Assessment of Schwanniomyces occidentalis as a host for protein production using the wide-range Xplor®2 expression platform

Álvaro-Benito, Miguel; Fernández-Lobato, María; Baronian, Keith; Kunze, Gotthard

doi:10.1007/s00253-012-4527-9

Cited by 17 publications

(12 citation statements)

References 37 publications

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“…The auxotrophic mutant, A. adeninivorans G1216 [ aleu2 ALEU2::aade2 ] (Alvaro-Benito et al 2012), was used as recipient strain. All strains were cultivated at 30 °C, 180 rpm in 200 mL of broth in a 1-L flask.…”

Section: Methodsmentioning

confidence: 99%

“…A set of primers (Table 1) and the above plasmids were used in PCR to amplify the DNA fragments with an ORF, promoter, terminator and additional restriction sites. A two-step cloning procedure was used for the construction of the final expression plasmid based on Xplor2.4 system (Alvaro-Benito et al 2012). The reductase gene ( phaB [LT608132]) was used to make the TEF1-phaB-PHO5 fragment flanked by 5′- Bsi WI/ Spe I and 3′- Mlu I/ Sac II sites and was introduced into the basic vector to create Xplor2.4-TEF1-phaB-PHO5.…”

Section: Methodsmentioning

confidence: 99%

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Production of (R)-3-hydroxybutyric acid by Arxula adeninivorans

Biernacki

Riechen²,

Hähnel

et al. 2017

AMB Expr

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(R)-3-hydroxybutyric acid can be used in industrial and health applications. The synthesis pathway comprises two enzymes, β-ketothiolase and acetoacetyl-CoA reductase which convert cytoplasmic acetyl-CoA to (R)-3-hydroxybutyric acid [(R)-3-HB] which is released into the culture medium. In the present study we used the non-conventional yeast, Arxula adeninivorans, for the synthesis enantiopure (R)-3-HB. To establish optimal production, we investigated three different endogenous yeast thiolases (Akat1p, Akat2p, Akat4p) and three bacterial thiolases (atoBp, thlp, phaAp) in combination with an enantiospecific reductase (phaBp) from Cupriavidus necator H16 and endogenous yeast reductases (Atpk2p, Afox2p). We found that Arxula is able to release (R)-3-HB used an existing secretion system negating the need to engineer membrane transport. Overexpression of thl and phaB genes in organisms cultured in a shaking flask resulted in 4.84 g L−1 (R)-3-HB, at a rate of 0.023 g L−1 h−1 over 214 h. Fed-batch culturing with glucose as a carbon source did not improve the yield, but a similar level was reached with a shorter incubation period [3.78 g L−1 of (R)-3-HB at 89 h] and the rate of production was doubled to 0.043 g L−1 h−1 which is higher than any levels in yeast reported to date. The secreted (R)-3-HB was 99.9% pure. This is the first evidence of enantiopure (R)-3-HB synthesis using yeast as a production host and glucose as a carbon source.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Production of (R)-3-hydroxybutyric acid by Arxula adeninivorans

Biernacki

Riechen²,

Hähnel

et al. 2017

AMB Expr

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“…The 25S ribosomal DNA (rDNA) target sequences were interrupted by the selection marker module (ALEU2 promoter -ATRP1m gene -ATRP1 terminator) and Eco47III, SpeI, SacII, SalI, ApaI multicloning restriction sites for insertion [32]. These rDNA sequences allow yeast rDNA integration of the ERBB2 gene in the genome of A. adeninivorans as single or multi-copy.…”

Section: Construction Of Her-2[neu] Expression Plasmidsmentioning

confidence: 99%

Purification and immunodetection of the complete recombinant HER-2[neu] receptor produced in yeast

Chamas

Giersberg

Friedrich

et al. 2015

Protein Expression and Purification

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“…The Arxula transformation/expression platform, Xplor s 2, which lacks resistance markers and Escherichia coli elements, was used to produce stable mitotic transformants using the method described by Aĺvaro-Benito et al (2013). A gene based on C. albicans EBP1 sequence was synthesised using the Arxula preferred codon usage with a hexahistine tag (his tag) incorporated at the 3′ end of the gene (EBP1-6H).…”

Section: Cellsmentioning

confidence: 99%