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Synthetic biology approaches to improve biocatalyst identification in metagenomic library screening.

Guazzaroni ME, Silva-Rocha R, Ward RJ - Microb Biotechnol (2014)

Bottom Line: There is a growing demand for enzymes with improved catalytic performance or tolerance to process-specific parameters, and biotechnology plays a crucial role in the development of biocatalysts for use in industry, agriculture, medicine and energy generation.Metagenomics takes advantage of the wealth of genetic and biochemical diversity present in the genomes of microorganisms found in environmental samples, and provides a set of new technologies directed towards screening for new catalytic activities from environmental samples with potential biotechnology applications.The bottleneck arising from inefficient screening of enzymatic activities has been addressed from several perspectives; however, the limitations related to biased expression in heterologous hosts cannot be overcome by using a single approach, but rather requires the synergetic implementation of multiple methodologies.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Química, FFCLRP, University of São Paulo, Ribeirão Preto, SP, Brazil.

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Synthetic Biology may overcome limiting steps in activity-based metagenomic library screening. Current bottlenecks in functional metagenomics are related to (A) limitations in the host capabilities, (B) the performance of the genetic tools and (C) the availability of efficient screening methods.A. In the case of the host, critical steps related to the recognition of transcriptional and translational signals, as well as the folding and modification of the expressed enzyme need to be enhanced. Host performance might be improved by reducing the metabolic burden related to the expression of unnecessary genes.B. The use of semi-synthetic, high-efficiency genetic tools is essential for the construction of metagenomic libraries that can be maintained and screened in a wide number of microorganisms. The example shows the pSEVA bacterial vector, which is endowed with several functional features such as terminators, origin for transfer and an extensive polylinker optimized for use in several bacterial hosts.C. Genetic circuits constructed by combining input modules (e.g. promoters and regulators) and output devices (such as reporter proteins) assembled with a standard format that uses the same sets of restriction enzymes (represented by X1, X2, etc.). Such circuits facilitate the screening of enzymatic activities in metagenomic libraries. The standardization of the assembly process facilitates the combination of several independent modules to construct sophisticated activity-trigged biosensors.
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fig02: Synthetic Biology may overcome limiting steps in activity-based metagenomic library screening. Current bottlenecks in functional metagenomics are related to (A) limitations in the host capabilities, (B) the performance of the genetic tools and (C) the availability of efficient screening methods.A. In the case of the host, critical steps related to the recognition of transcriptional and translational signals, as well as the folding and modification of the expressed enzyme need to be enhanced. Host performance might be improved by reducing the metabolic burden related to the expression of unnecessary genes.B. The use of semi-synthetic, high-efficiency genetic tools is essential for the construction of metagenomic libraries that can be maintained and screened in a wide number of microorganisms. The example shows the pSEVA bacterial vector, which is endowed with several functional features such as terminators, origin for transfer and an extensive polylinker optimized for use in several bacterial hosts.C. Genetic circuits constructed by combining input modules (e.g. promoters and regulators) and output devices (such as reporter proteins) assembled with a standard format that uses the same sets of restriction enzymes (represented by X1, X2, etc.). Such circuits facilitate the screening of enzymatic activities in metagenomic libraries. The standardization of the assembly process facilitates the combination of several independent modules to construct sophisticated activity-trigged biosensors.

Mentions: The previous sections have highlighted a number of bottlenecks facing metagenomic screening that need to be resolved in order to improve the discovery rate of target enzymes. These limitations can be grouped in three main categories (as shown in Fig. 2). First, there is a need for improvement in host organism capabilities with the aim of improving the expression of the target enzymes. Second, the development of new genetic tools is necessary in order to improve the construction of metagenomic libraries suitable for screening in different hosts. Finally, continuation of ongoing research to elaborate novel screening strategies that enhance the discovery rate of the enzymes of interest is needed. The advances in synthetic biology over the past decade could provide the framework to address these constrains, and a particularly promising approach is the analysis of biological systems in an analogous way as electronic devices, whereby cells can be reprogrammed to perform new tasks with high efficiency (Purnick and Weiss, 2009; Weber and Fussenegger, 2010). Synthetic biology relies on a conceptual framework more closely related to engineering than biology, such as design, modelling, implementation and debugging (Canton et al., 2008; Purnick and Weiss, 2009; Weber and Fussenegger, 2010). The design aspect focuses on the planning and construction of new gene circuits for the desired application (Canton et al., 2008). Modelling involves computational simulation of the proposed gene circuits in order to both evaluate performance and capabilities and to guide the selection of the suitable molecular components necessary for its construction (Koide et al., 2009). The implementation step encompasses the physical assembly of the DNA elements encoding the appropriate components (such as promoters, regulators, terminators, enzymes, transporters, etc.), and follows a specific assembly standard (Arkin, 2008). Finally, the debugging step requires the testing and validation of the circuit in vivo, and includes the correction of undesirable traits that have their origin in the emergent properties of biology systems (Gardner et al., 2000; Moon et al., 2011; Siuti et al., 2013). Several examples of new biological circuits that have been successfully designed and implemented are currently available (Gardner et al., 2000; Cox et al., 2007; 2010; Moon et al., 2011; Silva-Rocha and de Lorenzo, 2011), and in recent years, the field has developed at a remarkable speed (Weber and Fussenegger, 2010; Zhan et al., 2010; Regot et al., 2011; Siuti et al., 2013).


