Abstract
Methods that allow the assembly of genes in one single DNA segment are of great use in bioengineering and synthetic biology. The biosynthesis of plipastatin, a lipopeptide antibiotic synthesized non-ribosomally by Bacillus subtilis 168, requires three gene blocks at different genome loci, i.e. the peptide synthetase operon ppsABCDE (38-kb), degQ (0.6 kb), and sfp (1.0 kb). We applied a DNA assembly protocol in B. subtilis, named ordered gene assembly in B. subtilis (OGAB) method, to incorporate those three gene blocks into a one-unit plasmid via one ligation-reaction. High yields of correct assembly, above 87%, allowed us to screen for the plasmid that produced plipastatin at a level approximately 10-fold higher than in the wild-type. In contrast to that recombinogenic technologies used in E. coli require repetitive assembly steps and/or several selection markers, our method features high fidelity and efficiency, is completed in one ligation using only one selection marker associating with plasmid vector, and is applicable to DNA fragments larger than 40 kb.
| Original language | English |
|---|---|
| Pages (from-to) | 592-603 |
| Number of pages | 12 |
| Journal | Journal of Biotechnology |
| Volume | 129 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - 2007 May 10 |
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Keywords
- Bacillus subtilis
- Gene assembly
- NRPS
- OGAB
- Plasmid
ASJC Scopus subject areas
- Biotechnology
Cite this
Production of the non-ribosomal peptide plipastatin in Bacillus subtilis regulated by three relevant gene blocks assembled in a single movable DNA segment. / Tsuge, Kenji; Matsui, Kuniko; Itaya, Mitsuhiro.
In: Journal of Biotechnology, Vol. 129, No. 4, 10.05.2007, p. 592-603.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Production of the non-ribosomal peptide plipastatin in Bacillus subtilis regulated by three relevant gene blocks assembled in a single movable DNA segment
AU - Tsuge, Kenji
AU - Matsui, Kuniko
AU - Itaya, Mitsuhiro
PY - 2007/5/10
Y1 - 2007/5/10
N2 - Methods that allow the assembly of genes in one single DNA segment are of great use in bioengineering and synthetic biology. The biosynthesis of plipastatin, a lipopeptide antibiotic synthesized non-ribosomally by Bacillus subtilis 168, requires three gene blocks at different genome loci, i.e. the peptide synthetase operon ppsABCDE (38-kb), degQ (0.6 kb), and sfp (1.0 kb). We applied a DNA assembly protocol in B. subtilis, named ordered gene assembly in B. subtilis (OGAB) method, to incorporate those three gene blocks into a one-unit plasmid via one ligation-reaction. High yields of correct assembly, above 87%, allowed us to screen for the plasmid that produced plipastatin at a level approximately 10-fold higher than in the wild-type. In contrast to that recombinogenic technologies used in E. coli require repetitive assembly steps and/or several selection markers, our method features high fidelity and efficiency, is completed in one ligation using only one selection marker associating with plasmid vector, and is applicable to DNA fragments larger than 40 kb.
AB - Methods that allow the assembly of genes in one single DNA segment are of great use in bioengineering and synthetic biology. The biosynthesis of plipastatin, a lipopeptide antibiotic synthesized non-ribosomally by Bacillus subtilis 168, requires three gene blocks at different genome loci, i.e. the peptide synthetase operon ppsABCDE (38-kb), degQ (0.6 kb), and sfp (1.0 kb). We applied a DNA assembly protocol in B. subtilis, named ordered gene assembly in B. subtilis (OGAB) method, to incorporate those three gene blocks into a one-unit plasmid via one ligation-reaction. High yields of correct assembly, above 87%, allowed us to screen for the plasmid that produced plipastatin at a level approximately 10-fold higher than in the wild-type. In contrast to that recombinogenic technologies used in E. coli require repetitive assembly steps and/or several selection markers, our method features high fidelity and efficiency, is completed in one ligation using only one selection marker associating with plasmid vector, and is applicable to DNA fragments larger than 40 kb.
KW - Bacillus subtilis
KW - Gene assembly
KW - NRPS
KW - OGAB
KW - Plasmid
UR - http://www.scopus.com/inward/record.url?scp=34247111293&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34247111293&partnerID=8YFLogxK
U2 - 10.1016/j.jbiotec.2007.01.033
DO - 10.1016/j.jbiotec.2007.01.033
M3 - Article
C2 - 17376553
AN - SCOPUS:34247111293
VL - 129
SP - 592
EP - 603
JO - Journal of Biotechnology
JF - Journal of Biotechnology
SN - 0168-1656
IS - 4
ER -