Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing

The non-conventional yeast Pichia pastoris is a popular host for recombinant protein production in scientific research and industry. Typically, the expression cassette is integrated into the genome via homologous recombination. Due to unknown integration events, a large clonal variability is often encountered consisting of clones with different productivities as well as aberrant morphological or growth characteristics. In this study, we analysed several clones with abnormal colony morphology and discovered unpredicted integration events via whole genome sequencing. These include (i) the relocation of the locus targeted for replacement to another chromosome (ii) co-integration of DNA from the E. coli plasmid host and (iii) the disruption of untargeted genes affecting colony morphology. Most of these events have not been reported so far in literature and present challenges for genetic engineering approaches in this yeast. Especially, the presence and independent activity of E. coli DNA elements in P. pastoris is of concern. In our study, we provide a deeper insight into these events and their potential origins. Steps preventing or reducing the risk for these phenomena are proposed and will help scientists working on genetic engineering of P. pastoris or similar non-conventional yeast to better understand and control clonal variability.

[1]  M. Wubbolts,et al.  Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomena. , 2004, FEMS yeast research.

[2]  Y. Zhuang,et al.  Understanding the effect of foreign gene dosage on the physiology of Pichia pastoris by transcriptional analysis of key genes , 2011, Applied Microbiology and Biotechnology.

[3]  Vera Meyer,et al.  Highly efficient gene targeting in the Aspergillus niger kusA mutant. , 2007, Journal of biotechnology.

[4]  J. Cregg.  6 – EXPRESSION IN THE METHYLOTROPHIC YEAST Pichia pastoris , 1999 .

[5]  Martina Geier,et al.  Compact multi-enzyme pathways in P. pastoris. , 2015, Chemical communications.

[6]  Karen M Polizzi,et al.  Can too many copies spoil the broth? , 2013, Microbial Cell Factories.

[7]  A. Glieder,et al.  Deletion of the Pichia pastoris KU70 Homologue Facilitates Platform Strain Generation for Gene Expression and Synthetic Biology , 2012, PloS one.

[8]  G. Letchworth,et al.  High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. , 2004, BioTechniques.

[9]  S. Kohlwein,et al.  Interaction of Pik1p and Sjl proteins in membrane trafficking. , 2005, FEMS yeast research.

[10]  I. Yamashita,et al.  In vivo ligation of linear DNA molecules to circular forms in the yeast Saccharomyces cerevisiae , 1983, Journal of bacteriology.

[11]  C. Desmaze,et al.  Impact of the KU80 pathway on NHEJ-induced genome rearrangements in mammalian cells. , 2004, Molecular cell.

[12]  Jonathon T. Olesberg,et al.  Real-time monitoring of glycerol and methanol to enhance antibody production in industrial Pichia pastoris bioprocesses , 2015 .

[13]  Gerhard G Thallinger,et al.  A Toolbox of Diverse Promoters Related to Methanol Utilization: Functionally Verified Parts for Heterologous Pathway Expression in Pichia pastoris. , 2016, ACS synthetic biology.

[14]  Byung-Kwon Choi,et al.  Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Alexander Goesmann,et al.  High-quality genome sequence of Pichia pastoris CBS7435. , 2011, Journal of biotechnology.

[16]  Erich Leitner,et al.  Production of the sesquiterpenoid (+)-nootkatone by metabolic engineering of Pichia pastoris. , 2014, Metabolic engineering.

[17]  P. Green,et al.  Consed: a graphical tool for sequence finishing. , 1998, Genome research.

[18]  Oliver Spadiut,et al.  Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway , 2012, Microbial Cell Factories.

[19]  J. Nicaud,et al.  Efficient homologous recombination with short length flanking fragments in Ku70 deficient Yarrowia lipolytica strains , 2012, Biotechnology Letters.

[20]  T. Petes,et al.  Palindromic sequences in heteroduplex DNA inhibit mismatch repair in yeast , 1989, Nature.

[21]  Michael Sauer,et al.  Pichia pastoris: protein production host and model organism for biomedical research. , 2013, Future microbiology.

[22]  Hal S Alper,et al.  Frontiers of yeast metabolic engineering: diversifying beyond ethanol and Saccharomyces. , 2013, Current opinion in biotechnology.

[23]  N. He,et al.  Enhancement of the Gene Targeting Efficiency of Non-Conventional Yeasts by Increasing Genetic Redundancy , 2013, PloS one.

[24]  Y Jigami,et al.  OCH1 encodes a novel membrane bound mannosyltransferase: outer chain elongation of asparagine‐linked oligosaccharides. , 1992, The EMBO journal.

[25]  S. Rausch,et al.  Cloning and disruption of the Pichia pastoris ARG1, ARG2, ARG3, HIS1, HIS2, HIS5, HIS6 genes and their use as auxotrophic markers , 2005, Yeast.

[26]  T. Vogl,et al.  Restriction site free cloning (RSFC) plasmid family for seamless, sequence independent cloning in Pichia pastoris , 2015, Microbial Cell Factories.

[27]  J. Cregg,et al.  New selectable marker/auxotrophic host strain combinations for molecular genetic manipulation of Pichia pastoris. , 2001, Gene.

[28]  C. Kurtzman.  Biotechnological strains of Komagataella (Pichia) pastoris are Komagataellaphaffii as determined from multigene sequence analysis , 2009, Journal of Industrial Microbiology & Biotechnology.

[29]  D. Fotiadis,et al.  Cultivation strategies to enhance productivity of Pichia pastoris: A review. , 2015, Biotechnology advances.

[30]  Yuzhi Hong,et al.  Cloning of a Laccase Gene from a Novel Basidiomycete Trametes sp. 420 and Its Heterologous Expression in Pichia pastoris , 2007, Current Microbiology.

