Alternative PCR-Based Approaches for Generation of Komagataella phaffii Strains.

Komagataella phaffii (Pichia pastoris) is a widely known microbial host for recombinant protein production and an emerging model organism in fundamental research. The development of new materials and techniques on this yeast improves heterologous protein synthesis. One of the most prominent ways to enhance protein production efficiency is to select K. phaffii strains with multiple expression cassettes and generate MutS strains using various vectors. In this study, we demonstrate approaches to expand the applications of pPICZ series vectors. Procedures based on PCR amplification and in vivo cloning allow rapid exchange of selectable markers. The combination of PCR amplification with split-marker-mediated transformation allows the development of K. phaffii MutS strains with two expression cassettes using pPICZ vectors. Both PCR-based approaches were applied to efficiently produce interleukin-2 mimetic Neo-2/15 in K. phaffii. The described techniques provide alternative ways to generate and improve K. phaffii strains without the need for obtaining new specific vectors or additional cloning of expression cassettes.

Effect of Methionine on Gene Expression in Komagataella phaffii Cells.

Komagataella phaffii yeast plays a prominent role in modern biotechnology as a recombinant protein producer. For efficient use of this yeast, it is essential to study the effects of different media components on its growth and gene expression. We investigated the effect of methionine on gene expression in K. phaffii cells using RNA-seq analysis. Several gene groups exhibited altered expression when K. phaffii cells were cultured in a medium with methanol and methionine, compared to a medium without this amino acid. Methionine primarily affects the expression of genes involved in its biosynthesis, fatty acid metabolism, and methanol utilization. The AOX1 gene promoter, which is widely used for heterologous expression in K. phaffii, is downregulated in methionine-containing media. Despite great progress in the development of K. phaffii strain engineering techniques, a sensitive adjustment of cultivation conditions is required to achieve a high yield of the target product. The revealed effect of methionine on K. phaffii gene expression is important for optimizing media recipes and cultivation strategies aimed at maximizing the efficiency of recombinant product synthesis.

In Vivo Production of RNA Aptamers and Nanoparticles: Problems and Prospects.

RNA aptamers are becoming increasingly attractive due to their superior properties. This review discusses the early stages of aptamer research, the main developments in this area, and the latest technologies being developed. The review also highlights the advantages of RNA aptamers in comparison to antibodies, considering the great potential of RNA aptamers and their applications in the near future. In addition, it is shown how RNA aptamers can form endless 3-D structures, giving rise to various structural and functional possibilities. Special attention is paid to the Mango, Spinach and Broccoli fluorescent RNA aptamers, and the advantages of split RNA aptamers are discussed. The review focuses on the importance of creating a platform for the synthesis of RNA nanoparticles in vivo and examines yeast, namely Saccharomyces cerevisiae, as a potential model organism for the production of RNA nanoparticles on a large scale.

Transcriptome Analysis Unveils the Effects of Proline on Gene Expression in the Yeast Komagataella phaffii.

Komagataella phaffii yeast is one of the most important biocompounds producing microorganisms in modern biotechnology. Optimization of media recipes and cultivation strategies is key to successful synthesis of recombinant proteins. The complex effects of proline on gene expression in the yeast K. phaffii was analyzed on the transcriptome level in this work. Our analysis revealed drastic changes in gene expression when K. phaffii was grown in proline-containing media in comparison to ammonium sulphate-containing media. Around 18.9% of all protein-encoding genes were differentially expressed in the experimental conditions. Proline is catabolized by K. phaffii even in the presence of other nitrogen, carbon and energy sources. This results in the repression of genes involved in the utilization of other element sources, namely methanol. We also found that the repression of AOX1 gene promoter with proline can be partially reversed by the deletion of the KpPUT4.2 gene.

Relative Reactivity of Benzothiophene-Fused Enediynes in the Bergman Cyclization.

To find promising analogues of naturally occurring enediyne antibiotics with a sufficient reactivity in the Bergman cyclization and moderately stable under isolation and storage, a scale of relative enediynes reactivity was created on the basis of calculated free activation energies for the Bergman cyclization within 12 known and new benozothiophene, benzene, and cinnoline annulated 9- and 10-membered enediynes. To verify the predicted reactivity/stability balance, three new carbocyclic enediynes fused to a benzothiophene core bearing 3,4,5-trimethoxybenzene, fluoroisopropyl, and isopropenyl substituents were synthesized using the Nicholas-type macrocyclization. It was confirmed that annulation of a 3,4,5-trimethoxybenzene moiety to a 10-membered enediyne macrocycle imparts high reactivity to an enediyne while also conferring instability under ambient temperature. Fluoroisopropyl-substituted 10-membered enediyne from the opposite end of the scale was found to be stable while moderately reactive in the Bergman cyclization. Along with the experimentally confirmed moderate reactivity (DSC kinetic studies), (fluoroisopropyl)enediyne showed a significant DNA damaging activity in plasmid cleavage assays comparable with the known anticancer drug Zeocin.

Acid phosphatases of budding yeast as a model of choice for transcription regulation research.

Acid phosphatases of budding yeast have been studied for more than forty years. This paper covers biochemical characteristics of acid phosphatases and different aspects in expression regulation of eukaryotic genes, which were researched using acid phosphatases model. A special focus is devoted to cyclin-dependent kinase Pho85p, a negative transcriptional regulator, and its role in maintaining mitochondrial genome stability and to pleiotropic effects of pho85 mutations.