ABSTRACT
Komagataella phaffii (formerly Pichia pastoris) is a methylotrophic yeast widely used in laboratories around the world to produce recombinant proteins. Given its advantageous features, it has also gained much interest in the context of modern biotechnology. In this review, we present the utilization of K. phaffii as a platform to produce several products of economic interest such as biopharmaceuticals, renewable chemicals, fuels, biomaterials, and food/feed products. Finally, we present synthetic biology approaches currently used for strain engineering, aiming at the production of new bioproducts.
ABSTRACT
The yeast Komagataella phaffii is widely used as a microbial host for heterologous protein production. However, molecular tools for this yeast are basically restricted to a few integrative and replicative plasmids. Four sequences that have recently been proposed as the K. phaffii centromeres could be used to develop a new class of mitotically stable vectors. In this work, we designed a color-based genetic assay to investigate plasmid stability in K. phaffii and constructed vectors bearing K. phaffii centromeres and the ADE3 marker. These genetic tools were evaluated in terms of mitotic stability by transforming an ade2/ade3 auxotrophic strain and regarding plasmid copy number by quantitative PCR (qPCR). Our results confirmed that the centromeric plasmids were maintained at low copy numbers as a result of typical chromosome-like segregation during cell division. These features, combined with in vivo assembly possibilities, prompt these plasmids as a new addition to the K. phaffii genetic toolbox.
Subject(s)
Centromere/genetics , Colorimetry/methods , Pichia/genetics , Plasmids/analysis , DNA, Fungal/metabolism , Plasmids/genetics , Plasmids/metabolism , Real-Time Polymerase Chain ReactionABSTRACT
Komagataella phaffii (formerly Pichia pastoris) is a well-known fungal system for heterologous protein production in the context of modern biotechnology. To obtain higher protein titers in this system many researchers have sought to optimize gene expression by increasing the levels of transcription of the heterologous gene. This has been typically achieved by manipulating promoter sequences or by generating clones bearing multiple copies of the desired gene. The aim of this work is to describe how these different molecular strategies have been applied in K. phaffii presenting their advantages and drawbacks.
Subject(s)
Ascomycota/genetics , Ascomycota/metabolism , Genetic Enhancement/methods , Promoter Regions, Genetic/genetics , Protein Engineering/methods , Recombinant Proteins/biosynthesis , Transcription Factors/biosynthesis , Cloning, Molecular/methods , Gene Dosage/genetics , Gene Expression Regulation, Fungal/genetics , Recombinant Proteins/genetics , Transcription Factors/geneticsABSTRACT
DNA replication, together with repair mechanisms and cell cycle control, are the most important cellular processes necessary to maintain correct transfer of genetic information to the progeny. These processes are well conserved throughout the Eukarya, and the genes that are involved provide essential information for understanding the life cycle of an organism. We used computational tools for data mining of genes involved in these processes in the pathogenic fungus Paracoccidiodes brasiliensis. Data derived from transcriptome analysis revealed that the cell cycle of this fungus, as well as DNA replication and repair, and the recombination machineries, are highly similar to those of the yeast Saccharomyces cerevisiae. Among orthologs detected in both species, there are genes related to cytoskeleton structure and assembly, chromosome segregation, and cell cycle control genes. We identified at least one representative gene from each step of the initiation of DNA replication. Major players in the process of DNA damage and repair were also identified.