[31]  C. Scorer,et al.  Foreign gene expression in yeast: a review , 1992, Yeast.

[32]  J. Cregg,et al.  Mxr1p, a Key Regulator of the Methanol Utilization Pathway and Peroxisomal Genes in Pichia pastoris , 2006, Molecular and Cellular Biology.

[33]  Sophie Brachat,et al.  Contribution of Horizontal Gene Transfer to the Evolution of Saccharomyces cerevisiae , 2005, Eukaryotic Cell.

[34]  Yves Van de Peer,et al.  Genome sequence of the recombinant protein production host Pichia pastoris , 2009, Nature Biotechnology.

[35]  Narmada Thanki,et al.  CDD: a Conserved Domain Database for the functional annotation of proteins , 2010, Nucleic Acids Res..

[36]  T. G. Villa,et al.  Construction of new Pichia pastoris X-33 strains for production of lycopene and β-carotene , 2011, Applied Microbiology and Biotechnology.

[37]  J. Hansen.  Inactivation of MXR1 Abolishes Formation of Dimethyl Sulfide from Dimethyl Sulfoxide inSaccharomyces cerevisiae , 1999, Applied and Environmental Microbiology.

[38]  F. Nasar,et al.  Long Palindromic Sequences Induce Double-Strand Breaks during Meiosis in Yeast , 2000, Molecular and Cellular Biology.

[39]  Brigitte Gasser,et al.  Identification and deletion of the major secreted protein of Pichia pastoris , 2012, Applied Microbiology and Biotechnology.

[40]  Ju Chu,et al.  GAP Promoter Library for Fine-Tuning of Gene Expression in Pichia pastoris , 2011, Applied and Environmental Microbiology.

[41]  D. Wei,et al.  Metabolic engineering of Pichia pastoris for the production of dammarenediol-II. , 2015, Journal of biotechnology.

[42]  Karin Kovar,et al.  Promoter library designed for fine-tuned gene expression in Pichia pastoris , 2008, Nucleic acids research.

[43]  D. Botstein,et al.  Transformation of yeast with linearized plasmid DNA. Formation of inverted dimers and recombinant plasmid products. , 1985, Journal of molecular biology.

[44]  Sang Yup Lee,et al.  Model based engineering of Pichia pastoris central metabolism enhances recombinant protein production , 2014, Metabolic engineering.

[45]  E. Sambuk,et al.  Effect of Nitrogen Source and Inorganic Phosphate Concentration on Methanol Utilization and PEX Genes Expression in Pichia pastoris , 2014, TheScientificWorldJournal.

[46]  L. Ma,et al.  A visual method for direct selection of high-producing Pichia pastoris clones , 2011, BMC biotechnology.

[47]  X. Lei,et al.  Expression of the Aspergillus fumigatus phytase gene in Pichia pastoris and characterization of the recombinant enzyme. , 2000, Biochemical and biophysical research communications.

[48]  T. Vogl,et al.  Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris. , 2016, Journal of biotechnology.

[49]  Ming-qi Liu,et al.  Expression of recombinant Bacillus licheniformis xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it. , 2008, Protein expression and purification.

[50]  M. Romanos,et al.  High-Level Expression of Tetanus Toxin Fragment C in Pichia Pastoris Strains Containing Multiple Tandem Integrations of the Gene , 1991, Bio/Technology.

[51]  Brigitte Gasser,et al.  Induction without methanol: novel regulated promoters enable high-level expression in Pichia pastoris , 2013, Microbial Cell Factories.

[52]  C. Huynh,et al.  Identification and characterization of an essential family of inositol polyphosphate 5-phosphatases (INP51, INP52 and INP53 gene products) in the yeast Saccharomyces cerevisiae. , 1998, Genetics.

[53]  M. Sauer,et al.  Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris , 2008, Microbial cell factories.

[54]  J. Kalinowski,et al.  Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization , 2016, Microbial Cell Factories.

[55]  Arjen M. Krikken,et al.  Novel genetic tools for Hansenula polymorpha. , 2012, FEMS yeast research.

[56]  Robert C. Wolpert,et al.  A Review of the , 1985 .

[57]  A. Glieder,et al.  Knockout of an endogenous mannosyltransferase increases the homogeneity of glycoproteins produced in Pichia pastoris , 2013, Scientific Reports.

[58]  A. Kegel,et al.  Genome wide distribution of illegitimate recombination events in Kluyveromyces lactis , 2006, Nucleic acids research.

[59]  C. Kurtzman.  Description of Komagataella phaffii sp. nov. and the transfer of Pichia pseudopastoris to the methylotrophic yeast genus Komagataella. , 2005, International journal of systematic and evolutionary microbiology.

[60]  Jack Hoopes,et al.  Humanization of Yeast to Produce Complex Terminally Sialylated Glycoproteins , 2006, Science.

[61]  D. Maresch,et al.  Development of a fed-batch process for a recombinant Pichia pastoris Δoch1 strain expressing a plant peroxidase , 2015, Microbial Cell Factories.

[62]  J. Setubal,et al.  Complete genome sequencing of Agrobacterium sp. H13-3, the former Rhizobium lupini H13-3, reveals a tripartite genome consisting of a circular and a linear chromosome and an accessory plasmid but lacking a tumor-inducing Ti-plasmid. , 2011, Journal of biotechnology.

[63]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[64]  T. E. Wilson,et al.  Nonhomologous end joining in yeast. , 2005, Annual review of genetics.

[65]  Mudassar Ahmad,et al.  Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production , 2014, Applied Microbiology and Biotechnology.