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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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Comparison of the co-expression levels of eGFP and mCherry genes in C. glutamicum. (A) the genes were assembled in operons with the indicated RBSs controlling the expression. In all cases operons are transcribed from the tac promoter. (B) the genes were co-expressed from two transcriptional units and the indicated promoter drives the expression of each gene. Fluorescence intensity of eGFP and mCherry relative to OD after 24 h of incubation of cultures is shown. Cultures were grown in BHIS medium and supplemented with 0.5 mM IPTG when the tac promoter was used. 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 4: Comparison of the co-expression levels of eGFP and mCherry genes in C. glutamicum. (A) the genes were assembled in operons with the indicated RBSs controlling the expression. In all cases operons are transcribed from the tac promoter. (B) the genes were co-expressed from two transcriptional units and the indicated promoter drives the expression of each gene. Fluorescence intensity of eGFP and mCherry relative to OD after 24 h of incubation of cultures is shown. Cultures were grown in BHIS medium and supplemented with 0.5 mM IPTG when the tac promoter was used. Values shown are means of three independent determinations. The standard deviations were in all the cases less than 10% of the corresponding means.

Mentions: To demonstrate that the pTGR system allows the amount of both proteins to be altered by either using RBSs with different stregths in the case of operons, or promoters of different strengths in the case of multiple transcriptional units; a further set of experiments was carried out. First, the operon contained in plasmid pTGR8 was modified by exchanging the sod RBS that controls the translation of mCherry with the lacZ RBS to obtain the pTGR10 plasmid. As expected, when using this construct the amount of mCherry is lower than that obtained when the sod RBS is used (sod/sod and sod/LacZ, bars, Figure 4A). Next, a new plasmid designed pTGR11 was created where the sod RBS controlling the translation of eGFP is replaced by lacZ RBS, and the anticipated reduction in the expression was achieved confirming the feasibility of this approach to tune gene expression.


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)

Comparison of the co-expression levels of eGFP and mCherry genes in C. glutamicum. (A) the genes were assembled in operons with the indicated RBSs controlling the expression. In all cases operons are transcribed from the tac promoter. (B) the genes were co-expressed from two transcriptional units and the indicated promoter drives the expression of each gene. Fluorescence intensity of eGFP and mCherry relative to OD after 24 h of incubation of cultures is shown. Cultures were grown in BHIS medium and supplemented with 0.5 mM IPTG when the tac promoter was used. 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 4: Comparison of the co-expression levels of eGFP and mCherry genes in C. glutamicum. (A) the genes were assembled in operons with the indicated RBSs controlling the expression. In all cases operons are transcribed from the tac promoter. (B) the genes were co-expressed from two transcriptional units and the indicated promoter drives the expression of each gene. Fluorescence intensity of eGFP and mCherry relative to OD after 24 h of incubation of cultures is shown. Cultures were grown in BHIS medium and supplemented with 0.5 mM IPTG when the tac promoter was used. Values shown are means of three independent determinations. The standard deviations were in all the cases less than 10% of the corresponding means.
Mentions: To demonstrate that the pTGR system allows the amount of both proteins to be altered by either using RBSs with different stregths in the case of operons, or promoters of different strengths in the case of multiple transcriptional units; a further set of experiments was carried out. First, the operon contained in plasmid pTGR8 was modified by exchanging the sod RBS that controls the translation of mCherry with the lacZ RBS to obtain the pTGR10 plasmid. As expected, when using this construct the amount of mCherry is lower than that obtained when the sod RBS is used (sod/sod and sod/LacZ, bars, Figure 4A). Next, a new plasmid designed pTGR11 was created where the sod RBS controlling the translation of eGFP is replaced by lacZ RBS, and the anticipated reduction in the expression was achieved confirming the feasibility of this approach to tune gene expression.

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