A method for gene amplification and simultaneous deletion in Corynebacterium glutamicum genome without any genetic markers

Xu, Jianzhong; Xia, Xiuhua; Zhang, Junlan; Guo, Yanfeng; Qian, He; Zhang, Weiguo

doi:10.1016/j.plasmid.2014.02.001

Cited by 13 publications

(26 citation statements)

References 22 publications

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“…The targeted fragment with a selectable marker is constructed in vitro and then transferred into the host cell. The artificial fragment replaces the targeted wild-type gene by double-crossover homologous recombination (Tilly et al, 2000;Xu et al, 2014c). The key aspect of this method is that it requires a gene transfer mechanism and a selectable marker (Tilly et al, 2000).…”

Section: Gene Inactivation Based On Insertional Inactivationmentioning

confidence: 99%

“…Plasmids generally carry genetic markers, such as a drug resistance marker, which are used for highefficiency screening of target-transformants (Xu et al, 2014c). However, there are several disadvantages to the application of plasmid-mediated gene overexpression, such as the instability of the plasmid and negative effect on cell growth.…”

Section: Strategies For Gene Over-expressionmentioning

confidence: 99%

“…Recently we have developed a method for the simultaneous replacement of a targeted gene by a given gene cassette, leaving no genetic markers (Fig. 6) (Xu et al, 2014c). However, the methods mentioned above are only used in modifying the C. glutamicum chromosome.…”

Section: Strategies For Gene Over-expressionmentioning

confidence: 99%

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Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression

Zhang

2016

J. Zhejiang Univ. Sci. B

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Abstract:With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators.

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Section: Gene Inactivation Based On Insertional Inactivationmentioning

confidence: 99%

Section: Strategies For Gene Over-expressionmentioning

confidence: 99%

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Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression

Zhang

2016

J. Zhejiang Univ. Sci. B

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“…Metabolic engineering via genetic modification in C. glutamicum is based on intensifying or shifting carbon flux towards the biosynthetic pathway of the aimed product or on enhancing the degradation of some special substrates [3]. Main strategies used in remodeling the cell metabolic balance involved introducing heterologous genes, boosting the activity of key genes, relieving the regulation of enzymes, and reducing gene expression of byproduct biosynthesis [4][5][6][7]. However, genetic modification of C. glutamicum is mainly dependent on the application of antibiotic resistance genes that are used as positive markers for screening target-recombinant strains, especially for the introduction of heterologous genes and overexpression of key genes [8].…”

mentioning

confidence: 99%

“…Plasmid-mediated hetero-/homologous gene overexpression in amino acid producers has some shortcomings, such as high cost, changes in Corynebacteria's physiological function, problems in food safety, etc [7,8]. Although Hu et al [9] have constructed a novel expression system for gene overexpression using a plasmid system, the antibiotic resistance gene was also introduced into the cell.…”

mentioning

confidence: 99%

A method for simultaneous gene overexpression and inactivation in the Corynebacterium glutamicum genome

Zhang²,

Han

et al. 2016

Journal of Industrial Microbiology and Biotechnology

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The gene integration method is an important tool to stably express desirable genes in bacteria. To avoid heavy workload and cost, we constructed a rapid and efficient method for genome modification. This method depended on a mobilizable plasmid, which contains a P tac promoter, an introduced multiple cloning site (iMCS), and rrnBT1T2 terminator. Briefly, the mobilizable plasmid pK18-MBPMT with the P tac-iMCS-rrnBT1T2 cartridge derived from pK18mobsacB was prepared to directly integrate hetero-/homologous DNA into the Corynebacterium glutamicum genome. Like our previous method, this method was based on insertional inactivation and double-crossover homologous recombination, which simultaneously achieved gene overexpression and inactivation in the genome without the use of genetic markers. Compared to the previous method, this protocol omitted the construction of a recombinant expression plasmid and clone of the target gene(s) cassette, which significantly decreased the workload, cost, and operational time. Using this method, the heterologous gene amy and the homologous gene lysC (T311I) were successfully integrated into the C. glutamicum genome at alaT and avtA loci, respectively. Moreover, the operation time of this method was shorter than that of the previous method, especially for repeated integration. This method, which is based on the mobilizable plasmid pK18-MBPMT, thus represents a potentially attractive protocol for the integration of genes in the course of genetic modification of C. glutamicum.

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De novo biosynthesis of α‐aminoadipate via multi‐strategy metabolic engineering in Escherichia coli

Zhang

Cai

et al. 2022

MicrobiologyOpen

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As a non‐protein amino acid, α‐aminoadipate is used in the fields of medicine, chemical engineering, food science, and others. For example, α‐aminoadipate is an important precursor for the production of β‐lactam antibiotics. Currently, the synthesis of α‐aminoadipate depends on chemical catalysis that has the disadvantages of high cost, low yield, and serious pollution. In this study, we construct a biosynthesis pathway of α‐aminoadipate in Escherichia coli using lysine as a precursor. In addition, we regulate the cell metabolism to improve the titer of α‐aminoadipate via multi‐strategy metabolic engineering. First, a novel synthetic pathway was constructed to realize de novo synthesis of α‐aminoadipate with titers of 82 mg/L. Second, the key enzymes involved in enhancing precursor synthesis were overexpressed and the CO2 fixation process was introduced, and these led to 80% and 34% increases in the α‐aminoadipate concentration, reaching 147 and 110 mg/L, respectively. Third, cofactor regulation was used to maintain the coupling balance of material and energy, with the intracellular α‐aminoadipate concentration reaching 140 mg/L. Fourth, the weakening of the synthesis of acetic acid was used to strengthen the synthesis of α‐aminoadipate, and this resulted in the enhancement of the α‐aminoadipate concentration by 2.2 times, reaching 263 mg/L. Finally, combination optimization was used to promote the production of α‐aminoadipate. The titers of α‐aminoadipate reached 368 mg/L (strain EcN11#) and 415 mg/L (strain EcN11##), which was 3.5 and 4 times higher than that of the parent strain. With these efforts, 1.54 g/L of α‐aminoadipate was produced under fed‐batch conditions by strain EcN11#. This study is the first to present the effective biosynthesis of α‐aminoadipate in E. coli using multi‐strategy metabolic engineering.

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A method for gene amplification and simultaneous deletion in Corynebacterium glutamicum genome without any genetic markers

Cited by 13 publications

References 22 publications

Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression

Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression

A method for simultaneous gene overexpression and inactivation in the Corynebacterium glutamicum genome

De novo biosynthesis of α‐aminoadipate via multi‐strategy metabolic engineering in Escherichia coli

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