Cloning, targeted disruption and heterologous expression of theKluyveromyces marxianus endopolygalacturonase gene (EPG1 )

Šiekštelė, Rimantas; Bartkeviciūte, D; Sasnauskas, Kęstutis

doi:10.1002/(sici)1097-0061(19990315)15:4<311::aid-yea379>3.0.co;2-9

Cited by 27 publications

(14 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Maximal activities were obtained at pH 4.8, and over 80 % of maximum activity was maintained between pH 4.4 and 5.2. These values are within the range observed in pectinase enzymes, in the acidic region between pH 4 and 5.5, according to works with Saccharomyces paradoxus, K. marxianus CECT 1043, K. marxianus, K. wickerhamii IFO 1675, and S. cerevisiae (Blanco et al 1994;Yoshitake et al 1994;Siekstele et al 1999;Serrat et al 2002;Eschstruth and Divol 2011). At the typical pH of grape juice (pH 3.2-3.6) the enzymatic extract of K. marxianus NRRL-Y-7571 expressed approximately 70 % of its maximum activity.…”

Section: Resultssupporting

confidence: 85%

Comparison of a pectinolytic extract of Kluyveromyces marxianus and a commercial enzyme preparation in the production of Ives (Vitis labrusca) grape juice

Piemolini-Barreto

Antônio

Echeverrigaray

2015

World J Microbiol Biotechnol

View full text Add to dashboard Cite

This study analyses the effect of the crude enzymatic extract produced by Kluyveromyces marxianus (EEB) in the maceration and clarification of juice produced from Ives (Vitis labrusca) grapes compared to the commercial enzyme preparation Pectinex(®)Ultra Color (PEC). Treatments were conducted with a total pectinolytic activity of 1 U/mL of fruit juice, at 40 °C, for 60 min. After the enzymatic treatment, the juices were evaluated with respect to yield, viscosity, and degree of clarification, as well as the effect of the enzymes on polyphenol concentration, anthocyanins, and juice color. The results showed that both EEB and PEC increase yield, reduce viscosity and contribute to the clarification of grape juice. After enzyme treatment with the EEB preparation, the extraction yield increased 28.02 % and decreased 50.70 % in viscosity during the maceration of the pulp. During the juice production process clarification increased 11.91 %. With PEC, higher values for these parameters: 42.36, 63.20, and 26.81 % respectively, were achieved. The addition of EEB resulted in grape juice with better color intensity and extraction of phenolic compounds and anthocyanins. Considering all comparison criteria, the enzymatic extract of K. marxianus NRRL-Y-7571 can potentially be used in the production of juice.

show abstract

Section: Resultssupporting

confidence: 85%

Comparison of a pectinolytic extract of Kluyveromyces marxianus and a commercial enzyme preparation in the production of Ives (Vitis labrusca) grape juice

Piemolini-Barreto

Antônio

Echeverrigaray

2015

World J Microbiol Biotechnol

View full text Add to dashboard Cite

show abstract

“…Plasmid pKDU–HGH was used for investigation of secretion of human growth hormone (HGH). This plasmid was constructed on the basis of the pKDU7 vector (Šiekštelė et al , 1999) and included pUC19, the full‐size K. drosophilarum plasmid pKD1, the Kluyveromyces marxianus URA3 gene, and an expression cassette containing the K. lactis LAC4 gene promoter, S. cerevisae α‐factor signal sequence encoding DNA, the gene of HGH and the K. lactis LAC4 transcription terminator. The centromeric plasmid KCp491 was kindly provided by Dr M. Wesolowski‐Louvel (Wesolowski‐Louvel et al , 1996).…”

Section: Methodsmentioning

confidence: 99%

Studies of yeast Kluyveromyces lactis mutations conferring super‐secretion of recombinant proteins

