AUTHOR=Li Ding , Zhang Bo , Li Shuting , Zhou Jie , Cao Hui , Huang Yan , Cui Zhongli 

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

JOURNAL=Frontiers in Microbiology

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2017.01698

DOI=10.3389/fmicb.2017.01698

ISSN=1664-302X

ABSTRACT=<p><italic>Pichia pastoris</italic> 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/<italic>lox</italic> 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/<italic>lox</italic> recombination system for multiple integrations of target gene to construct multicopy expression strain of <italic>P. pastoris</italic>. P<sub>AOX1</sub> promoter was fused to <italic>cre</italic> to construct a methanol inducible Cre recombinase. The leakage expression of Cre recombinase in <italic>Escherichia coli</italic> was blocked by introducing the operator gene <italic>lacO</italic>. The expression vector designed pMCO-AOXα was stable in <italic>E. coli</italic> and could effectively rescue the Zeocin resistance gene for next round of integration in <italic>P. pastoris</italic>. Phytase AppA from <italic>E. coli</italic> was chosen as a reporter gene. Transformants with 2–16 copies of <italic>appA</italic> were constructed by using a single antibiotic. Expression of <italic>appA</italic> was gene dosage dependent when &lt;12 copies were integrated. The protein yield increased 4.45-folds when 12 copies of <italic>appA</italic> were integrated comparing with the single copy integration. Our results showed that pMCO-AOXα was highly effective for rational construction of multicopy transformat in <italic>P. pastoris</italic>.</p>