Identification and Application of Novel Autonomously Replicating Sequences (ARSs) for Promoter-Cloning and Co-Transformation in<i>Candida utilis</i>

Ikushima, Shigehito; Minato, Toshiko; Kondö, Keiji

doi:10.1271/bbb.80568

Cited by 8 publications

(10 citation statements)

References 28 publications

(21 reference statements)

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“…For example, a C. utilis co-transformation-strategy yielded positive clones at low frequency, 9) but makes it possible to breed yeasts corresponding to non-genetically modified organisms. The Cre/loxP method presented here inevitably leaves a single loxP element behind after excision of the selectable marker.…”

Section: Discussionmentioning

confidence: 99%

“…2) From the resulting plasmid, the 2.2-kb expression cassette of the cre ORF flanked by the PMA promoter and terminator was isolated as a NotI fragment and then inserted into partially NotIdigested pCARS7, 9) yielding plasmid pCU595, which conferred resistance to G418 via APT (Fig. 1A).…”

Section: )mentioning

confidence: 99%

“…It was based on the CuARS2-containing plasmid pCARS7, which should drop from cells at a frequency of about 80% in three to four generations. 9) Strain CUD1H was transformed with pCU595 for HPT marker excision, and 30 of about 1,600 G418-resistant colonies were transferred to new plates and analyzed for loss of the LHL module by replica-plating onto HygB-containing YPD. All these transformants lost their HygB-resistance, suggesting that cre-recombination via the loxP sites was successful.…”

Section: Expression Of Cre Recombinase and Marker Excisionmentioning

confidence: 99%

See 2 more Smart Citations

Efficient Gene Disruption in the High-Ploidy YeastCandida utilisUsing the Cre-loxPSystem

Ikushima¹,

Fujii²,

Kobayashi³

2009

Bioscience, Biotechnology, and Biochemistry

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Section: Discussionmentioning

confidence: 99%

Section: )mentioning

confidence: 99%

Section: Expression Of Cre Recombinase and Marker Excisionmentioning

confidence: 99%

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Efficient Gene Disruption in the High-Ploidy YeastCandida utilisUsing the Cre-loxPSystem

Ikushima¹,

Fujii²,

Kobayashi³

2009

Bioscience, Biotechnology, and Biochemistry

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“…High plasmid stability was observed for integrations in URA3 (Kondo et al 1997) and HIS3 (Kunigo et al 2013), while instability was shown for integrations in the rDNA (Kondo et al 1997) and both stability and instability were reported for integrations at the TDH3 locus (Kunigo et al 2013. Conceivably, mitotic recombination between homologous chromosomes may evict sectors of chromosomes carrying expression vectors in some cells, which may outcompete the parental cells in time, especially if the respective product is a Bbiosynthetic burden.T wo chromosomal autonomously replicating sequences (ARS) sequences have been described for C. utilis (Ikushima et al 2009c), although they provide only low copy numbers and their instability is undesirable for high-level production.…”

Section: Recombinant Dna Toolsmentioning

confidence: 99%

Candida utilis and Cyberlindnera (Pichia) jadinii: yeast relatives with expanding applications

Buerth

Tielker

Ernst

2016

Appl Microbiol Biotechnol

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The yeast Candida utilis is used as a food additive and as a host for heterologous gene expression to produce various metabolites and proteins. Reliable protocols for intracellular production of recombinant proteins are available for C. utilis and have now been expanded to secrete proteins into the growth medium or to achieve surface display by linkage to a cell wall protein. A recombinant C. utilis strain was recently shown to induce oral tolerance in a mouse model of multiple sclerosis suggesting future applications in autoimmune therapy. Whole genome sequencing of C. utilis and its presumed parent Cyberlindnera (Pichia) jadinii demonstrated different ploidy but high sequence identity, consistent with identical recombinant technologies for both yeasts. C. jadinii was recently described as an antagonist to the important human fungal pathogen Candida albicans suggesting its use as a probiotic agent. The review summarizes the status of recombinant protein production in C. utilis, as well as current and future biotechnological and medical applications of C. utilis and C. jadinii.

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“…[23][24][25][26] Recently, we developed new strategies for performing efficient multiple transformations of C. utilis. One is co-transformation, 27) and the other is based on the CreloxP recombination system. 28) Using the latter method, a four-round deletion of the CuURA3 gene, encoding orotidine-5 0 -phosphate decarboxylase, yielded an uracil auxotroph.…”

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confidence: 99%

Genetic Engineering ofCandida utilisYeast for Efficient Production ofL-Lactic Acid

Ikushima¹,

Fujii²,

Kobayashi³

et al. 2009

Bioscience, Biotechnology, and Biochemistry

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Polylactic acid is receiving increasing attention as a renewable alternative for conventional petroleum-based plastics. In the present study, we constructed a metabolically-engineered Candida utilis strain that produces L-lactic acid with the highest efficiency yet reported in yeasts. Initially, the gene encoding pyruvate decarboxylase (CuPDC1) was identified, followed by four CuPDC1 disruption events in order to obtain a null mutant that produced little ethanol (a by-product of L-lactic acid). Two copies of the L-lactate dehydrogenase (L-LDH) gene derived from Bos taurus under the control of the CuPDC1 promoter were then integrated into the genome of the CuPdc1-null deletant. The resulting strain produced 103.3 g/l of L-lactic acid from 108.7 g/l of glucose in 33 h, representing a 95.1% conversion. The maximum production rate of L-lactic acid was 4.9 g/l/h. The optical purity of the L-lactic acid was found to be more than 99.9% e.e.

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Identification and Application of Novel Autonomously Replicating Sequences (ARSs) for Promoter-Cloning and Co-Transformation inCandida utilis

Cited by 8 publications

References 28 publications

Efficient Gene Disruption in the High-Ploidy YeastCandida utilisUsing the Cre-loxPSystem

Efficient Gene Disruption in the High-Ploidy YeastCandida utilisUsing the Cre-loxPSystem

Candida utilis and Cyberlindnera (Pichia) jadinii: yeast relatives with expanding applications

Genetic Engineering ofCandida utilisYeast for Efficient Production ofL-Lactic Acid

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