Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker

Abstract

Background: A commonly used approach to improve recombinant protein production is to increase the levels of expression by providing extra-copies of a heterologous gene. In Komagataella phaffii (Pichia pastoris) this is usually accomplished by transforming cells with an expression vector carrying a drug-resistance marker following a screening for multicopy clones on plates with increasingly higher concentrations of an antibiotic. Alternatively, defective auxotrophic markers can be used for the same purpose. These markers are generally transcriptionally impaired genes lacking most of the promoter region. Among the defective markers commonly used in Saccharomyces cerevisiae is leu2-d, an allele of LEU2 which is involved in leucine metabolism. Cells transformed with this marker can recover prototrophy when they carry multiple copies of leu2-d in order to compensate the poor transcription from this defective allele.

Results: A K. phaffii strain auxotrophic for leucine (M12) was constructed by disrupting endogenous LEU2. The resulting strain was successfully transformed with a vector carrying leu2-d and an EGFP (enhanced green fluorescent protein) reporter gene. Vector copy numbers were determined from selected clones which grew to different colony sizes on transformation plates. A direct correlation was observed between colony size, number of integrated vectors and EGFP production. By using this approach we were able to isolate genetically stable clones bearing as many as 20 integrated copies of the vector and with no significant effects on cell growth.

Conclusions: In this work we have successfully developed a genetic system based on a defective auxotrophic which can be applied to improve heterologous protein production in K. phaffii. The system comprises a K. phaffii leu2 strain and an expression vector carrying the defective leu2-d marker which allowed the isolation of multicopy clones after a single transformation step. Because a linear correlation was observed between copy number and heterologous protein production, this system may provide a simple approach to improve recombinant protein productivity in K. phaffii.

Keywords: Auxotrophic marker; Expression system; Komagataella phaffii; Leucine biosynthesis; Multicopy integration.

Fig. 1

Strain phenotypic analysis. Strains LK and M12 were grown in different media to confirm drug resistance and leucine assimilation. X-33 was used as a control. a YPD containing G418 or hygromycin B (Hyg B). b MD lacking or not leucine in different concentrations. c MD with leucine replacing ammonium sulfate. d Buffered MD (pH 6.0) containing leucine. X-33 wild-type strain with intact LEU2 gene, LK leu2 strain disrupted with kan cassette, M12 strain obtained after marker removal with CreA recombinase

Fig. 2

Heterologous expression in K. phaffii M12. Intracellular expression of enhanced green fluorescent protein (EGFP) was visualized by epifluorescence microscopy. Untransformed (M12) and transformed (M12 + GFP) cells were viewed under differential interference contrast (DIC) or fluorescence (FM) microscopy after growth in glycerol or methanol containing media. All images were collected with the same exposure time

Fig. 3

Physical map of pKGFP-ld expression vector for multiple copy integration in K. phaffii. leu2-d defective LEU2 allele from S. cerevisiae. P _PGK1 and P _TEF1 promoters from the K. phaffii PGK1 and TEF1 genes, respectively, P _EM7 synthetic E. coli promoter, kan G418/kanamycin resistance gene, EGFP enhanced green fluorescence gene, CYCtt and AOX1tt transcription termination sequences from CYC1 and AOX1 genes, respectively, ori E. coli origin of replication

Fig. 4

Growth kinetic of selected clones transformed with pKGFP-ld. Cells were grown on MD medium at 30 °C during 72 h. Growth was expressed as the natural logarithm of OD₆₀₀ which was measured every 30 min. Initial OD₆₀₀ = 0.08. X-33 K. phaffii wild-type strain

Fig. 5

Vector copy number determination. a Schematic representation of genomic contexts and expected sizes of bands obtained after hybridization with a PGK probe (annealing positions are represented by a red line). Dark grey areas correspond to integrated P_PGK1 sequences derived from vector. b Result of Southern blot analysis. c Correlation between copy number and maximum growth rates of selected clones. Error bars represent standard error of the mean. M O’GeneRuler 1 kb DNA ladder, M12 K. phaffii leu2 strain, E EcoRI restriction site

Fig. 6

Intracellular EGFP production. a Flow cytometry analysis of the cells positive for EGFP production. b Correlation between copy number and EGFP production. M12 leu2 strain. Error bars represent standard error of the mean. Asterisks indicate significant difference between the evaluated clone and the M12 control strain according to ANOVA followed by Tukey’s post-test (p < 0.05)