High-level Production of Isoleucine and Fusel alcohol by expression of the Feedback Inhibition-insensitive Threonine deaminase in Saccharomyces cerevisiae

A variety of the yeast Saccharomyces cerevisiae with intracellular accumulation of isoleucine (Ile) would be a promising strain for developing a distinct kind of sake, a traditional Japanese alcoholic beverage, because Ile-derived volatile compounds have a great impact on the flavor and taste of fermented foods. In this study, we isolated an Ile-accumulating mutant (strain K9-I48) derived from a diploid sake yeast of S. cerevisiae by conventional mutagenesis. Strain K9-I48 carries a novel mutation in the ILV1 gene encoding the His480Tyr variant of threonine deaminase (TD). Interestingly, the TD activity of the His480Tyr variant was markedly insensitive to feedback inhibition by Ile, but was not upregulated by valine, leading to intracellular accumulation of Ile and extracellular overproduction of 2-methyl-1-butanol, a fusel alcohol derived from Ile, in yeast cells. The present study demonstrated for the first time that the conserved histidine residue located in a linker region between two regulatory domains is involved in allosteric regulation of TD. Moreover, sake brewed with strain K9-I48 contained 2-3 times more 2-methyl-1-butanol and 2-methylbutyl acetate than sake brewed with the parent strain. These findings are valuable for the engineering of TD to increase the productivity of Ile and its derived fusel alcohols. IMPORTANCE Fruit-like flavors of isoleucine-derived volatile compounds, 2-methyl-1-butanol (2MB) and its acetate ester, contribute to a variety of the flavors and tastes of alcoholic beverages. Besides its value as aroma components in foods and cosmetics, 2MB has attracted significant attention as second-generation biofuels. Threonine deaminase (TD) catalyzes the first step in isoleucine biosynthesis and its activity is subject to feedback inhibition by isoleucine. Here, we isolated an isoleucine-accumulating sake yeast mutant and identified a mutant gene encoding a novel variant of TD. The variant TD exhibited much less sensitivity to isoleucine, leading to higher production of 2MB as well as isoleucine than the wild-type TD. Furthermore, sake brewed with a mutant yeast expressing the variant TD contained more 2MB and its acetate ester than that brewed with the parent strain. These findings will contribute to the development of superior industrial yeast strains for high-level production of isoleucine and its related fusel alcohols.

Credit Constraints and Increased Firm-level Production Fragmentation: Evidence from India

Previous studies have underlined various rationales for production fragmentation from wage differentials, decreased trade costs, access to specialized skills and resources, access to new markets, and benevolent government policies, to technological advancement. However, the idea that a firm’s financing structure can influence its production structure remains less explored, more so empirically. Firms that are financially constrained find it difficult to complete the entire production process in-house and therefore tend to resort to production fragmentation. The current study investigates this link between the extent of credit constraints faced by firms and their outsourcing behavior using data from Indian manufacturing firms over a period of ten years. We also separately study this linkage for firms that tend to export more vis-à-vis firms that export less, to ascertain if increased exporting have relaxed the financial constraint of the firms. The results confirm the positive effect of credit constraints on outsourcing behavior. For a robustness check, subsample regressions and alternative measures of constraints are also analyzed. The study has important policy implications for developing countries such as India, where outsourcing may prove to be a profitable reorganization strategy for firms that are financially constrained.

Plant-Based Vaccines: Antigen Design, Diversity, and Strategies for High Level Production

Vaccines for human use have conventionally been developed by the production of (1) microbial pathogens in eggs or mammalian cells that are then inactivated, or (2) by the production of pathogen proteins in mammalian and insect cells that are purified for vaccine formulation, as well as, more recently, (3) by using RNA or DNA fragments from pathogens. Another approach for recombinant antigen production in the last three decades has been the use of plants as biofactories. Only have few plant-produced vaccines been evaluated in clinical trials to fight against diseases, of which COVID-19 vaccines are the most recent to be FDA approved. In silico tools have accelerated vaccine design, which, combined with transitory antigen expression in plants, has led to the testing of promising prototypes in pre-clinical and clinical trials. Therefore, this review deals with a description of immunoinformatic tools and plant genetic engineering technologies used for antigen design (virus-like particles (VLP), subunit vaccines, VLP chimeras) and the main strategies for high antigen production levels. These key topics for plant-made vaccine development are discussed and perspectives are provided.

Electronic Current Transformer Defects Statistical Analysis of Intelligent Substation

In order to master operation status of the electronic current transformer(ECT), application investigation and defects statistical analysis were conducted on ECT of 110(66)kV~1000kV applied in smart substation of the State Grid Corporation of China(SGCC). The defect location, defect cause and defect type of ECT with different principles are analyzed. The results indicate that the defects of ECT are mainly concentrated in the acquisition unit, the defect rate is closely related to the reliability of electronic components, the technical level, production process and quality control of the manufacturer. In view of the typical defect analysis, the corresponding solution measures are put forward to provide reference for the subsequent research and application of ECT.

A series-connected switched source and an H-bridge based multilevel inverter

An inverter circuit is promoted in this paper, using series-connected switched dc sources along with an H-bridge circuit with optimized circuit elements like switching devices and diode clamped (DC) sources. This configuration uses DC supplies that can be strung together in series to create a significant voltage level. This topology consists of two parts, namely: 1) level production part and 2) polarity production part. The combination of some of the dc sources and switching devices completes the level production part. The H-bridge in the presented structure produces the polarity generation part. The DC-link capacitors are not needed in this design. There is a full presentation of the operating modes and modeling process of the proposed converter. Finally, in the MATLAB/SIMULINK setting the proposed topology is simulated and output current and voltage results have been examined.