RESUMO
The methylotrophic yeast Hansenula polymorpha belongs to a limited number of non-Saccharomyces yeast species used as hosts for heterologous gene expression. It has successfully been applied for the production of hormones, antigens and enzymes. The system excells by mitotically stable recombinant strains, high productivity and faithful processing of the produced polypeptides. The favourable characteristics of this microorganism for protein production at an industrial scale are described in the following article focusing on some recent representative examples.
RESUMO
We have introduced the glucoamylase gene (GAM1) from Schwanniomyces occidentalis into the genome of the methylotrophic yeast Hansenula polymorpha to study the potential of this organism as a host for high-level expression of a heterologous gene encoding a secretory protein. Transformants of H. polymorpha containing GAM1 under control of the formate dehydrogenase (FMD) promoter are stable and efficiently secrete an active glucoamylase that is faithfully processed and modified. Yields of up to 1.4g/l of active enzyme were obtained at cell densities of 100-130 grams dry weight per liter.
Assuntos
Clonagem Molecular/métodos , Glucana 1,4-alfa-Glucosidase/genética , Pichia/genética , Saccharomycetales/enzimologia , Sequência de Aminoácidos , Fermentação , Genes Fúngicos , Glucana 1,4-alfa-Glucosidase/biossíntese , Glucana 1,4-alfa-Glucosidase/metabolismo , Dados de Sequência Molecular , Pichia/enzimologia , PlasmídeosRESUMO
Two chalcone synthase genes in maize have been cloned and molecularly characterized to be the C2 and the Whp (white pollen) locus. The two genes have highly homologous exon sequences but differ considerably in sequences 5' upstream and 3' downstream of the coding region, as well as in their introns. Northern and Western experiments of chalcone synthase expression in various tissues and in different genotypes indicated that C2 and Whp are differently regulated. The expression of Whp in maize aleurone is dependent on the presence of the recessive allele of the gene intensifier (in). The regulatory effect of in on Whp expression is not detectable at the transcriptional level, but seems to take place during translation.
Assuntos
Aciltransferases/genética , Antocianinas/genética , Regulação da Expressão Gênica , Biossíntese de Proteínas , Zea mays/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , Western Blotting , Clonagem Molecular , Dados de Sequência Molecular , Poli A/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA/genética , RNA Mensageiro , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
Plasmids without an origin of replication, but bearing the URA3 gene of Saccharomyces cerevisiae as a selective marker for transformation, are shown to replicate autonomously in Hansenula polymorpha, indicating that parts of the S. cerevisiae URA3 gene can fulfil an autonomous replication and stabilization function in H. polymorpha. Such plasmids, replicated in low copy number in monomeric conformation, could be rescued in E. coli, and showed a low mitotic stability under selective and non-selective conditions. Selective propagation of such transformants, however, led to the integration of plasmid sequences into the H. polymorpha genome. The integration event usually occurred in high copy number (approx. 30-50) at a single non-homologous site of the genome. The plasmid sequences were found to be present in tandem array and stable under non-selective conditions. In contrast, the use of homologous URA3 gene under similar conditions led to low-copy-number transformants.
Assuntos
Vetores Genéticos/genética , Orotidina-5'-Fosfato Descarboxilase/genética , Pichia/genética , Plasmídeos/genética , Transformação Genética/genética , Replicação do DNA/genética , DNA Fúngico/análise , Amplificação de Genes , Genes Fúngicos/genética , Marcadores Genéticos , Saccharomyces cerevisiae/genéticaRESUMO
A DNA sequence coding for a subtype of the hirudin variant HV1 was expressed in the methylotrophic yeast Hansenula polymorpha from a strongly inducible promoter element derived from a gene of the methanol metabolism pathway. For secretion, the coding sequence was fused to the KEX2 recognition site of three different prepro segments engineered from the MF alpha 1 gene of Saccharomyces cerevisiae, the glucoamylase (GAM1) gene of Schwanniomyces occidentalis and the gene for a crustacean hyperglycemic hormone from the shore crab Carcinus maenas. In all three cases, correct processing of the precursor molecule and efficient secretion of the mature protein were observed. In fermentations on a 10-1 scale of a transformant strain harbouring a MF alpha 1/hirudin-gene fusion yields in the range of grams per litre could be obtained. The majority of the secreted product was identified as the full-length 65-amino-acid hirudin. Only small amounts of a truncated 63-amino- acid product, frequently observed in S. cerevisiae-based expression systems, could be detected.
Assuntos
Hirudinas/metabolismo , Pichia/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes , Braquiúros/genética , Fermentação , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Glucana 1,4-alfa-Glucosidase/biossíntese , Glucana 1,4-alfa-Glucosidase/genética , Hirudinas/biossíntese , Hirudinas/genética , Hormônios de Invertebrado/biossíntese , Hormônios de Invertebrado/genética , Fator de Acasalamento , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Biossíntese Peptídica , Peptídeos/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genéticaRESUMO
This article describes the expression of the hirudin gene heterologously in the methylotrophic yeast Hansenula polymorpha, the establishment of an industrial-scale production process and the subsequent clinical development of polyethylene glycol (PEG)-hirudin. PEGylation increases the molecular weight of hirudin, thereby reducing its kidney filtration rate and immunogenicity and increasing its half-life in the circulation.
Assuntos
Hirudinas/síntese química , Microbiologia Industrial , Pichia/genética , Animais , Antitrombinas/síntese química , Antitrombinas/genética , Antitrombinas/uso terapêutico , Ensaios Clínicos como Assunto , Terapia com Hirudina , Hirudinas/análogos & derivados , Hirudinas/genética , Humanos , Farmacocinética , Transformação GenéticaRESUMO
The exploitation of recombinant DNA technology to engineer expression systems for heterologous proteins represented a major task within the field of biotechnology during the last decade. Yeasts attracted the attention of molecular biologists because of properties most favourable for their use as hosts in heterologous protein production. Yeasts follow the general eukaryotic posttranslational modification pattern of expressed polypeptides, exhibit the ability to secrete heterologous proteins and benefit from an established fermentation technology. Aside from the baker's yeast Saccharomyces cerevisiae, an increasing number of alternative non-Saccharomyces yeast species are used as expression systems in basic research and for an industrial application. In the following review a selection from the different yeast systems is described and compared.