Characterization and application of a putative transcription factor (SUT2) in Pichia pastoris

Pichia pastoris is able to metabolize methanol via a specific MUT (methanol utilization) pathway. Based on the powerful AOX1 (Alcohol Oxidase 1) promoter, the P. pastoris expression system has become one of the most widely used eukaryotic expression systems. The molecular mechanisms of methanol metabolic regulation remain unclearly understood, so it is important to identify and develop new transcriptional regulators. Our previous studies suggested that the expression of SUT2 could be induced by methanol but is repressed by glycerol, which indicates that SUT2 may be involved in methanol metabolism through an unknown mechanism. SUT2 encodes a putative transcription factor-like protein harboring a Gal4-like Zn₂Cys₆ DNA-binding domain in Pichia pastoris, and its homolog in Saccharomyces cerevisiae regulates sterol uptake and synthesis. This study shows that the overexpression of SUT2 promoted the expression of AOX1 and increases ergosterol content in cells. Furthermore, via truncation of the putative SUT2 promoter at diverse loci, the - 973 base pair (bp) to - 547 bp region to the ATG was shown to be the core element of the inducible promoter P_SUT2, which strongly responds to the methanol signal. The transcriptional start site of SUT2, "A" at the 22nd bp upstream of ATG, was determined with 5'-rapid amplification of cDNA ends. A forward-loop cassette was constructed with MXR1 (Methanol Expression Regulator 1, a positive transcription factor of P_AOX1) promoted by P_SUT2, enabling moderate elevation in the expression level of Mxr1 and high activity of P_AOX1 without damaging cellular robustness further boosting the production of heterologous proteins. The P_AOX1-driven expression of enhanced green fluorescent protein in this novel system was improved by 18%, representing a promising method for extrinsic protein production. SUT2 may play roles in methanol metabolism by participating in sterol biosynthesis. P_SUT2 was characterized as a novel inducible promoter in P. pastoris and a P_SUT2-driven MXR1 forward-loop cassette was constructed to enhance the P_AOX1 activity, laying a foundation for further development and application of P. pastoris expression system.