A Novel Vector for Construction of Markerless Multicopy Overexpression Transformants in Pichia pastoris

Abstract

Pichia pastoris is widely used as a platform for heterologous protein expression because of its high volumetric productivity. Multicopy integration of the target gene is commonly used to improve the production of the target protein. Cre/lox recombination system is a powerful tool for the marker rescue during multiple integrations with one selection marker. Here we reported a novel expression vector based on the Cre/lox recombination system for multiple integrations of target gene to construct multicopy expression strain of P. pastoris. P_AOX1 promoter was fused to cre to construct a methanol inducible Cre recombinase. The leakage expression of Cre recombinase in Escherichia coli was blocked by introducing the operator gene lacO. The expression vector designed pMCO-AOXα was stable in E. coli and could effectively rescue the Zeocin resistance gene for next round of integration in P. pastoris. Phytase AppA from E. coli was chosen as a reporter gene. Transformants with 2-16 copies of appA were constructed by using a single antibiotic. Expression of appA was gene dosage dependent when <12 copies were integrated. The protein yield increased 4.45-folds when 12 copies of appA were integrated comparing with the single copy integration. Our results showed that pMCO-AOXα was highly effective for rational construction of multicopy transformat in P. pastoris.

Keywords: Pichia pastoris expression system; dosage effect; markerless genetic manipulation; novel expression vector; phytase appA.

Figure 1

Identification of the leakage expression of Cre recombinase. (A) The map of plasmid pMC. The primer pairs lox-F/R are indicated by arrows, and the calculated molecular weight of the two detected fragments is shown on top. (B) Single-colony PCR analysis of E. coli (pMC). Line M, DNA marker; Line 1, the detected fragment of Cre-AOX1TT-P_TEF1-P_EM7-Zeo^R-CYC1TT-lox66; Line 2–7, the detected fragment of lox71-P_AOX1-Cre-AOX1TT-P_TEF1-P_EM7-Zeo^R-CYC1TT-lox66. (C) RT-PCR analysis of E. coli (pMC). Line M, DNA marker; Line 1, positive control of 16s rRNA gene; Line 2, cre gene; Line 3, negative control.

Figure 2

Prevention of Cre-mediated plasmid recombination. (A) Schematic map of the integration of uORF and lacO. The sequences of uORF and lacO are underlined; the downstream Xho I sites are in bold. (B) Single-colony PCR analysis. Line M, DNA marker; Line 1, the detected fragment of Cre-AOX1TT-P_TEF1-P_EM7-Zeo^R-CYC1TT-lox66; Line 2, the detected fragment between lox71 and lox66 of pMCO; Line 3, the detected fragment between lox71 and lox66 of pMCU. (C) Gel electrophoresis analysis of extracted recombinant plasmids from E. coli. Line M, DNA marker; Line 1, pMCO; Line 2, pMCU. (D) RT-PCR analysis of E. coli (pMCO). Line M, DNA marker; Line 1, positive control of 16s rRNA gene; Line 2, cre gene.

Figure 3

Scheme of the expression vector pMCO- AOXα.

Figure 4

Scheme for construction and identification of the expression vectors with 1, 2, 4, or 8 copies of the appA gene. (A) Schematic map of the construction of expression vectors pMCO-AOXα-A1, pMCO-AOXα-A2, pMCO-AOXα-A4, and pMCO-AOXα-A8. (B) Gel electrophoresis analysis of recombinant vectors digested with Spe I and Xba I. Line M, DNA marker; Line 1, pMCO-AOXα-A1; Line 2, pMCO-AOXα-A2; Line 3, pMCO-AOXα-A4; Line 4, pMCO-AOXα-A8.

Figure 5

Identification and expression analysis of the transformants that were resistant or sensitive to Zeocin. (A) Expression analysis of the Zeocin^R transformants. (B) Identification of the colonies that were sensitive toward Zeocin. (C) Single-colony PCR analysis of Zeocin^S strains using primer pairs lox-F/αf-R. Line M, DNA marker; Line 1, Zeocin^R transformants; Line 2-11, 10 randomly selected Zeocin^S transformants. (D) Expression analysis of the Zeocin^S transformants. The experiments were performed 10 or 20 times as described; the mean values ± SD are presented. Bars with the same letters are not significant (P < 0.05).

Figure 6

Expression analysis of the transformants that were resistant or sensitive to Zeocin. (A) Expression analysis of the Zeocin^R transformants. (B) Expression analysis of the Zeocin^S transformants. The experiments were performed 10 times as described; the mean values ± SD are presented. Bars with the same letters are not significant (P < 0.05).

Figure 7

Expression analysis of the Zeocin^S transformants. (A) SDS-PAGE analysis. Line M: protein marker; Line 1: GS115-pMCO-AOXα-A1-Zeocin^S #2; Line 2: GS115-pMCO-AOXα-A2-Zeocin^S #3; Line 3: GS115-pMCO-AOXα-A4-Zeocin^S #1; Line 4: GS115-pMCO-AOXα-A8-Zeocin^S #17; Line 5: GS115-pMCO-AOXα-A8A4-Zeocin^S #5; Line 6: GS115-pMCO-AOXα-A8A8-Zeocin^S #7. (B) Production of phytase with different copy number of the appA gene. The experiments were performed 3 times; the mean values ± SD are presented. Bars with the same letters are not significant (P < 0.05).