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Genome sequence, secretome and sugar transport of the protein production host Pichia pastoris
2. INDUSTRIAL BIOTECHNOLOGY 2.2 Microbial Genomics Oral
ple. Furthermore, we present an open access web-based genome browser. doi:10.1016/j.nbt.2009.06.680
Genome sequence, secretome and sugar transport of the protein production host Pichia pastoris D. Mattanovich 1,2,∗ , A. Graf 1,2 , J. Stadlmann 3 , M. Dragosits 1 , A. Redl 1,2 , M. Maurer 1,2 , M. Sauer 1,2 , F. Altmann 3 , B. Gasser 1 1
Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Vienna, Austria 2 School of Bioengineering, University of Applied Sciences FH-Campus Wien, Vienna, Austria 3 Department of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, Austria
Poster 2.2.002 Effects of unidirectional deletions on the KlCYC1 3 -UTR searching for sequences directing alternative RNA processing selection M.A. Freire-Picos ∗ , I. Vázquez Chico, A.M. Rodríguez-Torres, S. Seoane Rosende ˜ ˜ Spain Universidade da Coruna/Facultad de Ciencias, A Coruna,
The yeast Pichia pastoris is widely used as a production platform for heterologous proteins, and as a model organism for peroxisome metabolism and secretory organelle proliferation. As no published genome sequence is available, most data on genetic and physiological background for strain and process design rely on analogies to other, well-studied yeasts like Saccharomyces cerevisiae. To gain more insight into specific features of growth and protein secretion, we have sequenced the 9.4 Mb genome of the type strain DSMZ 70382 and analyzed secretome and sugar transporters. The computationally predicted secretome consists of 131 ORFs. When grown on glucose, only 20 different proteins were actually secreted at significant levels, as identified by LC—MS. The low amount of secreted protein highlights one major feature of P. pastoris, being the low contamination of heterologous proteins with host cell protein, and supports the development of glucose-based expression platforms for P. pastoris. The putative sugar transporters were identified and compared to those of related yeast species, which offers the basis to study and engineer carbon metabolism. Especially the efficient utilization of glycerol is highlighted by this analysis. Availability of the genome sequence allows for the development of genome scale analysis and engineering of P. pastoris as a protein production platform, as will be shown in an exam-
1871-6784/$ — see front matter
The alternative RNA processing (cleavage and polyadenylation) is an eukaryotic conserved mechanism. The obtained mRNA isoforms, with different 3 -UTR (3 -untranslated regions) length, are usually tissue-specific regulated alternatives, or growth-dependent variants with different stability. We work with KlCYC1 as a yeast gene with a 1.2 kb 3 -UTR that causes a long (L) and a short (S) transcript due to alternative RNA processing. The gene encodes for cytochrome c in the yeast Kluyveromyces lactis. When expressed in Saccharomyces cerevisiae the RNA processing positions are conserved. The L/S transcript ratio changes along the growth phase in K. lactis cultures or when expressed in S. cerevisiae mutants for RNA processing factors. We have previously shown that is possible to modulate KlCYC1 gene expression by splitting the KlCYC1 3 -UTR: the proximal 3 -UTR (1—713) is important for early logarithmic phase cytochrome c expression while the distal region (699—1194) enhances KlCYC1 expression at late logarithmic phase and consequently increases the biomass production under respiratory growth conditions. As part of the search for cis-elements causing the KlCYC1 alternative RNA processing we performed serial deletions of either the complete 3 -UTR or the splitted forms (proximal and distal). The
ple. Furthermore, we present an open access web-based genome browser. doi:10.1016/j.nbt.2009.06.680
Genome sequence, secretome and sugar transport of the protein production host Pichia pastoris D. Mattanovich 1,2,∗ , A. Graf 1,2 , J. Stadlmann 3 , M. Dragosits 1 , A. Redl 1,2 , M. Maurer 1,2 , M. Sauer 1,2 , F. Altmann 3 , B. Gasser 1 1
Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Vienna, Austria 2 School of Bioengineering, University of Applied Sciences FH-Campus Wien, Vienna, Austria 3 Department of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, Austria
Poster 2.2.002 Effects of unidirectional deletions on the KlCYC1 3 -UTR searching for sequences directing alternative RNA processing selection M.A. Freire-Picos ∗ , I. Vázquez Chico, A.M. Rodríguez-Torres, S. Seoane Rosende ˜ ˜ Spain Universidade da Coruna/Facultad de Ciencias, A Coruna,
The yeast Pichia pastoris is widely used as a production platform for heterologous proteins, and as a model organism for peroxisome metabolism and secretory organelle proliferation. As no published genome sequence is available, most data on genetic and physiological background for strain and process design rely on analogies to other, well-studied yeasts like Saccharomyces cerevisiae. To gain more insight into specific features of growth and protein secretion, we have sequenced the 9.4 Mb genome of the type strain DSMZ 70382 and analyzed secretome and sugar transporters. The computationally predicted secretome consists of 131 ORFs. When grown on glucose, only 20 different proteins were actually secreted at significant levels, as identified by LC—MS. The low amount of secreted protein highlights one major feature of P. pastoris, being the low contamination of heterologous proteins with host cell protein, and supports the development of glucose-based expression platforms for P. pastoris. The putative sugar transporters were identified and compared to those of related yeast species, which offers the basis to study and engineer carbon metabolism. Especially the efficient utilization of glycerol is highlighted by this analysis. Availability of the genome sequence allows for the development of genome scale analysis and engineering of P. pastoris as a protein production platform, as will be shown in an exam-
1871-6784/$ — see front matter
The alternative RNA processing (cleavage and polyadenylation) is an eukaryotic conserved mechanism. The obtained mRNA isoforms, with different 3 -UTR (3 -untranslated regions) length, are usually tissue-specific regulated alternatives, or growth-dependent variants with different stability. We work with KlCYC1 as a yeast gene with a 1.2 kb 3 -UTR that causes a long (L) and a short (S) transcript due to alternative RNA processing. The gene encodes for cytochrome c in the yeast Kluyveromyces lactis. When expressed in Saccharomyces cerevisiae the RNA processing positions are conserved. The L/S transcript ratio changes along the growth phase in K. lactis cultures or when expressed in S. cerevisiae mutants for RNA processing factors. We have previously shown that is possible to modulate KlCYC1 gene expression by splitting the KlCYC1 3 -UTR: the proximal 3 -UTR (1—713) is important for early logarithmic phase cytochrome c expression while the distal region (699—1194) enhances KlCYC1 expression at late logarithmic phase and consequently increases the biomass production under respiratory growth conditions. As part of the search for cis-elements causing the KlCYC1 alternative RNA processing we performed serial deletions of either the complete 3 -UTR or the splitted forms (proximal and distal). The