Gene knockout, an important technology in molecular biology, has been broadly applied in industrial microbial metabolic engineering. From the basic mechanism of DNA recombination, we summarized and compared in this review different gene knockout strategies. Three most hot and important approaches, including the lambda Red recombination system using the linear dsDNA as recombination substrate, the single or double crossover homologous recombination using the circular plasmid DNA as substrate, and the transposase mediated transposition recombination, were summarized in detail. Developing frontiers and application prospects of gene knockout were further discussed.
Biotechnology ; methods ; Gene Knockout Techniques ; methods ; Industrial Microbiology ; methods ; Metabolic Engineering ; methods ; Metabolic Networks and Pathways

1,2,4-Butanetriol (BT) is an important non-natural chemical with a variety of industrial applications. A recombinant Escherichia coli biosynthesizing BT from D-xylose was constructed by heterologously expressing xdh and mdlC, and knocking out competing pathway genes including xylA, xylB, yjhE, yagH and ycdW. To optimize BT synthesis pathway, the third catalytic step that catalyzes the decarboxylation reaction of 3-deoxy-D-glycero-pentulosonic acid was identified as a potential bottleneck. Consequently, 2-keto acid decarboxylases from three different microorganisms were screened, and the kivD gene from Lactococcus lactis was found to increase BT titer by 191%. The improved strain BW-025 reached a final BT titer of 2.38 g/L under optimized transformation conditions. Attempts on synthetic pathway optimization were also made by fine-tuning the expression levels of each enzyme involved in the whole pathway based on BW-025. As a result, an xdh overexpressed recombinant strain, BW-074 was finally generated, with 48.62% higher BT production than that of BW-025.
Butanols ; metabolism ; Escherichia coli ; metabolism ; Gene Knockout Techniques ; Genetic Engineering ; Industrial Microbiology ; methods ; Metabolic Networks and Pathways

Streptomycetes are Gram-positive bacteria of Actinomycetales. These organisms can produce many economically important secondary metabolites. With the development of molecular biology, gene sequencing technology and synthetic biology, people gained a better understanding of the Streptomyces family. The means to transform genome on the molecular level is also increasing. By simplifying the Streptomyces genome rationally and efficiently, it will improve the yield and quality of the metabolites as well as reduce the consumption of the substrates. Markerless knockout is an important way to carry out genetic modification. Here we describe novel genome modification techniques developed for Streptomyces in recent years with focus on the markerless knockout technologies.
Chromosomes, Bacterial ; genetics ; Gene Knockout Techniques ; methods ; Genes, Bacterial ; genetics ; Streptomyces ; genetics

pBR322-Red is a newly constructed recombineering plasmid, which contains a part of the pBR322 vector, a series of regulatory elements of lambda-prophage and Red recombination genes. In the beginning, we studied the best working conditions of pBR322-Red, and then modified lac operon in E. coli W3110 chromosome using the plasmid as follow: Firstly, we knockout the lacI gene using Red-mediated recombineering with overlapping single stranded DNA oligonucleotides. Secondly, we substituded the lacA and lacY genes with lacZ, a report gene, by Red-mediated linearized double strands DNA homologous recombination. Finally, we detected the expression of lacZ on these loci for the first time. The results suggested that pBR322-Red system is suitable for modifying W3110 chromosome with various recombination strategies.
Bacteriophage lambda ; genetics ; Chromosomes, Bacterial ; genetics ; Escherichia coli ; genetics ; Gene Knock-In Techniques ; methods ; Gene Knockout Techniques ; methods ; Humans ; Lac Operon ; genetics ; Plasmids ; genetics ; Recombination, Genetic

Shikimic acid (SA), as a hydroaromatic intermediate in the common pathway of aromatic amino acid biosynthesis, is the starting material for the synthesis of neuraminidase inhibitors and other useful compounds. The fermentative production of SA by metabolically engineered microorganisms is an excellent alternative to the extraction from fruits of the Illicium plant. In this study, Escherichia coli was metabolically engineered by rational design and genetic manipulation for fermentative production of SA. Firstly, blocking the aromatic amino acid pathway after the production of SA was carried out by deletion of aroL and aroK genes encoding SA kinase. Secondly, the ptsG gene encoding protein EIICBglc were removed in the aroL/aroK mutant strain to make the phosphotransferase system (PTS) system default. In the resulting strain, the phosphoenolpyruvate-dependent PTS pathway, a main pathway for glucose transport, were replaced by ATP-dependent GalP (galactose permease). Thus, more PEP flux was used to produce SA as a critical precursor of SA. Furthermore, ydiB gene (encoding quinic acid/SA dehydrogenase) was deleted to prevent SA precursors of 3-dehyroquinic acid into the byproduct of quinic acid. Thus, the engineered strain with four genes deletion was constructed and 576 mg/L SA was produced in the shake flask fermentation. Results show that SA produciton was increased 90 times compared to the parent strain E. coli CICIM B0013.
Escherichia coli ; enzymology ; genetics ; metabolism ; Gene Knockout Techniques ; Metabolic Engineering ; methods ; Recombinant Proteins ; genetics ; metabolism ; Shikimic Acid ; metabolism

