Enlarging the synthetic biology toolbox for Pichia pastoris: Golden Gate cloning and CRISPR/Cas9

State-of-the-art strain engineering techniques for the protein producing yeast host Pichia pastoris include overexpression of homologous and heterologous genes, and deletion of host genes. For this purpose overexpression vectors and gene deletion methods such as the split marker technique have been established. For metabolic and cell engineering purposes, the simultaneous overexpression of more than one gene is often needed. Previous approaches employing subsequent steps of overexpression and marker recycling were time- and labor-consuming. Therefore, efficient systems allowing multiple gene overexpression are required, that can be stably integrated into the P. pastoris genome. To this end, we developed a synthetic biology toolbox based on Golden Gate cloning to enable efficient construction of complex and versatile over-expression vectors. Up to five different expression cassettes, employing a library of promoters and terminators can be combined into one vector, and successfully integrated into the genomic DNA of P. pastoris at targeted loci in one step. Recent trends in synthetic biology, however, go into the direction of building up large and complex reaction networks. To allow for clean and unscarred genetic engineering, a CRISPR/Cas9 based method for gene insertions, deletions and replacements was developed, which paves the way for precise genomic rearrangements in P. pastoris. By using this technique precise genomic integrations were performed efficiently without integrative selection markers. The repertoire of genetic techniques developed so far, will provide a wide variety of possibilities to engineer P. pastoris. Applications for these synthetic biology tools in cell engineering of recombinant P. pastoris will be presented

Synthetic two-species allodiploid and three-species allotetraploid Saccharomyces hybrids with euploid (complete) parental subgenomes

Abstract Combination of the genomes of Saccharomyces species has great potential for the construction of new industrial strains as well as for the study of the process of speciation. However, these species are reproductively isolated by a double sterility barrier. The first barrier is mainly due to the failure of the chromosomes to pair in allodiploid meiosis. The second barrier ensures that the hybrid remains sterile even after genome duplication, an event that can restore fertility in plant interspecies hybrids. The latter is attributable to the autodiploidisation of the allotetraploid meiosis that results in sterile allodiploid spores (return to the first barrier). Occasionally, mating-competent alloaneuploid spores arise by malsegregation of MAT-carrying chromosomes. These can mate with cells of a third species resulting in aneuploid zygotes having at least one incomplete subgenome. Here we report on the construction of euploid three-species hybrids by making use of “rare mating” between a sterile S. kudriavzevii x S. uvarum allodiploid hybrid and a diploid S. cerevisiae strain. The hybrids have allotetraploid 2nScnSk nSu genomes consisting of complete sets of parental chromosomes. This is the first report on the production of euploid three-species Saccharomyces hybrids by natural mating, without genetic manipulation. The hybrids provide possibilities for studying the interactions of three allospecific genomes and their orthologous genes present in the same cell

Genotypic and phenotypic diversity among Komagataella species reveals a hidden pathway for xylose utilization

Background The yeast genus Komagataella currently consists of seven methylotrophic species isolated from tree environments. Well-characterized strains of K. phaffii and K. pastoris are important hosts for biotechnological applications, but the potential of other species from the genus remains largely unexplored. In this study, we characterized 25 natural isolates from all seven described Komagataella species to identify interesting traits and provide a comprehensive overview of the genotypic and phenotypic diversity available within this genus. Results Growth tests on different carbon sources and in the presence of stressors at two different temperatures allowed us to identify strains with differences in tolerance to high pH, high temperature, and growth on xylose. As Komagataella species are generally not considered xylose-utilizing yeasts, xylose assimilation was characterized in detail. Growth assays, enzyme activity measurements and 13C labeling confirmed the ability of K. phaffii to utilize D-xylose via the oxidoreductase pathway. In addition, we performed long-read whole-genome sequencing to generate genome assemblies of all Komagataella species type strains and additional K. phaffii and K. pastoris isolates for comparative analysis. All sequenced genomes have a similar size and share 83–99% average sequence identity. Genome structure analysis showed that K. pastoris and K. ulmi share the same rearrangements in difference to K. phaffii, while the genome structure of K. kurtzmanii is similar to K. phaffii. The genomes of the other, more distant species showed a larger number of structural differences. Moreover, we used the newly assembled genomes to identify putative orthologs of important xylose-related genes in the different Komagataella species. Conclusions By characterizing the phenotypes of 25 natural Komagataella isolates, we could identify strains with improved growth on different relevant carbon sources and stress conditions. Our data on the phenotypic and genotypic diversity will provide the basis for the use of so-far neglected Komagataella strains with interesting characteristics and the elucidation of the genetic determinants of improved growth and stress tolerance for targeted strain improvement.ISSN:1475-285

From strain engineering to process development: monoclonal antibody production with an unnatural amino acid in Pichia pastoris

Background Expansion of the genetic code is a frequently employed approach for the modification of recombinant protein properties. It involves reassignment of a codon to another, e.g., unnatural, amino acid and requires the action of a pair of orthogonal tRNA and aminoacyl tRNA synthetase modified to recognize only the desired amino acid. This approach was applied for the production of trastuzumab IgG carrying p-azido-l-phenylalanine (pAzF) in the industrial yeast Pichia pastoris. Combining the knowledge of protein folding and secretion with bioreactor cultivations, the aim of the work was to make the production of monoclonal antibodies with an expanded genetic code cost-effective on a laboratory scale. Results Co-translational transport of proteins into the endoplasmic reticulum through secretion signal prepeptide change and overexpression of lumenal chaperones Kar2p and Lhs1p improved the production of trastuzumab IgG and its Fab fragment with incorporated pAzF. In the case of Fab, a knockout of vacuolar targeting for protein degradation further increased protein yield. Fed-batch bioreactor cultivations of engineered P. pastoris strains increased IgG and IgGpAzF productivity by around 50- and 20-fold compared to screenings, yielding up to 238 mg L−1 and 15 mg L−1 of fully assembled tetrameric protein, respectively. Successful site-specific incorporation of pAzF was confirmed by mass spectrometry. Conclusions Pichia pastoris was successfully employed for cost-effective laboratory-scale production of a monoclonal antibody with an unnatural amino acid. Applying the results of this work in glycoengineered strains, and taking further steps in process development opens great possibilities for utilizing P. pastoris in the development of antibodies for subsequent conjugations with, e.g., bioactive payloads.ISSN:1475-285

Yeasts from temperate forests

Yeasts are ubiquitous in temperate forests. While this broad habitat is well-defined, the yeasts inhabiting it and their life cycles, niches, and contributions to ecosystem functioning are less understood. Yeasts are present on nearly all sampled substrates in temperate forests worldwide. They associate with soils, macroorganisms, and other habitats and no doubt contribute to broader ecosystem-wide processes. Researchers have gathered information leading to hypotheses about yeasts' niches and their life cycles based on physiological observations in the laboratory as well as genomic analyses, but the challenge remains to test these hypotheses in the forests themselves. Here, we summarize the habitat and global patterns of yeast diversity, give some information on a handful of well-studied temperate forest yeast genera, discuss the various strategies to isolate forest yeasts, and explain temperate forest yeasts' contributions to biotechnology. We close with a summary of the many future directions and outstanding questions facing researchers in temperate forest yeast ecology. Yeasts present an exciting opportunity to better understand the hidden world of microbial ecology in this threatened and global habitat