High-level expression of the phenylalanine ammonia lyase-encoding gene from Rhodosporidium toruloides in Saccharomyces cerevisiae and Escherichia coli using a bifunctional expression system.
A chimeric yeast promoter (pPGK::REP2), capable of directing high-level gene expression in both Saccharomyces cerevisiae and Escherichia coli, has been constructed. It was derived by fusing the promoter of the yeast PGK gene (encoding phosphoglycerate kinase) to a region residing immediately 5' to the yeast 2 mu plasmid REP2 gene (encoding a trans-acting plasmid maintenance protein). In S. cerevisiae, transcripts initiated within the REP2-derived moiety of the promoter, but the transcription start point was dictated by the PGK determinator sequence. Promoter function in E. coli was due to the presence of consensus prokaryotic -35 and -10 motifs in the REP2 moiety. To facilitate expression studies, the promoter was incorporated into a versatile series of S. cerevisiae/E. coli shuttle vectors which provided a choice of selectable marker and copy number in S. cerevisiae. To maximise translational efficiency, a novel cloning strategy was devised which allows the juxtaposition of genes to the promoter such that the heterologous AUG replaces that of the REP2 AUG, without any alteration in the surrounding nucleotide (nt) context. This strategy was used to place both the Tn903 neo gene and the Rhodosporidium toruloides phenylalanine ammonia lyase (PAL)-encoding gene under the transcriptional control of pPGK::REP2. In the former case, cells became resistant to extremely high levels of Geneticin (> 3 mg/ml in the case of S. cerevisiae). In the case of the latter, PAL was shown to accumulate to approx. 9 and 10% of total soluble protein in S. cerevisiae and E. coli, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Faulkner
J D
JD
Division of Biotechnology, Centre for Applied Microbiology and Research, Salisbury, Wiltshire, UK.
Anson
J G
JG
Tuite
M F
MF
Minton
N P
NP
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004271:DNA, Fungal; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005800:Genes, Fungal; D005819:Genetic Markers; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010650:Phenylalanine Ammonia-Lyase / Q000096:biosynthesis* / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014158:Transcription, Genetic; D014587:Ustilaginales / Q000201:enzymology / Q000235:genetics*
D004271:DNA, Fungal; D005819:Genetic Markers; D011993:Recombinant Fusion Proteins; D010650:Phenylalanine Ammonia-Lyase