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
A cloned cDNA, generated from mRNA isolates of phosphate-derepressed H. polymorpha cells, was identified to harbour an incomplete sequence of the coding region for a repressible acid phosphatase. The cDNA fragment served as a probe to screen a plasmid library of H. polymorpha genomic DNA. A particular clone, p606, of a 1.9-kb insert contained a complete copy of the PHO1 gene. Sequencing revealed the presence of a 1329-nucleotide open reading frame encoding a protein of 442 amino acids with a calculated M(r) of 49400. The encoded protein has an N-terminal 17-amino-acid secretory leader sequence and seven potential N-glycosylation sites. The leader cleavage site was confirmed by N-terminal sequencing of the purified enzyme. The nucleotide sequence is 48.9% homologous, the derived amino acid sequence 36% homologous to its Saccharomyces cerevisiae counterpart. The derived amino acid sequence harbours a consensus sequence RHGXRXP, previously identified as a sequence involved in active-site formation of acid phosphatases. The PHO1 promoter and the secretion leader sequence present promising new tools for heterologous gene expression.
Assuntos
Fosfatase Ácida/genética , Sequência Consenso , Regulação Fúngica da Expressão Gênica , Pichia/enzimologia , Homologia de Sequência de Aminoácidos , Fosfatase Ácida/química , Fosfatase Ácida/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Antifúngicos/biossíntese , Sequência de Bases , Western Blotting , Cromatografia em Gel , Cromatografia por Troca Iônica , Clonagem Molecular , DNA Complementar/química , Eletroforese em Gel de Poliacrilamida , Escherichia coli/química , Regulação Enzimológica da Expressão Gênica , Biblioteca Gênica , Dados de Sequência Molecular , Pichia/genética , RNA Mensageiro/química , Coelhos , Mapeamento por Restrição , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
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.