultural resources. One of the most cultivated cyanobacteria is Arthrospira (“Spirulina”) and its production is also already at industrial scale. Depending on the cultivation conditions, this cyanobacteria is able to accumulate up to 65% DW (dry weight) of glycogen, making it a feasible feedstock for bioethanol production. The aim of this review is to provide a clear overview of these operating conditions for glycogen accumulation.