Bartkeviciūte¹,

Sasnauskas²

2002

Yeast

View full text Add to dashboard Cite

We have isolated mutants responsible for a super-secretion phenotype in Kluyveromyces lactis using the gene coding for a Bacillus amyloliquefaciens α-amylase as a marker for secretion. These mutations defined two groups, dominant and recessive. The recessive mutant strain, which secreted the heterologous protein in five-fold excess compared to the wild-type strain, was used for the cloning of genes, restraining the super-secreting phenotype. In screening for genes affecting supersecreting phenotype, we found that multiple copies of 10 different independently isolated DNA sequences suppressed the super-secreting phenotype. The first among the genes characterized, named KlSEL1 ('secretion lowering') showed homology to Saccharomyces cerevisiae ORF YML013w. The KlSEL1 gene is predicted to encode a polypeptide of 620 amino acid residues containing a putative transmembrane domain and UBX domain, characteristic for the ubiquitin-regulatory proteins. We demonstrated that the disruption of the SEL1 orthologues in K. lactis and S. cerevisiae conferred the super-secreting phenotype. SEL1 isolated from S. cerevisiae suppressed the super-secretion phenotype in K. lactis klsel1 strain, likewise homologous KlSEL1 . No other phenotypic features for strains lacking the SEL1 gene were noticed except for the S. cerevisiae mutant growth being notably slower than in a wt strain. No growth changes were observed in the K. lactis klsel1 mutant. The set of genes (suppressors of over-secreting phenotype) could be attractive for further analysis of gene functions, super-secreting mechanisms and construction of new strains. This collection could be useful for the expedient construction of reduced yeast genomes, optimized for heterologous protein secretion. The KlSEL1 gene has been assigned EMBL Accession No. AJ488285.

show abstract

“…Siekstele et al overexpressed the endopolygalacturonase (EPG1) gene of K. marxianus in an EPG1 deletion strain of the same background [23]. In contrast to native EPG1 expression, overexpression of this gene led to an additional band on the protein gel, of higher molecular weight, suggesting that a more glycosylated form of the enzyme is secreted during overexpression, together with the native form.…”

Section: Introductionmentioning

confidence: 99%

Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus

et al. 2010

View full text Add to dashboard Cite

BackgroundIn spite of its advantageous physiological properties for bioprocess applications, the use of the yeast Kluyveromyces marxianus as a host for heterologous protein production has been very limited, in constrast to its close relative Kluyveromyces lactis. In the present work, the model protein glucose oxidase (GOX) from Aspergillus niger was cloned into K. marxianus CBS 6556 and into K. lactis CBS 2359 using three different expression systems. We aimed at verifying how each expression system would affect protein expression, secretion/localization, post-translational modification, and biochemical properties.ResultsThe highest GOX expression levels (1552 units of secreted protein per gram dry cell weight) were achieved using an episomal system, in which the INU1 promoter and terminator were used to drive heterologous gene expression, together with the INU1 prepro sequence, which was employed to drive secretion of the enzyme. In all cases, GOX was mainly secreted, remaining either in the periplasmic space or in the culture supernatant. Whereas the use of genetic elements from Saccharomyces cerevisiae to drive heterologous protein expression led to higher expression levels in K. lactis than in K. marxianus, the use of INU1 genetic elements clearly led to the opposite result. The biochemical characterization of GOX confirmed the correct expression of the protein and showed that K. marxianus has a tendency to hyperglycosylate the protein, in a similar way as already observed for other yeasts, although this tendency seems to be smaller than the one of e.g. K. lactis and S. cerevisiae. Hyperglycosylation of GOX does not seem to affect its affinity for the substrate, nor its activity.ConclusionsTaken together, our results indicate that K. marxianus is indeed a good host for the expression of heterologous proteins, not only for its physiological properties, but also because it correctly secretes and folds these proteins.

show abstract

Cloning, targeted disruption and heterologous expression of theKluyveromyces marxianus endopolygalacturonase gene (EPG1 )

Cited by 27 publications

References 52 publications

Comparison of a pectinolytic extract of Kluyveromyces marxianus and a commercial enzyme preparation in the production of Ives (Vitis labrusca) grape juice

Comparison of a pectinolytic extract of Kluyveromyces marxianus and a commercial enzyme preparation in the production of Ives (Vitis labrusca) grape juice

Studies of yeast Kluyveromyces lactis mutations conferring super‐secretion of recombinant proteins

Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus

Contact Info

Product

Resources

About