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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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pTGR platform features. (A) Map of the generic pTGR synthetic plasmid where all parts are flanked by a standard set of unique restriction sites. (B) The two types of constructions for the expression of multiple genes. Sequential insertion of cassettes containing RBS-ORF for the assembly of operons or cassettes comprising Promoter-RBS-ORF-transcriptional terminator for the assembly of gene clusters.
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Figure 1: pTGR platform features. (A) Map of the generic pTGR synthetic plasmid where all parts are flanked by a standard set of unique restriction sites. (B) The two types of constructions for the expression of multiple genes. Sequential insertion of cassettes containing RBS-ORF for the assembly of operons or cassettes comprising Promoter-RBS-ORF-transcriptional terminator for the assembly of gene clusters.

Mentions: The series of synthetic plasmids was named pTGR, the generic vector is illustrated in Figure 1A and the all the derivatives used in this study are listed in Table 1 and a schematic representation is shown in Additional file 1. The plasmid posses the following eight parts: (i) a replication origin for the host, (ii) a replication origin for E. coli, (iii) a selectable marker, and a transcriptional unit cassette containing: (iv) a transcriptional regulator, (v) a promoter with an corresponding operator, (vi) a RBS, (vii) the gene to be expressed and (viii) a transcriptional terminator.


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)

pTGR platform features. (A) Map of the generic pTGR synthetic plasmid where all parts are flanked by a standard set of unique restriction sites. (B) The two types of constructions for the expression of multiple genes. Sequential insertion of cassettes containing RBS-ORF for the assembly of operons or cassettes comprising Promoter-RBS-ORF-transcriptional terminator for the assembly of gene clusters.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: pTGR platform features. (A) Map of the generic pTGR synthetic plasmid where all parts are flanked by a standard set of unique restriction sites. (B) The two types of constructions for the expression of multiple genes. Sequential insertion of cassettes containing RBS-ORF for the assembly of operons or cassettes comprising Promoter-RBS-ORF-transcriptional terminator for the assembly of gene clusters.
Mentions: The series of synthetic plasmids was named pTGR, the generic vector is illustrated in Figure 1A and the all the derivatives used in this study are listed in Table 1 and a schematic representation is shown in Additional file 1. The plasmid posses the following eight parts: (i) a replication origin for the host, (ii) a replication origin for E. coli, (iii) a selectable marker, and a transcriptional unit cassette containing: (iv) a transcriptional regulator, (v) a promoter with an corresponding operator, (vi) a RBS, (vii) the gene to be expressed and (viii) a transcriptional terminator.

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