Effect of Biotin Starvation on Gene Expression in Komagataella phaffii Cells.

Methylotrophic yeast Komagataella phaffii is widely used in biotechnology for recombinant protein production. Due to the practical significance of these yeasts, it is extremely important to properly select cultivation conditions and optimize the media composition. In this study the effect of biotin starvation on gene expression in K. phaffii at transcriptome level was investigated. It was demonstrated, that the response of K. phaffii cell to biotin deficiency strongly depends on the carbon source in the medium. In the media containing glycerol, biotin deficiency led to activation of the genes involved in biotin metabolism, glyoxylate cycle, and synthesis of acetyl-CoA in cytoplasm, as well as repression of the genes, involved in lipo- and gluconeogenesis. In the methanol-containing media, biotin deficiency primarily led to repression of the genes, involved in protein synthesis, and activation of cell response to oxidative stress.

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.

GADMA2: more efficient and flexible demographic inference from genetic data.

BACKGROUND: Inference of complex demographic histories is a source of information about events that happened in the past of studied populations. Existing methods for demographic inference typically require input from the researcher in the form of a parameterized model. With an increased variety of methods and tools, each with its own interface, the model specification becomes tedious and error-prone. Moreover, optimization algorithms used to find model parameters sometimes turn out to be inefficient, for instance, by being not properly tuned or highly dependent on a user-provided initialization. The open-source software GADMA addresses these problems, providing automatic demographic inference. It proposes a common interface for several likelihood engines and provides global parameters optimization based on a genetic algorithm. RESULTS: Here, we introduce the new GADMA2 software and provide a detailed description of the added and expanded features. It has a renovated core code base, new likelihood engines, an updated optimization algorithm, and a flexible setup for automatic model construction. We provide a full overview of GADMA2 enhancements, compare the performance of supported likelihood engines on simulated data, and demonstrate an example of GADMA2 usage on 2 empirical datasets. CONCLUSIONS: We demonstrate the better performance of a genetic algorithm in GADMA2 by comparing it to the initial version and other existing optimization approaches. Our experiments on simulated data indicate that GADMA2's likelihood engines are able to provide accurate estimations of demographic parameters even for misspecified models. We improve model parameters for 2 empirical datasets of inbred species.

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.