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Design and testing of a synthetic biology framework for genetic engineering of Corynebacterium glutamicum.

Ravasi P, Peiru S, Gramajo H, Menzella HG - Microb. Cell Fact. (2012)

Bottom Line: Synthetic biology approaches can make a significant contribution to the advance of metabolic engineering by reducing the development time of recombinant organisms.We anticipate that the pTGR platform will contribute to explore the potential of novel parts to regulate gene expression, and to facilitate the assembly of genetic circuits for metabolic engineering of C. glutamicum.The standardization provided by this approach may provide a means to improve the productivity of biosynthetic pathways in microbial factories for the production of novel compounds.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genetic Engineering & Fermentation Technology, Instituto de Biología Celular y Molecular de Rosario-CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario, 2000, República Argentina.

ABSTRACT

Background: Synthetic biology approaches can make a significant contribution to the advance of metabolic engineering by reducing the development time of recombinant organisms. However, most of synthetic biology tools have been developed for Escherichia coli. Here we provide a platform for rapid engineering of C. glutamicum, a microorganism of great industrial interest. This bacteria, used for decades for the fermentative production of amino acids, has recently been developed as a host for the production of several economically important compounds including metabolites and recombinant proteins because of its higher capacity of secretion compared to traditional bacterial hosts like E. coli. Thus, the development of modern molecular platforms may significantly contribute to establish C. glutamicum as a robust and versatile microbial factory.

Results: A plasmid based platform named pTGR was created where all the genetic components are flanked by unique restriction sites to both facilitate the evaluation of regulatory sequences and the assembly of constructs for the expression of multiple genes. The approach was validated by using reporter genes to test promoters, ribosome binding sites, and for the assembly of dual gene operons and gene clusters containing two transcriptional units. Combinatorial assembly of promoter (tac, cspB and sod) and RBS (lacZ, cspB and sod) elements with different strengths conferred clear differential gene expression of two reporter genes, eGFP and mCherry, thus allowing transcriptional "fine-tuning"of multiple genes. In addition, the platform allowed the rapid assembly of operons and genes clusters for co-expression of heterologous genes, a feature that may assist metabolic pathway engineering.

Conclusions: We anticipate that the pTGR platform will contribute to explore the potential of novel parts to regulate gene expression, and to facilitate the assembly of genetic circuits for metabolic engineering of C. glutamicum. The standardization provided by this approach may provide a means to improve the productivity of biosynthetic pathways in microbial factories for the production of novel compounds.

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Effect of IPTG concentration and plasmid copy number on eGFP expression in C. glutamicum using the pTGR system. (A) Fluorescence intensity of eGFP relative to OD during the growth of C. glutamicum using different IPTG concentration to drive the expression of the reporter gene. (B) Fluorescence intensity of eGFP relative to OD testing three different replicons. pGA1 (medium copy number), pCRY4 (low copy number) and pNG2 (low copy number.) Cultures were grown in BHIS medium and supplemented with the indicated concentration of IPTG at OD590 = 0.5. Values shown are means of three independent determinations. The standard deviations were in all the cases less than 10% of the corresponding means.
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Figure 5: Effect of IPTG concentration and plasmid copy number on eGFP expression in C. glutamicum using the pTGR system. (A) Fluorescence intensity of eGFP relative to OD during the growth of C. glutamicum using different IPTG concentration to drive the expression of the reporter gene. (B) Fluorescence intensity of eGFP relative to OD testing three different replicons. pGA1 (medium copy number), pCRY4 (low copy number) and pNG2 (low copy number.) Cultures were grown in BHIS medium and supplemented with the indicated concentration of IPTG at OD590 = 0.5. Values shown are means of three independent determinations. The standard deviations were in all the cases less than 10% of the corresponding means.

Mentions: In order to explore further alternatives to tune gene expression using the pTGR platform, the level of expression of eGFP was tested under different inducer concentrations. Using pTGR5 vector, eGFP expression driven by tac promoter was induced with IPTG concentrations ranging 0.025-0,5 mM. The obtained results (Figure 5A) show a variation in gene expression between 0.025-0,25 mM, and no further increase in fluorescence relative to OD was obtained above the higher concentration, suggesting a saturation effect. These results indicate that a new instance of regulation can be exploited when using the tac promoter to drive the expression of heterologous genes with the pTGR system.


