Kluyveromyces as a Host for Heterologous Gene Expression: Expression and Secretion of Prochymosin

Berg, Johan; Laken, K.J. van der; Ooyen, A.J.J. van; Renniers, Ton C.; Rietveld, Krijn; Schaap, A. Paul; Brake, Anthony J.; Bishop, Robert; Schultz, Konrad; Moyer, Donna L.

doi:10.1038/nbt0290-135

Cited by 159 publications

(77 citation statements)

References 24 publications

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“…Interest in K. lactis originated from its ability to utilize lactose as carbon source and from the possibility of its use for the production of b-galactosidase. New interest in this yeast came from its good properties in secretion of heterologous proteins (Fleer et al, 1991a, b;Swinkels et al, 1993;Van De Berg et al, 1990).…”

Section: Introductionmentioning

confidence: 99%

Isolation and study of KlLSM4 , a Kluyveromyces lactis gene homologous to the essential gene LSM4 of Saccharomyces cerevisiae

Mazzoni

Falcone

2001

Yeast

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We have isolated the KlLSM4 gene as a multicopy suppressor of a Kluyveromyces lactis mutant which shows a rag x phenotype (resistance to antimycin A on glucose). This gene is homologous to the ScLSM4 of Saccharomyces cerevisiae, which codes for an essential 187 amino acid protein containing Sm-like domains. These motifs are present in the evolutionarily conserved family of the Sm-like proteins, which are involved in a large number of cellular processes, including pre-mRNA splicing and mRNA decapping. We demonstrated that the first 72 amino acids of KlLsm4p, which contain the Sm-like domains, can restore cell viability in both K. lactis and S. cerevisiae cells lacking the wild-type protein. However, the absence of the carboxy-terminal region resulted in a remarkable loss of cell viability in the stationary phase. The KlLSM4 sequence has been deposited in the EMBL Data library under Accession No. AJ311719.

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Section: Introductionmentioning

confidence: 99%

Isolation and study of KlLSM4 , a Kluyveromyces lactis gene homologous to the essential gene LSM4 of Saccharomyces cerevisiae

Mazzoni

Falcone

2001

Yeast

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“…Heterologous expression is now also performed in P. angusta, Y. lipolytica, and K. lactis. The aspartyl protease chymosin, for example, which is the active constituent of the cheese rennet, is overproduced and secreted from K. lactis [47]. The number of sophisticated vectors speci¢cally designed for appropriate expression in various yeast backgrounds is rapidly extending [48].…”

Section: Yeasts As Cell Factoriesmentioning

confidence: 99%

Genomic Exploration of the Hemiascomycetous Yeasts: 1. A set of yeast species for molecular evolution studies¹

et al. 2000

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The identification of molecular evolutionary mechanisms in eukaryotes is approached by a comparative genomics study of a homogeneous group of species classified as Hemiascomycetes. This group includes Saccharomyces cerevisiae, the first eukaryotic genome entirely sequenced, back in 1996. A random sequencing analysis has been performed on 13 different species sharing a small genome size and a low frequency of introns. Detailed information is provided in the 20 following papers. Additional tables available on websites describe the ca. 20 000 newly identified genes. This wealth of data, so far unique among eukaryotes, allowed us to examine the conservation of chromosome maps, to identify the`yeast-specific' genes, and to review the distribution of gene families into functional classes. This project conducted by a network of seven French laboratories has been designated`Gënolevures'. ß

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“…These yeasts have a GRAS status and are now well accessible for molecular genetic techniques. They are used for the production of a number of heterologous proteins (Romanos et al 1992), including bovine chymosin, a milk-clotting enzyme used in cheese manufacturing (van den Berg et al 1990). Large-scale industrial processes have also been developed in the past for Yarrowia lipolytica, in particular for the production of citric acid and SCP.…”

Section: Hostsmentioning

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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Kluyveromyces as a Host for Heterologous Gene Expression: Expression and Secretion of Prochymosin

Cited by 159 publications

References 24 publications

Isolation and study of KlLSM4 , a Kluyveromyces lactis gene homologous to the essential gene LSM4 of Saccharomyces cerevisiae

Isolation and study of KlLSM4 , a Kluyveromyces lactis gene homologous to the essential gene LSM4 of Saccharomyces cerevisiae

Genomic Exploration of the Hemiascomycetous Yeasts: 1. A set of yeast species for molecular evolution studies¹

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

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Kluyveromyces as a Host for Heterologous Gene Expression: Expression and Secretion of Prochymosin

Cited by 159 publications

References 24 publications

Isolation and study of KlLSM4 , a Kluyveromyces lactis gene homologous to the essential gene LSM4 of Saccharomyces cerevisiae

Isolation and study of KlLSM4 , a Kluyveromyces lactis gene homologous to the essential gene LSM4 of Saccharomyces cerevisiae

Genomic Exploration of the Hemiascomycetous Yeasts: 1. A set of yeast species for molecular evolution studies1

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

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Genomic Exploration of the Hemiascomycetous Yeasts: 1. A set of yeast species for molecular evolution studies¹