Stable Multicopy Vectors for High–Level Secretion of Recombinant Human Serum Albumin by Kluyveromyces Yeasts

Fleer, Reinhard; Yeh, Patrice; Amellal, N.; Maury, Isabelle; Fournier, Alain; Bacchetta, F.; Baduel, P.; Jung, Gérard; L'Hôte, H.; Becquart, Jérôme; Fukuhara, Hiroshi; Mayaux, Jean‐François

doi:10.1038/nbt1091-968

Cited by 146 publications

(83 citation statements)

References 34 publications

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“…To test whether the P LAC4 variants were able to direct the expression of a heterologous gene in K. lactis, the gene encoding HSA was cloned into each of the pGBN vectors. HSA was chosen as a reporter protein due to its high-level expression and efficient secretion from K. lactis when expressed from wild-type P LAC4 (7). K. lactis strains containing each of the integrated pGBN1-HSA expression vectors were grown to saturation in YPGal medium, and secreted proteins in the spent culture medium (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Kluyveromyces lactis LAC4 Promoter Variants That Lack Function in Bacteria but Retain Full Function in K. lactis

Colussi

Taron

2005

Appl Environ Microbiol

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The strong LAC4 promoter (P LAC4 ) from Kluyveromyces lactis has been extensively used to drive expression of heterologous proteins in this industrially important yeast. A drawback of this expression method is the serendipitous ability of P LAC4 to promote gene expression in Escherichia coli. This can interfere with the process of assembling expression constructs in E. coli cells prior to their introduction into yeast cells, especially if the cloned gene encodes a protein that is detrimental to bacteria. In this study, we created a series of P LAC4 variants by targeted mutagenesis of three DNA sequences (PBI, PBII, and PBIII) that resemble the E. coli Pribnow box element of bacterial promoters and that reside immediately upstream of two E. coli transcription initiation sites associated with P LAC4 . Mutation of PBI reduced the bacterial expression of a reporter protein (green fluorescent protein [GFP]) by ϳ87%, whereas mutation of PBII and PBIII had little effect on GFP expression. Deletion of all three sequences completely eliminated GFP expression. Additionally, each promoter variant expressed human serum albumin in K. lactis cells to levels comparable to wild-type P LAC4 . We created a novel integrative expression vector (pKLAC1) containing the P LAC4 variant lacking PBI and used it to successfully clone and express the catalytic subunit of bovine enterokinase, a protease that has historically been problematic in E. coli cells. The pKLAC1 vector should aid in the cloning of other potentially toxic genes in E. coli prior to their expression in K. lactis.

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Section: Resultsmentioning

confidence: 99%

“…In K. lactis, expression of heterologous genes has been achieved using various promoters isolated from native K. lactis genes (7,10,11,15) or using promoters originating from other yeasts (1,2,8,15,17). However, the K. lactis LAC4 promoter (P LAC4 ) is often used because of its strength and inducible expression (14).…”

mentioning

confidence: 99%

Kluyveromyces lactis LAC4 Promoter Variants That Lack Function in Bacteria but Retain Full Function in K. lactis

Colussi

Taron

2005

Appl Environ Microbiol

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“…Under non-selective conditions, stability of pKDl-derived plasmids depends strongly on the host strain (Fleer et al 1991). After ten generations of non-selective growth, plasmid loss varied between 10 and 80%.…”

Section: Episomal and Integrating Expression Vectorsmentioning

confidence: 99%

Physiological and technological aspects of large-scale heterologous-protein production with yeasts

Hensing

Rouwenhorst

Heijnen

et al. 1995

Antonie van Leeuwenhoek

138

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Commercial production of heterologous proteins by yeasts has gained considerable interest. Expression systems have been developed for Saccharomyces cerevisiae and a number of other yeasts. Generally, much attention is paid to the molecular aspects of heterologous-gene expression. The success of this approach is indicated by the high expression levels that have been obtained in shake-flask cultures. For large-scale production however, possibilities and restrictions related to host-strain physiology and fermentation technology also have to be considered. In this review, these physiological and technological aspects have been evaluated with the aid of numerical simulations. Factors that affect the choice of a carbon substrate for large-scale production involve price, purity and solubility. Since oxygen demand and heat production (which are closely linked) limit the attainable growth rate in large-scale processes, the biomass yield on oxygen is also a key parameter. Large-scale processes impose restrictions on the expression system. Many promoter systems that work well in small-scale systems cannot be implemented in industrial environments. Furthermore, large-scale fed-batch fermentations involve a substantial number of generations. Therefore, even low expression-cassette instability has a profound effect on the overall productivity of the system. Multicopy-integration systems may provide highly stable expression systems for industrial processes. Large-scale fed-batch processes are typically performed at a low growth rate. Therefore, effects of a low growth rate on the physiology and product formation rates of yeasts are of key importance. Due to the low growth rates in the industrial process, a substantial part of the substrate carbon is expended to meet maintenance-energy requirements. Factors that reduce maintenance-energy requirements will therefore have a positive effect on product yield. The relationship between specific growth rate and specific product formation rate (kg product-[kg biomass]-1.h-I) is the main factor influencing production levels in large-scale production processes. Expression systems characterized by a high specific rate of product formation at low specific growth rates are highly favourable for large-scale heterologous-protein production.

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“…lactis. The A. adeninivorans glucoamylase gene was fused to the lactose-inducible LAC4 promoter and cloned into a multicopy pKD-1-based expression vector (Fleer et al, 1991). Expression plasmids pKN18 (no insert) and pKNH6 (glucoamylase-encoding) were used to transform two different K. lactis strains, MW98-8C and FB05.…”

Section: Secretion Ofmentioning

confidence: 99%

Secretion of active anti-Ras single-chain Fv antibody by the yeasts Yarrowia lipolytica and Kluyveromyces lactis

Swennen

Paul²,

Vernis

et al. 2002

Microbiology

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Stable Multicopy Vectors for High–Level Secretion of Recombinant Human Serum Albumin by Kluyveromyces Yeasts

Cited by 146 publications

References 34 publications

Kluyveromyces lactis LAC4 Promoter Variants That Lack Function in Bacteria but Retain Full Function in K. lactis

Kluyveromyces lactis LAC4 Promoter Variants That Lack Function in Bacteria but Retain Full Function in K. lactis

Physiological and technological aspects of large-scale heterologous-protein production with yeasts

Secretion of active anti-Ras single-chain Fv antibody by the yeasts Yarrowia lipolytica and Kluyveromyces lactis

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