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Engineering an ABC Transporter for Enhancing Resistance to Caffeine in Saccharomyces cerevisiae
- Source :
- Journal of Agricultural and Food Chemistry. 64:7973-7978
- Publication Year :
- 2016
- Publisher :
- American Chemical Society (ACS), 2016.
-
Abstract
- In addressing caffeine toxicity to the producing cells, engineering a transporter that can move caffeine from cytoplasm across the cell membrane to the extracellular space, thus enhancing caffeine resistance and potentially increasing the yield in yeast, is important. An ABC-transporter bfr1 from Schizosaccharomyces pombe was cloned and transformed into S. cerevisiae, resulting in enhancing caffeine resistance. Afterward, a library of randomly mutagenized bfr1 mutants through error-prone PCR was generated. One mutant was identified with drastically increased caffeine resistance (15 mg/mL). Sequencing and structural analysis illustrated that many of the mutations occurred at the cytosolic domain. Site-directed mutagenesis of these mutations confirmed at least one amino acid that conferred enhancing caffeine resistance in the mutated bfr1. These data demonstrated engineering ABC-transporters can be an efficient way to reduce product toxicity in heterologous systems.
- Subjects :
- 0301 basic medicine
030102 biochemistry & molecular biology
biology
Saccharomyces cerevisiae
Mutant
Mutagenesis (molecular biology technique)
ATP-binding cassette transporter
General Chemistry
biology.organism_classification
Yeast
Metabolic engineering
03 medical and health sciences
chemistry.chemical_compound
030104 developmental biology
Biochemistry
chemistry
Schizosaccharomyces pombe
General Agricultural and Biological Sciences
Caffeine
Subjects
Details
- ISSN :
- 15205118 and 00218561
- Volume :
- 64
- Database :
- OpenAIRE
- Journal :
- Journal of Agricultural and Food Chemistry
- Accession number :
- edsair.doi.dedup.....41855392e98772043d7b4044b7db550b