کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10234680 | 44870 | 2005 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Directed evolution of copy number of a broad host range plasmid for metabolic engineering
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
مهندسی شیمی
بیو مهندسی (مهندسی زیستی)
پیش نمایش صفحه اول مقاله
![عکس صفحه اول مقاله: Directed evolution of copy number of a broad host range plasmid for metabolic engineering Directed evolution of copy number of a broad host range plasmid for metabolic engineering](/preview/png/10234680.png)
چکیده انگلیسی
Random mutagenesis and directed evolution has been successfully used to improve desired properties of enzymes for biocatalysis and metabolic engineering. Here we employ the method to increase copy number of a pBBR-based broad host range plasmid, which can be used to express desired enzymes in a variety of microbial hosts. Localized random mutagenesis was performed in the replication control region of a pBBR-derived plasmid containing a β-carotene reporter. Mutant plasmids were isolated that showed increased β-carotene production. Real-time PCR analysis confirmed that the copy number of the mutant plasmids increased 3-7 fold. Sequence of the 10 mutant plasmids indicated that each plasmid contained single or multiple mutations in the rep gene or the flanking regions. Single amino acid change of serine to leucine at codon 100 of the replication protein and single nucleotide change of C to T at 46 bp upstream of the rep gene caused the increase of plasmid copy number. The utility of the mutant plasmids for metabolic engineering were further demonstrated by increased β-carotene production, when an isoprenoid pathway gene (dxs) was co-expressed on a compatible plasmid. The mutant plasmids were tested in Agrobacterium tumefaciens. Increase of plasmid copy number and β-carotene production was also observed in the non-Escherichia coli host.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Metabolic Engineering - Volume 7, Issue 1, January 2005, Pages 10-17
Journal: Metabolic Engineering - Volume 7, Issue 1, January 2005, Pages 10-17
نویسندگان
Luan Tao, Raymond E. Jackson, Qiong Cheng,