Abstract
Background Bacteria are versatile living systems that enhance our understanding of nature and enable biosynthesis of valuable molecules. Long fragment editing techniques are of great importance for accelerating bacterial chromosome engineering to obtain desirable and genetically stable strains. However, the existing genomic editing methods cannot meet the needs of researchers.
Results We herein report an efficient long fragment editing technique for complex chromosomal engineering in Escherichia coli. The technique enabled us to integrate DNA fragments up to 12 kb into the chromosome, and to knock out DNA fragments up to 187 kb from the chromosome, with over 95% positive rates. We applied this technique for E. coli chromosomal simplification, resulting in twelve individual deletion mutants and four cumulative deletion mutants. The simplest chromosome lost a 370.6 kb DNA sequence containing 364 open reading frames. In addition, we applied the technique to metabolic engineering and constructed a genetically stable plasmid-independent isobutanol production strain that produced 1.3 g/L isobutanol via shake-flask micro-aerobic fermentation.
Conclusions These results suggested that the technique is a powerful chromosomal engineering tool, highlighting its potential to be applied in different fields of synthetic biology.
List of abbreviations
- PCR
- polymerase chain reaction
- DSB
- double-strand break
- dsDNA
- double-stranded DNA
- ZFN
- zinc-finger nuclease
- TALEN
- transcription activator-like effector nuclease
- CRISPR
- clustered regularly interspaced short palindromic repeats
- Cas9
- CRISPR-associated protein 9
- sgRNA
- single-guide RNA
- PAM
- protospacer-adjacent motif
- CARS
- CRISPR/Cas9-assisted recombination system
- IPTG
- isopropyl-β-D-thiogalactopyranoside
- KanR
- kanamycin-resistant
- AmpR
- ampicillin-resistant
- LB
- Luria-Bertani
- ORF
- open reading frame
- UTR
- untranslated region
- NHEJ
- non-homologous end-joining
- ATCC
- American Type Culture Collection
- SOC
- super optimal broth with catabolite repression
- OD600
- optical density at a wavelength of 600 nm
- TS
- target site
- LHA
- left homologous arm
- RHA
- right homologous arm
- F
- forward primer
- R
- reverse primer.