Extending homologous sequence based on the single gene mutants by one-step PCR for efficient multiple gene knockouts

Li, Mingji; Gu, Pengfei; Kang, Junhua; Wang, Yang; Wang, Qin; Qi, Qingsheng

doi:10.1007/s12223-012-0111-z

Cited by 11 publications

(6 citation statements)

References 18 publications

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“…E. coli genes were deleted by the Wanner method (Datsenko & Wanner, 2000). Its efficiency was enhanced by 300‐bp homologous arms according to an optimized gene deletion method (Li et al., 2012). For polA and ligA knockout, additional efforts were required.…”

Section: Methodsmentioning

confidence: 99%

The pathway of recombining short homologous ends in Escherichia coli revealed by the genetic study

Yang

Wang

et al. 2021

Molecular Microbiology

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The recombination of short homologous ends in Escherichia coli has been known for 30 years, and it is often used for both site‐directed mutagenesis and in vivo cloning. For cloning, a plasmid and target DNA fragments were converted into linear DNA fragments with short homologous ends, which are joined via recombination inside E. coli after transformation. Here this mechanism of joining homologous ends in E. coli was determined by a linearized plasmid with short homologous ends. Two 3ʹ‐5ʹ exonucleases ExoIII and ExoX with nonprocessive activity digested linear dsDNA to generate 5ʹ single‐strand overhangs, which annealed with each other. The polymerase activity of DNA polymerase I (Pol I) was exclusively employed to fill in the gaps. The strand displacement activity and the 5ʹ‐3ʹ exonuclease activity of Pol I were also required, likely to generate 5ʹ phosphate termini for subsequent ligation. Ligase A (LigA) joined the nicks to finish the process. The model involving 5ʹ single‐stranded overhangs is different from established recombination pathways that all generate 3ʹ single‐stranded overhangs. This recombination is likely common in bacteria since the involved enzymes are ubiquitous.

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Section: Methodsmentioning

confidence: 99%

The pathway of recombining short homologous ends in Escherichia coli revealed by the genetic study

Yang

Wang

et al. 2021

Molecular Microbiology

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“…The gene pflB which encodes pyruvate formate lyase was knocked out by the one-step inactivation method as described previously [40] and poxB encoding pyruvate oxidase was knocked out by linearized DNA fragments with extending homologous sequence [41]. First, the linerized DNA fragments with the FLP recognition target sites and 39 bp homologous sequences were obtained via PCR using pKD4 (Km R ) as a template and pflB-F/pflB-R as primers.…”

Section: Methodsmentioning

confidence: 99%

Engineering the pathway in Escherichia coli for the synthesis of medium-chain-length polyhydroxyalkanoates consisting of both even- and odd-chain monomers

Zhuang

2019

Microb Cell Fact

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Background Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) containing various chain length monomers from C6 to C14 have more applications besides sustainable and environmental-friendly biomaterials owing to their superior physical and mechanical properties. We engineered a reversed fatty acid β-oxidation pathway in Escherichia coli that can synthesize mcl-PHA directly from glucose and achieved high yield. However, there were only even-chain monomers in the biosynthetic polymers. The need for mcl-PHA harboring both even- and odd-chain monomers with better and wider utility impels us to develop the biosynthetic routes for the production of the novel and unnatural mcl-PHA through rewiring the basic metabolism. Results In the present study, a propionate assimilation and metabolic route was integrated into the reversed fatty acid β-oxidation in order to produce mcl-PHA consisting of both even- and odd-numbered monomers. The content of odd-numbered monomers in mcl-PHA was improved with the increased propionate addition. After further deletion of pyruvate oxidase (PoxB) and pyruvate formate-lyase (PflB), the metabolically engineered chassis E. coli LZ08 harboring pQQ05 and pZQ06 (overexpression of prpP and prpE genes from R alstonia eutropha H16) innovatively accumulated 6.23 wt% mcl-PHA containing odd-chain monomers ranging from 7 to 13 carbon atoms about 20.03 mol%. Conclusions This is the first successful report on production of mcl-PHA harboring both even- and odd-chain monomers (C6–C14) synthesized from glucose and propionate in recombinant E. coli . This present study achieved the highest yield of de novo production of mcl-PHA containing odd-numbered monomers in E. coli at shake-flask fermentation level. Continued engineering of host strains and pathway enzymes will ultimately lead to more economical production of odd-chain monomers based on market demand. The synthetic pathway can provide a promising platform for production of other value-added chemicals and biomaterials that use acetyl-CoA and propionyl-CoA as versatile precursors and can be extended to other microorganisms as intelligent cell factories. Electronic supplementary material The online version of this article (10.1186/s12934-019-1186-x) contains supplementary material, which is available to authorized users.

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“…Primers araC-JF/araC-JR were then selected to verify the positive clones. After generation of strain P-1, the following genes were deleted in turn by the optimised one-step inactivation method 33 : pta , ptsG , aroL , trpR and pykF , respectively encoding phosphate acetyltransferase, fused phosphoenolpyruvate: carbohydrate phosphotransferase system IIBC components, shikimate kinase II, tryptophan pathway transcriptional repressor and pyruvate kinase I. To construct a control strain, aroK was also deleted.…”

Section: Methodsmentioning

confidence: 99%

“…Accumulation of L-phenylalanine, L-tyrosine, and L-tryptophan in strains P-8 and P-9 was determined with a Venusil AA analysis kit (Bonna-Agela Technologies, Tianjin, China). The fluorescence of recombinant strains was determined in 96-well microtitre plates as described previously 33 .…”

Section: Methodsmentioning

confidence: 99%

Tunable switch mediated shikimate biosynthesis in an engineered non-auxotrophic Escherichia coli

Wang

et al. 2016

Sci Rep

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Shikimate is a key intermediate in the synthesis of neuraminidase inhibitors. Compared with traditional methods, microbial production of shikimate has the advantages of environmental friendliness, low cost, feed stock renewability, and product selectivity and diversity. Despite these advantages, shikimate kinase I and II respectively encoded by aroK and aroL are inactivated in most shikimate microbial producers, thus requiring the addition of aromatic compounds during the fermentation process. To overcome this problem, we constructed a non-auxotrophic, shikimate-synthesising strain of Escherichia coli. By inactivation of repressor proteins, blocking of competitive pathways and overexpression of key enzymes, we increased the shikimate production of wild-type E. coli BW25113 to 1.73 g/L. We then designed a tunable switch that can conditionally decrease gene expression and substituted it for the original aroK promoters. Expression of aroK in the resulting P-9 strain was maintained at a high level during the growth phase and then reduced at a suitable time by addition of an optimal concentration of inducer. In 5-L fed-batch fermentation, strain P-9 produced 13.15 g/L shikimate without the addition of any aromatic compounds. The tunable switch developed in this study is an efficient tool for regulating indispensable genes involved in critical metabolic pathways.

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Extending homologous sequence based on the single gene mutants by one-step PCR for efficient multiple gene knockouts

Cited by 11 publications

References 18 publications

The pathway of recombining short homologous ends in Escherichia coli revealed by the genetic study

The pathway of recombining short homologous ends in Escherichia coli revealed by the genetic study

Engineering the pathway in Escherichia coli for the synthesis of medium-chain-length polyhydroxyalkanoates consisting of both even- and odd-chain monomers

Tunable switch mediated shikimate biosynthesis in an engineered non-auxotrophic Escherichia coli

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