Controlling AOX1 promoter strength in Pichia pastoris by manipulating poly (dA:dT) tracts

Abstract

Alcohol oxidase I (AOX1) promoter is the most popular but strictly-regulated methanol inducible promoter for heterologous protein expression in Pichia pastoris. In recent years, AOX1 promoter libraries have been developed with deletion or insertion methods. The present research manipulated poly (dA:dT) tracts in this promoter to control promoter strength, which hadn't been tried before. There were 34 variants derived from the native AOX1 promoter constructed. And variants were integrated into the same genomic location and upstream of the same reporter gene porcine growth hormone (pGH). To test the transferability of the results obtained from reporter gene pGH, the variants were connected to reporter gene Lac Z. The resulted promoter library spanned an activity range between 0.25 and 3.5 fold of the wild-type promoter activity. In addition, activities of variants correlated with their predicted nucleosome architecture, which were directed by poly (dA:dT) tracts. The cumulative sum of predicted nucleosome affinity across the region (-820 to -540) was related to promoters strength in single deletion variants on a proportional basis. Overall, the research promotes understanding of the regulatory patterns for AOX1 promoter and suggested that varying promoter expression of engineering nucleosome architecture was also a feasible approach in P. pastoris.