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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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Related in: MedlinePlus

Evaluation of the co-expression of two reporter genes from pTGR constructs. (A) Time course of the co-expression of eGFP and mCherry genes contained in an operon relative to OD. (B) Time course of the co-expression of eGFP and mCherry genes contained in two transcriptional units relative to OD. 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 3: Evaluation of the co-expression of two reporter genes from pTGR constructs. (A) Time course of the co-expression of eGFP and mCherry genes contained in an operon relative to OD. (B) Time course of the co-expression of eGFP and mCherry genes contained in two transcriptional units relative to OD. 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: The pTGR system was designed to facilitate constructions for the expression of multiple genes. For this, two possible formats are possible: gene assembly into operons, or clusters containing more than one transcriptional unit. In order to validate the system, an operon was initially created with two reporter genes, one encoding for eGFP and the second for mCherry fluorescent protein [24]. The operon was constructed in just two cloning steps to obtain the pTGR8 plasmid. First the synthetic ORFs encoding both fluorescent proteins were inserted into the NdeI-EcoRI sites of a pTGR vector containing the tac promoter and the sod RBS and second, the NheI-AvrII fragment containing sod RBS-eGFP ORF was mobilized to the AvrII site of the plasmid containing the mCherry gene. Expression of the resulting operon successfully co-produced the two fluorescent proteins (Figure 3A).


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)

Evaluation of the co-expression of two reporter genes from pTGR constructs. (A) Time course of the co-expression of eGFP and mCherry genes contained in an operon relative to OD. (B) Time course of the co-expression of eGFP and mCherry genes contained in two transcriptional units relative to OD. 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 3: Evaluation of the co-expression of two reporter genes from pTGR constructs. (A) Time course of the co-expression of eGFP and mCherry genes contained in an operon relative to OD. (B) Time course of the co-expression of eGFP and mCherry genes contained in two transcriptional units relative to OD. 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: The pTGR system was designed to facilitate constructions for the expression of multiple genes. For this, two possible formats are possible: gene assembly into operons, or clusters containing more than one transcriptional unit. In order to validate the system, an operon was initially created with two reporter genes, one encoding for eGFP and the second for mCherry fluorescent protein [24]. The operon was constructed in just two cloning steps to obtain the pTGR8 plasmid. First the synthetic ORFs encoding both fluorescent proteins were inserted into the NdeI-EcoRI sites of a pTGR vector containing the tac promoter and the sod RBS and second, the NheI-AvrII fragment containing sod RBS-eGFP ORF was mobilized to the AvrII site of the plasmid containing the mCherry gene. Expression of the resulting operon successfully co-produced the two fluorescent proteins (Figure 3A).

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