Selection of Promoter Fragments Involved in Peroxisomal Biosynthesis and Metabolic Pathway Using Glucose Oxidase as a Marker

Authors: Mehmet Önder YAVUZ

Abstract: This work is concerned with the development of Hansenula polymorpha as a host for the expression of heterologous gene, glucose oxidase (GOD), from Aspergillus niger as well as searching for any promoter which may act in relation to peroxisomes. Expression of GOD was used to isolate promoter fragments from the genome of H. polymorpha. A promoterless plasmid (MERPROp 10) was constructed and Bam II-Bg/II digested total H. polymorpha DNA was ligated to the unique BamHI site of this promoterless plasmid. Transformants which contain methanol inducible promoters were screened using the plate assay which was designed for glucose oxidase enzyme (Godp) secretion. Positive colonies were hybridised against three methanol metabolic pathway promoters, MOX (Methanol oxidase), DHAS (Dihydroxyacetone synthase) and FMDH (Formate dihydrogenase). Then some of the best promoter fragments were chosen for further analyisis. Western blots to ensure secretion of Godp and Northern blots to show production of GOD mRNA under inducive and repressive conditions for the methanol metabolic pathway were carried out. Two of the inserts were sequenced and their sequences were compared to the sequences in UWGCG database The first insert is in the construct MEHPROp10-21 which is 137 base pairs (bp). This insert does not show any significant homology to the sequences in the database and contains multiple T residues. The second insert is in MEHPROPp10-43 which is 1160 bp. This insert contains two different fragments which were ligated fortuitously during ligation of totol DNA fragments. One of the inserts is a mitochondrial DNA and the other is genomic. The genomic DNA fragment of this insert is 260 bp long and shows 81 % identity with the Candida albicans carboxypeptidase Y gene and 78 % identity with the Saccharomyces cerevisiae carboxypeptidase Y gene.

Keywords: Hansenula, polymorpha, promoter, peroxisome, methanol, library