RESUMO
Ectoine and 5-hydroxyectoine belong to the family of compatible solutes which are known to mainly contribute to the adaptation of the cell to osmotic stress by mediation of a constant turgor. In addition the cell's essential functions are maintained under stress conditions like high salinity, heat or aridity stress. Hansenula polymorpha was engineered to catalyze the transformation of monomeric substrates to 5-hydroxyectoine. For this purpose four genes encoding the enzymes of the 5-hydroxyectoine biosynthesis pathway of Halomonas elongata, EctA, EctB, EctC, and EctD, were inserted into the genome of H. polymorpha. Subsequently the syntheses of ectoine and 5-hydroxyectoine were analyzed and optimized. We showed that H. polymorpha is a suitable system for recombinant 5-hydroxyectoine synthesis in gram per liter scale (2.8 g L⻹ culture supernatant, 365 µmol/g dcw) in which almost 100% conversion of ectoine to 5-hydroxyectoine without necessity of high salinity were achieved.
Assuntos
Diamino Aminoácidos/biossíntese , Diamino Aminoácidos/genética , Pichia/genética , Pichia/metabolismo , Diamino Aminoácidos/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Genes Bacterianos , Halomonas/genética , Halomonas/metabolismo , Concentração de Íons de Hidrogênio , SalinidadeRESUMO
The literature as well as databases are ambiguous about the exact start of human interleukin-6 (IL-6)--three possibilities for the initiation of the mature protein are described. These three variants of IL-6, different in the exact initiation of the mature protein (A28, P29, or V30), were expressed in Hansenula polymorpha using the Saccharomyces cerevisiae MFα prepro sequence instead of the homologous pre sequence. All three IL-6 variants were secreted but the processing by the Kex2 protease showed significant differences. V30-IL-6 showed correctly processed material but also a molecule species of higher molecular weight indicating incomplete processing of the MFα pro peptide. P29-IL-6 did not yield any correctly processed IL-6, instead only the unprocessed pro form was found in the culture supernatant. Only A28-IL-6 led to 100% correctly processed material. N-terminal sequencing of this material revealed a start at V30--obviously the first two amino acids (Ala28-Pro29) have been removed by a so far unknown protease. Thus expression of both A28-IL-6 and V30-IL-6 as MFα prepro fusion proteins resulted in the very same mature V30-IL-6, however, the ratio of correctly processed molecules was significantly higher in the case of A28-IL-6. The expression of an MFα prepro-interferon α-2a (IFNα-2a) fusion protein in H. polymorpha leads to about 50% correctly processed molecules and 50% misprocessed forms which contain part of the pro peptide at the N-termini. The insertion of A28 and P29 of IL-6 between the pro peptide and the start of the mature IFNα-2a led to correct processing and elimination of all high molecular weight isoforms observed in earlier experiments.
Assuntos
Biotecnologia/métodos , Proteínas Fúngicas/genética , Peptídeos/genética , Pichia/genética , Proteínas Recombinantes de Fusão/genética , Sequência de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Proteínas Fúngicas/metabolismo , Variação Genética , Interferon alfa-2 , Interferon-alfa/genética , Interferon-alfa/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Fator de Acasalamento , Dados de Sequência Molecular , Peptídeos/metabolismo , Pichia/metabolismo , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The iron storage protein ferritin is a member of the non-heme iron protein family. It can store and release iron, therefore it prevents the cell from damage caused by iron-dioxygen reactions as well as it provides iron for biological processing. To study whether the human ferritin heavy chain (FTH1) can be expressed in Hansenula polymorpha, we integrated an expression cassette for FTH1 and analyzed the protein expression. We found very efficient expression of FTH1 and obtained yields up to 1.9 g/L under non-optimized conditions. Based on this result we designed a FTH1-PTH fusion protein to successfully express the parathyroid hormone fragment 1-34 (PTH) for the first time intracellular in H. polymorpha.