Design and testing of a synthetic biology framework for genetic engineering of Corynebacterium glutamicum.

Ravasi P, Peiru S, Gramajo H, Menzella HG - Microb. Cell Fact. (2012)

Effect of IPTG concentration and plasmid copy number on eGFP expression in C. glutamicum using the pTGR system. (A) Fluorescence intensity of eGFP relative to OD during the growth of C. glutamicum using different IPTG concentration to drive the expression of the reporter gene. (B) Fluorescence intensity of eGFP relative to OD testing three different replicons. pGA1 (medium copy number), pCRY4 (low copy number) and pNG2 (low copy number.) Cultures were grown in BHIS medium and supplemented with the indicated concentration of IPTG at OD590 = 0.5. Values shown are means of three independent determinations. The standard deviations were in all the cases less than 10% of the corresponding means.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539996&req=5

Figure 5: Effect of IPTG concentration and plasmid copy number on eGFP expression in C. glutamicum using the pTGR system. (A) Fluorescence intensity of eGFP relative to OD during the growth of C. glutamicum using different IPTG concentration to drive the expression of the reporter gene. (B) Fluorescence intensity of eGFP relative to OD testing three different replicons. pGA1 (medium copy number), pCRY4 (low copy number) and pNG2 (low copy number.) Cultures were grown in BHIS medium and supplemented with the indicated concentration of IPTG at OD590 = 0.5. Values shown are means of three independent determinations. The standard deviations were in all the cases less than 10% of the corresponding means.
Mentions: In order to explore further alternatives to tune gene expression using the pTGR platform, the level of expression of eGFP was tested under different inducer concentrations. Using pTGR5 vector, eGFP expression driven by tac promoter was induced with IPTG concentrations ranging 0.025-0,5 mM. The obtained results (Figure 5A) show a variation in gene expression between 0.025-0,25 mM, and no further increase in fluorescence relative to OD was obtained above the higher concentration, suggesting a saturation effect. These results indicate that a new instance of regulation can be exploited when using the tac promoter to drive the expression of heterologous genes with the pTGR system.

Bottom Line: Synthetic biology approaches can make a significant contribution to the advance of metabolic engineering by reducing the development time of recombinant organisms.We anticipate that the pTGR platform will contribute to explore the potential of novel parts to regulate gene expression, and to facilitate the assembly of genetic circuits for metabolic engineering of C. glutamicum.The standardization provided by this approach may provide a means to improve the productivity of biosynthetic pathways in microbial factories for the production of novel compounds.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genetic Engineering & Fermentation Technology, Instituto de Biología Celular y Molecular de Rosario-CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario, 2000, República Argentina.

ABSTRACT

Background: Synthetic biology approaches can make a significant contribution to the advance of metabolic engineering by reducing the development time of recombinant organisms. However, most of synthetic biology tools have been developed for Escherichia coli. Here we provide a platform for rapid engineering of C. glutamicum, a microorganism of great industrial interest. This bacteria, used for decades for the fermentative production of amino acids, has recently been developed as a host for the production of several economically important compounds including metabolites and recombinant proteins because of its higher capacity of secretion compared to traditional bacterial hosts like E. coli. Thus, the development of modern molecular platforms may significantly contribute to establish C. glutamicum as a robust and versatile microbial factory.

Results: A plasmid based platform named pTGR was created where all the genetic components are flanked by unique restriction sites to both facilitate the evaluation of regulatory sequences and the assembly of constructs for the expression of multiple genes. The approach was validated by using reporter genes to test promoters, ribosome binding sites, and for the assembly of dual gene operons and gene clusters containing two transcriptional units. Combinatorial assembly of promoter (tac, cspB and sod) and RBS (lacZ, cspB and sod) elements with different strengths conferred clear differential gene expression of two reporter genes, eGFP and mCherry, thus allowing transcriptional "fine-tuning"of multiple genes. In addition, the platform allowed the rapid assembly of operons and genes clusters for co-expression of heterologous genes, a feature that may assist metabolic pathway engineering.

Conclusions: We anticipate that the pTGR platform will contribute to explore the potential of novel parts to regulate gene expression, and to facilitate the assembly of genetic circuits for metabolic engineering of C. glutamicum. The standardization provided by this approach may provide a means to improve the productivity of biosynthetic pathways in microbial factories for the production of novel compounds.

Show MeSH
Related in: MedlinePlus