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High-level, recombinant production of membrane-integrated proteins in Escherichia coli is extremely relevant for many purposes, but has also been proven challenging. Here we study a combination of transcriptional fine-tuning in E. coli LEMO21(DE3) with different codon usage algorithms for heterologous production of membrane proteins. The overexpression of 6 different membrane proteins is compared for the wild-type gene codon usage variant, a commercially codon-optimized variant, and a codon-harmonized variant. We show that transcriptional fine-tuning plays a major role in improving the production of all tested proteins. Moreover, different codon usage variants significantly improved production of some of the tested proteins. However, not a single algorithm performed consistently best for the membrane-integrated production of the 6 tested proteins. In conclusion, for improving heterologous membrane protein production in E. coli, the major effect is accomplished by transcriptional tuning. In addition, further improvements may be realized by attempting different codon usage variants, such as codon harmonized variants, which can now be easily generated through our online Codon Harmonizer tool.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components and many products in biotechnology. The efficient synthesis of many of these products, however, is limited by the rate of NADPH regeneration. Hence, a thorough understanding of the reactions involved in the generation of NADPH is required to increase its turnover through rational strain improvement. Traditionally, the main engineering targets for increasing NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA) cycle. However, the importance of alternative NADPH-generating reactions has recently become evident. In the current review, the major canonical and non-canonical reactions involved in the production and regeneration of NADPH in prokaryotes are described, and their key enzymes are discussed. In addition, an overview of how different enzymes have been applied to increase NADPH availability and thereby enhance productivity is provided.

Novel microbial cultivation platforms are of increasing interest to researchers in academia and industry. The development of materials with specialized chemical and geometric properties has opened up new possibilities in the study of previously unculturable microorganisms and has facilitated the design of elegant, high-throughput experimental set-ups. Within the context of the international Genetically Engineered Machine (iGEM) competition, we set out to design, manufacture, and implement a flow device that can accommodate multiple growth platforms, that is, a silicon nitride based microsieve and a porous aluminium oxide based microdish. It provides control over (co-)culturing conditions similar to a chemostat, while allowing organisms to be observed microscopically. The device was designed to be affordable, reusable, and above all, versatile. To test its functionality and general utility, we performed multiple experiments with Escherichia coli cells harboring synthetic gene circuits and were able to quantitatively study emerging expression dynamics in real-time via fluorescence microscopy. Furthermore, we demonstrated that the device provides a unique environment for the cultivation of nematodes, suggesting that the device could also prove useful in microscopy studies of multicellular microorganisms.

Reduced nicotinamide adenine nucleotide phosphate (NADPH), which is one of the key cofactors in the metabolic network, plays an important role in the biochemical reactions, and physiological function of amino acid-producing strains. The manipulation of NADPH availability and form is an efficient and easy method of redirecting the carbon flux to the amino acid biosynthesis in industrial strains. In this review, we survey the metabolic mode of NADPH. Furthermore, we summarize the research developments in the understanding of the relationship between NADPH metabolism and amino acid biosynthesis. Detailed strategies to manipulate NADPH availability are addressed based on this knowledge. Finally, the uses of NADPH manipulation strategies to enhance the metabolic function of amino acid-producing strains are discussed. [ABSTRACT FROM AUTHOR]

High-level, recombinant production of membrane-integrated proteins in Escherichia coli is extremely relevant for many purposes, but has also been proven challenging. Here we study a combination of transcriptional fine-tuning in E. coli LEMO21(DE3) with different codon usage algorithms for heterologous production of membrane proteins. The overexpression of 6 different membrane proteins is compared for the wild-type gene codon usage variant, a commercially codon-optimized variant, and a codon-harmonized variant. We show that transcriptional fine-tuning plays a major role in improving the production of all tested proteins. Moreover, different codon usage variants significantly improved production of some of the tested proteins. However, not a single algorithm performed consistently best for the membrane-integrated production of the 6 tested proteins. In conclusion, for improving heterologous membrane protein production in E. coli, the major effect is accomplished by transcriptional tuning. In addition, further improvements may be realized by attempting different codon usage variants, such as codon harmonized variants, which can now be easily generated through our online Codon Harmonizer tool. [ABSTRACT FROM AUTHOR]

The euryarchaeon Thermococcus kodakarensis is a well-characterized anaerobic hyperthermophilic heterotroph and due to the availability of genetic engineering systems it has become one of the model organisms for studying Archaea. Despite this prominent role among the Euryarchaeota, no data about the ploidy level of this species is available. While polyploidy has been shown to exist in various Euryarchaeota, especially Halobacteria, the chromosome copy number of species belonging to one of the major orders within that phylum, i.e., the Thermococcales (including Thermococcus spp. and Pyrococcus spp.), has never been determined. This prompted us to investigate the chromosome copy number of T. kodakarensis. In this study, we demonstrate that T. kodakarensis is polyploid with a chromosome copy number that varies between 7 and 19 copies, depending on the growth phase. An apparent correlation between the presence of histones and polyploidy in Archaea is observed. [ABSTRACT FROM AUTHOR]