Enhanced production of anticoagulant hirudin in recombinant Saccharomyces cerevisiae by chromosomal δ-integration

Kim, Myoung Dong; Rhee, Shin Hyung; Seo, Jin Ho

doi:10.1016/s0168-1656(00)00376-x

Cited by 33 publications

(30 citation statements)

References 26 publications

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“…Strain CB2I20 was shown to be the best FAEE producer, and its FAEE production reached 34 mg/L, which was sixfold higher compared to the one achieved using a 2 µm based plasmid. The improvement is significant as the production of interesting chemicals or proteins in previous integration strains only reached levels comparable to the multicopy plasmid system (Kim et al, ; Lee and Silva, ; Oliveira et al, ; Tyo et al, ).…”

Section: Discussionmentioning

confidence: 99%

“…Several strategies have been explored to obtain multicopy integration of target gene(s) both in E. coli and S. cerevisiae by using repetitive chromosomal DNA sequences (Balbás and Gosset, 2001;Boeke et al, 1988;Cho et al, 1999;Fujii et al, 1990;Kim et al, 2001;Kimura et al, 1995;Lee and Da Silva, 2006;Lee and Silva, 1997;Wang et al, 1996;Yamada et al, 2010), and the concept was extended to other industrially relevant microorganisms (Pecota et al, 2007;Reeves et al, 2007;Yeh et al, 2010). In the case of S. cerevisiae, the integration targets were, for example, rRNA genes (Fujii et al, 1990) and sigma (Kudia and Nicolas, 1992) or deltasequences (Cho et al, 1999;Kim et al, 2001;Kimura et al, 1995;Lee and Da Silva, 2006;Lee and Silva, 1997;Wang et al, 1996;Yamada et al, 2010;Yuan and Ching, 2013). The delta-sequences represent long terminal repeats of Ty retrotransposons positioned throughout the genome of S. cerevisiae (Boeke et al, 1988), and the delta-integrated yeast showed integration of the target gene in several copies.…”

Section: Introductionmentioning

confidence: 99%

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Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters

Shi

Valle-Rodríguez

Siewers

et al. 2014

Biotech & Bioengineering

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In recent years, significant advances have been made to engineer robust microbes for overproducing biochemical products from renewable resources. These accomplishments have to a large extend been based on plasmid based methods. However, plasmid maintenance may cause a metabolic burden on the host cell and plasmid-based overexpression of genes can result in genetically unstable strains, which contributes to loss in productivity. Here, a chromosome engineering method based on delta integration was applied in Saccharomyces cerevisiae for the production of fatty acid ethyl esters (FAEEs), which can be directly used as biodiesel and would be a possible substitute for conventional petroleum-based diesel. An integration construct was designed and integrated into chromosomal delta sequences by repetitive transformation, which resulted in 1-6 copies of the integration construct per genome. The corresponding FAEE production increased up to 34 mg/L, which is an about sixfold increase compared to the equivalent plasmid-based producer. The integrated cassette in the yeast genome was stably maintained in nonselective medium after deletion of RAD52 which is essential for efficient homologous recombination. To obtain a further increase of FAEE production, genes encoding endogenous acyl-CoA binding protein (ACB1) and a bacterial NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (gapN) were overexpressed in the final integration strain, which resulted in another 40% percent increase in FAEE production. Our integration strategy enables easy engineering of strains with adjustable gene copy numbers integrated into the genome and this allows for an easy evaluation of the effect of the gene copy number on pathway flux. It therefore represents a valuable tool for introducing and expressing a heterologous pathway in yeast.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters

Shi

Valle-Rodríguez

Siewers

et al. 2014

Biotech & Bioengineering

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“…Production levels by the YEp‐type expression vectors were significantly higher than levels obtained with the YIp‐type vector both in batch and continuous culture. The increased gene dosage of the YEp‐type vectors thus dramatically improved heterologous protein production, as has been frequently reported (Smith et al , 1985; Bitter et al , 1987; Denis and Drouin, 1987; Kaisho et al , 1989; Janes et al , 1990; Weber et al , 1992; Mendoza‐Vega et al , 1994; Compagno et al , 1996; Lopes et al , 1996; Nacken et al , 1996; Gellissen and Hollenberg, 1997; Ljubijankic et al , 1999; Park et al , 2000; Vassileva et al , 2001; Kim et al , 2001). The previously reported saturation of the protein secretory capacity of the host strain by high‐level foreign gene expression from the multicopy YEp‐type vector (Wittrup et al , 1994; Tuite and Freedman, 1994; Parekh et al , 1995; Parekh and Wittrup, 1997) was not observed.…”