Synthetic biology approaches to improve biocatalyst identification in metagenomic library screening.

Guazzaroni ME, Silva-Rocha R, Ward RJ - Microb Biotechnol (2014)

Synthetic Biology may overcome limiting steps in activity-based metagenomic library screening. Current bottlenecks in functional metagenomics are related to (A) limitations in the host capabilities, (B) the performance of the genetic tools and (C) the availability of efficient screening methods.A. In the case of the host, critical steps related to the recognition of transcriptional and translational signals, as well as the folding and modification of the expressed enzyme need to be enhanced. Host performance might be improved by reducing the metabolic burden related to the expression of unnecessary genes.B. The use of semi-synthetic, high-efficiency genetic tools is essential for the construction of metagenomic libraries that can be maintained and screened in a wide number of microorganisms. The example shows the pSEVA bacterial vector, which is endowed with several functional features such as terminators, origin for transfer and an extensive polylinker optimized for use in several bacterial hosts.C. Genetic circuits constructed by combining input modules (e.g. promoters and regulators) and output devices (such as reporter proteins) assembled with a standard format that uses the same sets of restriction enzymes (represented by X1, X2, etc.). Such circuits facilitate the screening of enzymatic activities in metagenomic libraries. The standardization of the assembly process facilitates the combination of several independent modules to construct sophisticated activity-trigged biosensors.
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Related In: Results  -  Collection

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fig02: Synthetic Biology may overcome limiting steps in activity-based metagenomic library screening. Current bottlenecks in functional metagenomics are related to (A) limitations in the host capabilities, (B) the performance of the genetic tools and (C) the availability of efficient screening methods.A. In the case of the host, critical steps related to the recognition of transcriptional and translational signals, as well as the folding and modification of the expressed enzyme need to be enhanced. Host performance might be improved by reducing the metabolic burden related to the expression of unnecessary genes.B. The use of semi-synthetic, high-efficiency genetic tools is essential for the construction of metagenomic libraries that can be maintained and screened in a wide number of microorganisms. The example shows the pSEVA bacterial vector, which is endowed with several functional features such as terminators, origin for transfer and an extensive polylinker optimized for use in several bacterial hosts.C. Genetic circuits constructed by combining input modules (e.g. promoters and regulators) and output devices (such as reporter proteins) assembled with a standard format that uses the same sets of restriction enzymes (represented by X1, X2, etc.). Such circuits facilitate the screening of enzymatic activities in metagenomic libraries. The standardization of the assembly process facilitates the combination of several independent modules to construct sophisticated activity-trigged biosensors.
Mentions: The previous sections have highlighted a number of bottlenecks facing metagenomic screening that need to be resolved in order to improve the discovery rate of target enzymes. These limitations can be grouped in three main categories (as shown in Fig. 2). First, there is a need for improvement in host organism capabilities with the aim of improving the expression of the target enzymes. Second, the development of new genetic tools is necessary in order to improve the construction of metagenomic libraries suitable for screening in different hosts. Finally, continuation of ongoing research to elaborate novel screening strategies that enhance the discovery rate of the enzymes of interest is needed. The advances in synthetic biology over the past decade could provide the framework to address these constrains, and a particularly promising approach is the analysis of biological systems in an analogous way as electronic devices, whereby cells can be reprogrammed to perform new tasks with high efficiency (Purnick and Weiss, 2009; Weber and Fussenegger, 2010). Synthetic biology relies on a conceptual framework more closely related to engineering than biology, such as design, modelling, implementation and debugging (Canton et al., 2008; Purnick and Weiss, 2009; Weber and Fussenegger, 2010). The design aspect focuses on the planning and construction of new gene circuits for the desired application (Canton et al., 2008). Modelling involves computational simulation of the proposed gene circuits in order to both evaluate performance and capabilities and to guide the selection of the suitable molecular components necessary for its construction (Koide et al., 2009). The implementation step encompasses the physical assembly of the DNA elements encoding the appropriate components (such as promoters, regulators, terminators, enzymes, transporters, etc.), and follows a specific assembly standard (Arkin, 2008). Finally, the debugging step requires the testing and validation of the circuit in vivo, and includes the correction of undesirable traits that have their origin in the emergent properties of biology systems (Gardner et al., 2000; Moon et al., 2011; Siuti et al., 2013). Several examples of new biological circuits that have been successfully designed and implemented are currently available (Gardner et al., 2000; Cox et al., 2007; 2010; Moon et al., 2011; Silva-Rocha and de Lorenzo, 2011), and in recent years, the field has developed at a remarkable speed (Weber and Fussenegger, 2010; Zhan et al., 2010; Regot et al., 2011; Siuti et al., 2013).

Bottom Line: There is a growing demand for enzymes with improved catalytic performance or tolerance to process-specific parameters, and biotechnology plays a crucial role in the development of biocatalysts for use in industry, agriculture, medicine and energy generation.Metagenomics takes advantage of the wealth of genetic and biochemical diversity present in the genomes of microorganisms found in environmental samples, and provides a set of new technologies directed towards screening for new catalytic activities from environmental samples with potential biotechnology applications.The bottleneck arising from inefficient screening of enzymatic activities has been addressed from several perspectives; however, the limitations related to biased expression in heterologous hosts cannot be overcome by using a single approach, but rather requires the synergetic implementation of multiple methodologies.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Química, FFCLRP, University of São Paulo, Ribeirão Preto, SP, Brazil.

Show MeSH
Related in: MedlinePlus