It is one of the frequently utilized strategies for positive-negative selection to elevate the gene targeting efficiency in somatic cells by enriching targeted colonies. Knocking out prnp in animals by gene targeting can prevent it from expressing Prion protein (Pathogenic protein of transmissible spongiform encephalopathy), which enables it to resist infection of Prion. We constructed a bovine prnp biallelic targeting vector via the positive-negative selection strategy, and transfected the linearized vector into the bovine fetal fibroblasts through electroporation. Then, we selected cells in cell culture medium with G418 under a concentration of 600 microg/mL followed by Ganciclovir (GCV) under a concentration of 200 nmol/mL. In the end, we successfully obtained 176 cell clones. All these clones were identified by means of sequencing, immunofluorescence and western blotting, respectively, confirming that there existed 9 positive cell clones. The results showed that the bovine prnp gene was successfully knocked out. Conclusively, we provide an effective way to knockout bovine prnp gene, which could serve as the basis for producing prion protein gene knockout transgenic cloned cattle.
Animals ; Cattle ; Electroporation ; Encephalopathy, Bovine Spongiform ; genetics ; Fetus ; cytology ; Fibroblasts ; cytology ; metabolism ; Gene Knockout Techniques ; methods ; Gene Targeting ; Genetic Vectors ; genetics ; Prions ; genetics ; Transfection

Based on carbon metabolic pathway analysis of Escherichia coli MG1655, an aerobic succinate fermentation platform was constructed by knocking out five genes (ptsG, poxB, pta, iclR and sdhA), which was named E. coli QZ1111. Flask cultivation results showed that E. coli QZ1111 could accumulate succinate with a concentration of 26.4 g/L under aerobic conditions. The byproduct acetate was only 2.3 g/L. The production ratio of succinate and acetate reached 11.5:1.
Aerobiosis ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Gene Expression Regulation, Bacterial ; Gene Knockout Techniques ; Protein Engineering ; methods ; Recombinant Proteins ; biosynthesis ; genetics ; Succinic Acid ; metabolism

Construction and ethanol production effects of SNF4 gene knockout in Saccharomyces cerevisiae were described in this paper. For knockout of SNF4 gene in S. cerevisiae YS2, a PCR-amplified disruption cassette was used, encoding the short flanking homologous regions to the SNF4 gene and Kan(r) as selectable marker. The SNF4 gene disruption cassette was transformed into S. cerevisiae YS2 through LiAc/SS Carrier DNA/PEG. The positive transformants were grown on G418 plates and verified by PCR. The Kan(r) marker was rescued by transforming plasmid pSH65 into positive transformants and inducing expression of Cre recombinase in galactose-containing medium. Lastly, the YS2-deltaSNF4 strain, in which SNF4 allele gene were completely knocked out, was obtained by repeating the same procedure. The result of anaerobic fermentation showed that ethanol production of the SNF4 gene knockout strain had increased by 7.57 percent as compared with the original strain YS2. The experiment indicated ethanol production could be improved significantly with the approach ofSNF4 gene knockout by Cre-LoxP system.
AMP-Activated Protein Kinases ; genetics ; Ethanol ; metabolism ; Fermentation ; Gene Knockout Techniques ; methods ; Mutation ; Saccharomyces cerevisiae ; genetics ; Saccharomyces cerevisiae Proteins ; genetics ; Transcription Factors ; genetics

Hapten antibodies are active components of traditional Chinese medicines, have been widely applied in all of study fields of traditional Chinese medicine. First, hapten monoclonal antibodies could be designed into ELISA kits for quantitative analysis on the content of effective components in plant crude extracts or biological samples, which be applied for quality control and studies on pharmacokenetics of traditional Chinese medicines. Second, hapten monoclonal antibodies could be coupled with solid-phase carriers to generate immunoaffinity chromatography column, which could be used for knock-out extract preparation or pre-treatment of complicated sampless. Finally, a single-chain variable fragment antibody (scFV) gene segment of effective components of hapten monoclonal antibodies could be transformed into relative plant cells to gain new varieties with high-enrichment effective components, and thus achieve the molecular breeding of medicinal plants.
Animals ; Antibodies ; genetics ; immunology ; Chromatography, Affinity ; Enzyme-Linked Immunosorbent Assay ; Gene Knockout Techniques ; Haptens ; immunology ; metabolism ; Humans ; Medicine, Chinese Traditional ; methods

OBJECTIVETo construct a gene knock-out mutant of response regulator named RevS in Streptococcus suis serotype 2 virulent strain 05ZYH33, and to investigate the effects of its deletion on the biological characters of this pathogen and the pathogenesis to mice and piglets.

METHODSRecombinant gene knock-out vector consisting of Spc(r) cassette was constructed and flanking was constructed consisting of Spc(r) cassette with flanking homology regions to the RevS genes while the isogenic RevS-deficient mutant was screened by allelic replacement. The effects of RevS deletion on the basic biological characters of 05ZYH33 including growth stability, colonial morphology, haemolysis, Gram staining, growth curve and protein expression were examined in vitro. The mice and piglets were infected with 10(8) CFU wild virulent and mutant isolates.

RESULTSPCR analysis confirmed that the coding genes of RevS were replaced completely by Spc(r) cassette and the basic biological characters of 05ZYH33 did not undergo any apparent change. Balb/c mice infection assay indicated that RevS play a role in the pathogenesis of Streptococcus suis infections, while no remarkable difference was observed in the piglets' pathogenesis infection rates between mutant isolates deltaA05ZYH33 and wild-type isolates 05ZYH33.

CONCLUSIONThe mutant of Streptococcus suis 05ZYH33 response regulator was successfully constructed, while the mutation did not obviously affect the bacterial biological characters, while the knock-out mutant of RevS was shown to be attenuated in pathogenesis to mice and piglets.

Animals ; Bacterial Proteins ; genetics ; Gene Knockout Techniques ; methods ; Mice ; Mice, Inbred BALB C ; Models, Genetic ; Polymerase Chain Reaction ; Streptococcal Infections ; microbiology ; Streptococcus suis ; genetics ; pathogenicity