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Secretion of human serum albumin by Kluyveromyces lactis overexpressing KlPDI1 and KlERO1.

ABSTRACT: The control of protein conformation during translocation through the endoplasmic reticulum is often a bottleneck for heterologous protein production. The core pathway of the oxidative folding machinery includes two conserved proteins: Pdi1p and Ero1p. We increased the dosage of the genes encoding these proteins in the yeast Kluyveromyces lactis and evaluated the secretion of heterologous proteins. KlERO1, an orthologue of Saccharomyces cerevisiae ERO1, was cloned by functional complementation of the ts phenotype of an Scero1 mutant. The expression of KlERO1 was induced by treatment of the cells with dithiothreitol and by overexpression of human serum albumin (HSA), a disulfide bond-rich protein. Duplication of either PDI1 or ERO1 led to a similar increase in HSA yield. Duplication of both genes accelerated the secretion of HSA and improved cell growth rate and yield. Increasing the dosage of KlERO1 did not affect the production of human interleukin 1beta, a protein that has no disulfide bridges. The results confirm that the ERO1 genes of S. cerevisiae and K. lactis are functionally similar even though portions of their coding sequence are quite different and the phenotypes of mutants overexpressing the genes differ. The marked effects of KlERO1 copy number on the expression of heterologous proteins with a high number of disulfide bridges suggests that control of KlERO1 and KlPDI1 is important for the production of high levels of heterologous proteins of this type.

SUBMITTER: Lodi T

PROVIDER: S-EPMC1183311 | biostudies-literature | 2005 Aug

REPOSITORIES: biostudies-literature

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Secretion of human serum albumin by Kluyveromyces lactis overexpressing KlPDI1 and KlERO1.

Lodi Tiziana T Neglia Barbara B Donnini Claudia C

Applied and environmental microbiology 20050801 8

The control of protein conformation during translocation through the endoplasmic reticulum is often a bottleneck for heterologous protein production. The core pathway of the oxidative folding machinery includes two conserved proteins: Pdi1p and Ero1p. We increased the dosage of the genes encoding these proteins in the yeast Kluyveromyces lactis and evaluated the secretion of heterologous proteins. KlERO1, an orthologue of Saccharomyces cerevisiae ERO1, was cloned by functional complementation of ...[more]

PMID: 16085825

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Project description:BACKGROUND: Even before having its genome sequence published in 2004, Kluyveromyces lactis had long been considered a model organism for studies in genetics and physiology. Research on Kluyveromyces lactis is quite advanced and this yeast species is one of the few with which it is possible to perform formal genetic analysis. Nevertheless, until now, no complete metabolic functional annotation has been performed to the proteins encoded in the Kluyveromyces lactis genome. RESULTS: In this work, a new metabolic genome-wide functional re-annotation of the proteins encoded in the Kluyveromyces lactis genome was performed, resulting in the annotation of 1759 genes with metabolic functions, and the development of a methodology supported by merlin (software developed in-house). The new annotation includes novelties, such as the assignment of transporter superfamily numbers to genes identified as transporter proteins. Thus, the genes annotated with metabolic functions could be exclusively enzymatic (1410 genes), transporter proteins encoding genes (301 genes) or have both metabolic activities (48 genes). The new annotation produced by this work largely surpassed the Kluyveromyces lactis currently available annotations. A comparison with KEGG's annotation revealed a match with 844 (~90%) of the genes annotated by KEGG, while adding 850 new gene annotations. Moreover, there are 32 genes with annotations different from KEGG. CONCLUSIONS: The methodology developed throughout this work can be used to re-annotate any yeast or, with a little tweak of the reference organism, the proteins encoded in any sequenced genome. The new annotation provided by this study offers basic knowledge which might be useful for the scientific community working on this model yeast, because new functions have been identified for the so-called metabolic genes. Furthermore, it served as the basis for the reconstruction of a compartmentalized, genome-scale metabolic model of Kluyveromyces lactis, which is currently being finished.

Project description:The transcription factor KlPdr1p, belonging to the Zn2Cys6 family, is a central regulator of efflux pump expression in Kluyveromyces lactis. To better understand how KlPDR1-mediated drug resistance is achieved in K. lactis, we used DNA microarrays to identify genes whose expression was affected by deletion or overexpression of the KlPDR1 gene. All microarray experiments were performed using the 30K Kluyveromyces lactis NRRL Y-1140 microarray (MYcroarray, 5692 Plymouth Road, Ann Arbor, MI 48105, USA). Exponentially growing (1 x 107 cells ml-1) K. lactis PM6-7A cells (wild-type, PM6-7A/pdr1∆ and the wild-type transformed with multicopy plasmid carrying the gain-of-function allele of KlPDR1* gene) (Balazfyova et al. 2013), were collected and total RNA was isolated using RNeasy midi kit (Qiagen GmbH, Germany). 1 mg of total RNA was linearly amplified and labelled using Amino allyl MessageAmpII aRNA Amplification kit (Ambion, USA) with two different fluorescent dyes; AlexaFluor647 and AlexaFluor555 (Life Technologies, Germany). 4 µg of labelled RNA was hybridized (18 h at 45°C) in 6x SSPE with addition of formamide (10%), tween-20 (0,01%) and microarray-specofoc control oligos (1%, MYcroarray, USA). After washing, microarray images and two-color GPR output files were obtained using the microarray scanner InnoScan 900 and Mapix software version 7.3.1 (Inopsys, France). The two-color GPR files were processed using the R version 3.0.2 (R Core Team (2014). R: A language and enviroment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org) and functions available in the limma package (Smyth 2005). Briefly, the two-color GPR files were omported using the read.maimages() function, background-substracted using the “minimum“ method and within-array-normalized using the “loess“ method. The between-array normalisation was achieved using the “Aquantile“ method. For further analysis, only genes with log2FC ˃2 were selected and confirmed using qPCR.

Dataset Information

Secretion of human serum albumin by Kluyveromyces lactis overexpressing KlPDI1 and KlERO1.

Publications

Secretion of human serum albumin by Kluyveromyces lactis overexpressing KlPDI1 and KlERO1.

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