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.
Highlights
The methylotrophic yeast Pichia pastoris (Komagataella phaffi) has been commonly used for production of heterologous protein[1,2]
Portela RM et al.[4] designed 112 synthetic promoters based on sequence/function relationship of natural core promoters, nucleosome occupancy and the presence of short motifs and fused synthetic core promoters to AOX1 cis-regulatory modules (CRMs)
This study indicates that deletion or lengthening native poly tracts can alter the variants activities ranging from ~0.25 to ~3.5 fold of wild-type promoter activity, proving that the method is more effective than traditional methods for improving AOX1 promoter activities
Summary
The methylotrophic yeast Pichia pastoris (Komagataella phaffi) has been commonly used for production of heterologous protein[1,2]. Previous studies of optimizing AOX1 promoter have focused on deletion and duplication of putative transcription factor-binding sites and identification of upstream activation sequence[5,6]. Portela RM et al.[4] designed 112 synthetic promoters based on sequence/function relationship of natural core promoters, nucleosome occupancy and the presence of short motifs and fused synthetic core promoters to AOX1 cis-regulatory modules (CRMs). These studies provided new idea for engineering promoters including AOX1 promoter. Altering the presence and length of native poly (dA:dT) tracts can increase accessibility to the nearby transcription factor-binding sites which are covered by nucleosomes, regulating promoter activities[13,14]. Research shows that occupancy and affinity of nucleosomes in certain regions of AOX1 promoter are correlated with promoter activities
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