Basal transcription profiles of the rhamnose-inducible promoter PLRA3 and the development of efficient PLRA3-based systems for markerless gene deletion and a mutant library in Pichia pastoris.

An ideal inducible promoter presents inducibility with an inducer and no basal transcription without inducer. Previous studies have shown that PLRA3 in Pichia pastoris is a strong rhamnose-inducible promoter for driving the industrial production of recombinant proteins. However, another important profile of PLRA3, the basal transcription, was not investigated yet. In this study, the basal transcription of PLRA3 was assessed according to the profiles of two test strains grown in media lacking rhamnose: (1) the production of secretory β-galactosidase in P. pastoris GS115/PLRA3-LacB, in which lacB expression was regulated by PLRA3, and (2) growth in P. pastoris GS115/PLRA3-MazF, in which the expression of mazF, which encodes an intracellular toxic protein, was controlled by PLRA3. Analyses revealed low β-galactosidase production and non-obviously inhibited growth of the test strains, which suggests that there was a low basal transcription level of PLRA3 when rhamnose was absent. Thus, PLRA3 was an excellent candidate for genetic manipulation in P. pastoris due to its strict regulation, a strong and a low transcriptional activity with and without rhamnose, respectively. Subsequently, two systems were developed based on PLRA3 in P. pastoris: (1) an efficient markerless gene deletion system for single or multiple genes and (2) a high efficient piggyBac transposase-mediated mutation system for investigating the functions of unknown genes, as well as for the screening of expected mutants.