Basal transcription profiles of the rhamnose-inducible promoter P LRA3 and the development of efficient P LRA3 -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.