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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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Assessment of promoter and RBS activities in C. glutamicum with the pTGR system. (A) Fluorescence intensity of eGFP relative to OD during the growth of C. glutamicum using different promoters to drive the expression of the reporter gene. (B) Fluorescence intensity of eGFP relative to OD testing three different RBSs. Cultures were grown in BHIS medium and supplemented with 0.5 mM IPTG when indicated. 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 2: Assessment of promoter and RBS activities in C. glutamicum with the pTGR system. (A) Fluorescence intensity of eGFP relative to OD during the growth of C. glutamicum using different promoters to drive the expression of the reporter gene. (B) Fluorescence intensity of eGFP relative to OD testing three different RBSs. Cultures were grown in BHIS medium and supplemented with 0.5 mM IPTG when indicated. Values shown are means of three independent determinations. The standard deviations were in all the cases less than 10% of the corresponding means.

Mentions: Figure 2A shows eGFP expression under the control of three different promoters in C. glutamicum ATCC 13869 cultivated in BHIS medium. In experiments where the expression was driven by the tac promoter, 0.5 mM IPTG was added to the cultures. The expression from sod and cspB does not require the addition of an exogenous inducer since these promoters are growth phase dependant [23]. A similar rate of induction was observed for the three promoters, and the maximum amount of eGFP per cell was obtained after 24 hours; when cultures reached stationary phase. Background expression of eGFP was not detected in the absence of promoter. Clearly, the three promoters used to test the pTGR system provide different levels of expression under the tested conditions: high (tac), medium (cspB) and low (sod), showing that the pTGR may serve to rapidly evaluate and classify promoter parts.


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)

Assessment of promoter and RBS activities in C. glutamicum with the pTGR system. (A) Fluorescence intensity of eGFP relative to OD during the growth of C. glutamicum using different promoters to drive the expression of the reporter gene. (B) Fluorescence intensity of eGFP relative to OD testing three different RBSs. Cultures were grown in BHIS medium and supplemented with 0.5 mM IPTG when indicated. 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 2: Assessment of promoter and RBS activities in C. glutamicum with the pTGR system. (A) Fluorescence intensity of eGFP relative to OD during the growth of C. glutamicum using different promoters to drive the expression of the reporter gene. (B) Fluorescence intensity of eGFP relative to OD testing three different RBSs. Cultures were grown in BHIS medium and supplemented with 0.5 mM IPTG when indicated. Values shown are means of three independent determinations. The standard deviations were in all the cases less than 10% of the corresponding means.
Mentions: Figure 2A shows eGFP expression under the control of three different promoters in C. glutamicum ATCC 13869 cultivated in BHIS medium. In experiments where the expression was driven by the tac promoter, 0.5 mM IPTG was added to the cultures. The expression from sod and cspB does not require the addition of an exogenous inducer since these promoters are growth phase dependant [23]. A similar rate of induction was observed for the three promoters, and the maximum amount of eGFP per cell was obtained after 24 hours; when cultures reached stationary phase. Background expression of eGFP was not detected in the absence of promoter. Clearly, the three promoters used to test the pTGR system provide different levels of expression under the tested conditions: high (tac), medium (cspB) and low (sod), showing that the pTGR may serve to rapidly evaluate and classify promoter parts.

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