A rapid and simple procedure for high‐efficiency lithium acetate transformation of cryopreserved <i>Schizosaccharomyces pombe</i> cells

Suga, Minoru; Hatakeyama, Tatsuko

doi:10.1002/yea.1247

Cited by 56 publications

(43 citation statements)

References 26 publications

(38 reference statements)

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“…The resulting clones were tested for correct integration into the leu1 locus by replica plating on EMM dishes containing thiamine and phloxine B. One of the correct integrands was designated CAD62 and subjected to the preparation of cryocompetent cells [27]. Transformation of CAD62 with autosomally replicating pREP1 plasmids bearing cDNAs that code for the human microsomal P450s yielded strains CAD64 to CAD68 (see Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…Fission Yeast Strain Construction Transformation of fission yeast was done either using cryopreserved cells [27] or by the lithium acetate method [28]. Strain NCYC2036 (h − ura4-D18) was transformed with pCAD1-CPR to yield strain CAD62 (all strains are listed in Table 1).…”

Section: Methodsmentioning

confidence: 99%

“…Correct chromosomal integration of the pCAD1-CPR construct into the leu1 locus was confirmed by plating colonies on EMM lacking leucine. The resultant strain CAD62 was subsequently subjected to the cryopreservation method [27]. Cryopreserved CAD62 cells were then transformed with pREP1 expression vectors bearing the respective P450 cDNAs to yield five different strains that coexpress CPR and one of the P450s.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Convenient Gram-Scale Metabolite Synthesis by Engineered Fission Yeast Strains Expressing Functional Human P450 Systems

Drăgan

Peters

Bour

et al. 2010

Appl Biochem Biotechnol

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The growing need for the characterization of cytochrome P450 (P450) metabolites often necessitates their synthesis up to Gram-scale. This task may in principle be achieved by using various techniques including chemical synthesis, the use of laboratory animals, in vitro P450 systems or microbial biotransformation. However, these approaches are in many instances unfavorable due to low yields, laborious purification, costs of cofactors, or the formation of non-physiologic metabolites. The fission yeast Schizosaccharomyces pombe has previously been shown by others and us to be very well suited for the heterologous expression of human P450s. In this study, we demonstrate whole-cell biotransformation reactions carried out with fission yeast strains that coexpress human cytochrome P450 reductase (CPR) and one of the following P450 isoforms: CYP2B6, CYP2C9, CYP2C19, CYP2D6, or CYP3A4, respectively. These strains could successfully convert their respective standard substrates but showed different responses with respect to incubation pH, the presence of glucose, and temperature, respectively. In addition, the preparative of synthesis of 2.8 g of 4'-hydroxydiclofenac was achieved by whole-cell biotransformation of diclofenac using a CPR-CYP2C9 coexpressing fission yeast strain.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Convenient Gram-Scale Metabolite Synthesis by Engineered Fission Yeast Strains Expressing Functional Human P450 Systems

Drăgan

Peters

Bour

et al. 2010

Appl Biochem Biotechnol

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“…pCAD1-UGT constructs were prepared, before transformation, as reported previously (Drȃgan et al, 2005), and used to transform yeast strain NCYC 2036 (h Ϫ ura4-D18). Transformation was done, using competent cells prepared as described elsewhere (Suga and Hatakeyama, 2005), and yielded strains CAD200, DB1, DB3, DB5, DB23, DB24, DB25, DB26, DB32, and DB33. Correct integration into the leu1 locus was verified by selection of leucine auxotrophs on EMM dishes containing 5 M thiamine but no leucine.…”

Section: Methodsmentioning

confidence: 99%

Glucuronide Production by Whole-Cell Biotransformation Using Genetically Engineered Fission YeastSchizosaccharomyces pombe

Drăgan¹,

Buchheit²,

Bischoff³

et al. 2009

Drug Metab Dispos

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ABSTRACT:Drug metabolites generated by UDP glycosyltransferases (UGTs) are needed for drug development and toxicity studies, especially in the context of safety testing of metabolites during drug development. Because chemical metabolite synthesis can be arduous, various biological approaches have been developed; however, no whole-cell biotransformation with recombinant microbes that express human UGTs was yet achieved. In this study we expressed human UDP glucose-6-dehydrogenase together with several human or rat UGT isoforms in the fission yeast Schizosaccharomyces pombe and generated strains that catalyze the whole-cell glucuronidation of standard substrates. Moreover, we established two methods to obtain stable isotope-labeled glucuronide metabolites: the first uses a labeled aglycon, whereas the second uses 13 C 6 -glucose as a metabolic precursor of isotope-labeled UDP-glucuronic acid and yields a 6-fold labeled glucuronide. The system described here should lead to a significant facilitation in the production of both labeled and unlabeled drug glucuronides for industry and academia.

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“…Fission yeast strain NCYC2036 [34] with the genotype h -ura4.dl18 was used for transformation by pJMN6. Transformation was done by the lithium acetate method [35] to yield the new strain JMN6. Transformed cells were plated on Edinburgh minimal media (EMM) with 5 μM thiamine and incubated at 30°C.…”

Section: Strains and Mediamentioning

confidence: 99%

Expression and Secretion of a CB4-1 scFv–GFP Fusion Protein by Fission Yeast

Naumann¹,

Küttner

Bureik³

2010

Appl Biochem Biotechnol

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There is a rapidly growing demand for fluorescent single-chain Fv (scFv) antibody fragments for many applications. Yeasts have developed into attractive hosts for recombinant production of these functionalized proteins because they provide several advantages over prokaryotes and higher eukaryotes as expression systems, e.g., being capable of high-level secretion of heterologous proteins. In this study, we report Schizosaccharomyces pombe as a new host organism for secretory production of scFv-green fluorescent protein (GFP) fusions and compare it with previously described yeast expression systems. We cloned a plasmid for the expression and secretion of the anti-p24 (human immunodeficiency virus 1) CB4-1 scFv fused to GFP. After expression of the scFv-GFP fused to an N-terminal Cpy1 secretion signal sequence, fluorescence microscopy of living yeast cells indicated that the heterologous protein entered the secretory pathway. Western blot analysis of cell-free culture supernatants confirmed that the scFv-GFP was efficiently secreted with yields up to 5 mg/L. In addition, fluorescence measurements of culture supernatants demonstrated that the GFP moiety of the scFv-GFP protein is fully functional after secretion. Our data suggest that S. pombe has the potential for being used as alternative expression host in recombinant antibody fragment production by ensuring efficient protein processing and secretion.

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A rapid and simple procedure for high‐efficiency lithium acetate transformation of cryopreserved Schizosaccharomyces pombe cells

Cited by 56 publications

References 26 publications

Convenient Gram-Scale Metabolite Synthesis by Engineered Fission Yeast Strains Expressing Functional Human P450 Systems

Convenient Gram-Scale Metabolite Synthesis by Engineered Fission Yeast Strains Expressing Functional Human P450 Systems

Glucuronide Production by Whole-Cell Biotransformation Using Genetically Engineered Fission YeastSchizosaccharomyces pombe

Expression and Secretion of a CB4-1 scFv–GFP Fusion Protein by Fission Yeast

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