Section: Discussionmentioning

confidence: 99%

Comparison of three expression systems for heterologous xylanase production byS. cerevisiaein defined medium

Görgens

Planas

Zyl

et al. 2004

Yeast

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The influence of the auxotrophic deficiencies of the host strain and expression vector selection on the production of a heterologous protein was investigated. Heterologous xylanase production by two prototrophic S. cerevisiae transformants, containing either a plasmid-based, YEp-type expression system or an integrative, YIp-type expression system, were compared with production by an auxotrophic transformant, containing an identical YEp-type expression system, in batch and continuous cultivation, using a chemically defined medium. Heterologous xylanase production by the auxotrophic strains in defined medium was critically dependent on the availability of amino acids, as extracellular xylanase production increased dramatically when amino acids were over-consumed from the medium to the point of saturating the cell. Saturation with amino acids, indicated by an increased leakage of amino acids from the cell, was thus a prerequisite for high level of heterologous protein production by the auxotrophic strain. Maximal xylanase production levels by the auxotrophic strain corresponded to the levels obtained with a similar prototrophic strain during cultivation in defined medium without amino acids. Superfluous auxotrophic markers thus had a strong deleterious effect on heterologous protein production by recombinant yeasts, and the use of such strains should be limited to initial exploratory investigations. The increased copy number and foreign gene dosage of the YEp-based expression vector, stabilized by the ura3 fur1 autoselection system, significantly improved production levels of heterologous xylanase, compared to the YIp system, which is based on a single integration into the yeast genome. No evidence was found of the possible saturation of the host secretory capacity by multicopy overexpression. Stable production of heterologous xylanase at high levels by the prototrophic YEp-based recombinant strain, compared to the YIp system, was demonstrated.

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“…Briefly, identical amounts (about 20 μg of each plasmid) of three co-marked δ-integrative plasmids [19,20] (pδW-PGAGBGL, pδW-PGAGEG and pδW-PGAGCBH, which allow expression of BGL, EG and CBH, respectively, on the cell surface) were mixed and co-transformed into NBRC1440ΔHUWL. The transformants were spread on SPASC medium for selection, and several transformants were selected, based on colony size.…”

Section: Methodsmentioning

confidence: 99%

Direct ethanol production from cellulosic materials using a diploid strain of Saccharomyces cerevisiaewith optimized cellulase expression

Yamada

Taniguchi

Tanaka

et al. 2011

Biotechnol Biofuels

116

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BackgroundHydrolysis of cellulose requires the action of the cellulolytic enzymes endoglucanase, cellobiohydrolase and β-glucosidase. The expression ratios and synergetic effects of these enzymes significantly influence the extent and specific rate of cellulose degradation. In this study, using our previously developed method to optimize cellulase-expression levels in yeast, we constructed a diploid Saccharomyces cerevisiae strain optimized for expression of cellulolytic enzymes, and attempted to improve the cellulose-degradation activity and enable direct ethanol production from rice straw, one of the most abundant sources of lignocellulosic biomass.ResultsThe engineered diploid strain, which contained multiple copies of three cellulase genes integrated into its genome, was precultured in molasses medium (381.4 mU/g wet cell), and displayed approximately six-fold higher phosphoric acid swollen cellulose (PASC) degradation activity than the parent haploid strain (63.5 mU/g wet cell). When used to ferment PASC, the diploid strain produced 7.6 g/l ethanol in 72 hours, with an ethanol yield that achieved 75% of the theoretical value, and also produced 7.5 g/l ethanol from pretreated rice straw in 72 hours.ConclusionsWe have developed diploid yeast strain optimized for expression of cellulolytic enzymes, which is capable of directly fermenting from cellulosic materials. Although this is a proof-of-concept study, it is to our knowledge, the first report of ethanol production from agricultural waste biomass using cellulolytic enzyme-expressing yeast without the addition of exogenous enzymes. Our results suggest that combining multigene expression optimization and diploidization in yeast is a promising approach for enhancing ethanol production from various types of lignocellulosic biomass.

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Enhanced production of anticoagulant hirudin in recombinant Saccharomyces cerevisiae by chromosomal δ-integration

Cited by 33 publications

References 26 publications

Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters

Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters

Comparison of three expression systems for heterologous xylanase production byS. cerevisiaein defined medium

Direct ethanol production from cellulosic materials using a diploid strain of Saccharomyces cerevisiaewith optimized cellulase expression

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