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Victor De Lorenzo - One of the best experts on this subject based on the ideXlab platform.
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digitalizing Heterologous Gene expression in gram negative bacteria with a portable on off module
Molecular Systems Biology, 2019Co-Authors: Belen Calles, Angel Gonimoreno, Victor De LorenzoAbstract:While prokaryotic promoters controlled by signal-responding regulators typically display a range of input/output ratios when exposed to cognate inducers, virtually no naturally occurring cases are known to have an OFF state of zero transcription-as ideally needed for synthetic circuits. To overcome this problem, we have modelled and implemented a simple digitalizer module that completely suppresses the basal level of otherwise strong promoters in such a way that expression in the absence of induction is entirely impeded. The circuit involves the interplay of a translation-inhibitory sRNA with the translational coupling of the Gene of interest to a repressor such as LacI. The digitalizer module was validated with the strong inducible promoters Pm (induced by XylS in the presence of benzoate) and PalkB (induced by AlkS/dicyclopropyl ketone) and shown to perform effectively in both Escherichia coli and the soil bacterium Pseudomonas putida. The distinct expression architecture allowed cloning and conditional expression of, e.g. colicin E3, one molecule of which per cell suffices to kill the host bacterium. Revertants that escaped ColE3 killing were not found in hosts devoid of insertion sequences, suggesting that mobile elements are a major source of circuit inactivation in vivo.
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digitalizing Heterologous Gene expression in gram negative bacteria with a portable on off module
bioRxiv, 2019Co-Authors: Belen Calles, Angel Gonimoreno, Victor De LorenzoAbstract:ABSTRACT While prokaryotic promoters controlled by signal-responding regulators typically display a range of input/output ratios when exposed to cognate inducers, virtually no naturally occurring cases are known to have an off state of zero transcription—as ideally needed for synthetic circuits. To overcome this problem we have modelled and implemented simple digitalizer module that completely suppresses the basal level of otherwise strong promoters in such a way that expression in the absence of induction is entirely impeded. The circuit involves the interplay of a translation-inhibitory sRNA with the translational coupling of the Gene of interest to a repressor such as LacI. The digitalizer module was validated with the strong inducible promoters Pm (induced by XylS in the presence of benzoate) and PalkB (induced by AlkS/dicyclopropylketone) and shown to perform effectively both in E. coli and the soil bacterium Pseudomonas putida. The distinct expression architecture allowed cloning and conditional expression of e.g. colicin E3, one molecule of which per cell suffices to kill the host bacterium. Revertants that escaped ColE3 killing were not found in hosts devoid of insertion sequences, suggesting that mobile elements are a major source of circuit inactivation in vivo.
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tn7 based device for calibrated Heterologous Gene expression in pseudomonas putida
ACS Synthetic Biology, 2015Co-Authors: Sebastian Zobel, Victor De Lorenzo, Ilaria Benedetti, Lara Eisenbach, Nick Wierckx, Lars M BlankAbstract:The soil bacterium Pseudomonas putida is increasingly attracting considerable interest as a platform for advanced metabolic engineering through synthetic biology approaches. However, genomic context, Gene copy number, and transcription/translation interplay often introduce considerable uncertainty to the design of reliable Genetic constructs. In this work, we have established a standardized Heterologous expression device in which the promoter strength is the only variable; the remaining parameters of the flow have stable default values. To this end, we tailored a mini-Tn7 delivery transposon vector that inserts the constructs in a single genomic locus of P. putida’s chromosome. This was then merged with a promoter insertion site, an unvarying translational coupler, and a downstream location for placing the Gene(s) of interest under fixed assembly rules. This arrangement was exploited to benchmark a collection of synthetic promoters with low transcriptional noise in this bacterial host. Growth experiments ...
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genome reduction boosts Heterologous Gene expression in pseudomonas putida
Microbial Cell Factories, 2015Co-Authors: Sarah Lieder, Pablo I Nikel, Victor De Lorenzo, Ralf TakorsAbstract:Background The implementation of novel platform organisms to be used as microbial cell factories in industrial applications is currently the subject of intense research. Ongoing efforts include the adoption of Pseudomonas putida KT2440 variants with a reduced genome as the functional chassis for biotechnological purposes. In these strains, dispensable functions removed include flagellar motility (1.1% of the genome) and a number of open reading frames expected to improve genotypic and phenotypic stability of the cells upon deletion (3.2% of the genome).
Mervyn J. Bibb - One of the best experts on this subject based on the ideXlab platform.
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Heterologous expression of natural product biosynthetic Gene clusters in streptomyces coelicolor from genome mining to manipulation of biosynthetic pathways
Journal of Industrial Microbiology & Biotechnology, 2014Co-Authors: Juan Pablo Gomezescribano, Mervyn J. BibbAbstract:Heterologous Gene expression is one of the main strategies used to access the full biosynthetic potential of actinomycetes, as well as to study the metabolic pathways of natural product biosynthesis and to create unnatural pathways. Streptomyces coelicolor A3(2) is the most studied member of the actinomycetes, bacteria renowned for their prolific capacity to synthesize a wide range of biologically active specialized metabolites. We review here the use of strains of this species for the Heterologous production of structurally diverse actinomycete natural products.
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Streptomyces coelicolor as an expression host for Heterologous Gene clusters.
Methods in enzymology, 2012Co-Authors: Juan Pablo Gomez-escribano, Mervyn J. BibbAbstract:The expression of a Gene or a set of Genes from one organism in a different species is known as "Heterologous expression." In actinomycetes, prolific producers of natural products, Heterologous Gene expression has been used to confirm the clustering of secondary metabolite biosynthetic Genes, to analyze natural product biosynthesis, to produce variants of natural products by Genetic engineering, and to discover new compounds by screening genomic libraries. Recent advances in DNA sequencing have enabled the rapid and affordable sequencing of actinomycete genomes and revealed a large number of secondary metabolite Gene clusters with no known products. Heterologous expression of these cryptic Gene clusters combined with comparative metabolic profiling provides an important means to identify potentially novel compounds. In this chapter, the methods and strategies used to Heterologously express actinomycete Gene clusters, including the techniques used for cloning secondary metabolite Gene clusters, the Streptomyces hosts used for their expression, and the techniques employed to analyze their products by metabolic profiling, are described.
Hal S. Alper - One of the best experts on this subject based on the ideXlab platform.
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Short Synthetic Terminators for Improved Heterologous Gene Expression in Yeast
ACS synthetic biology, 2015Co-Authors: Kathleen A. Curran, Nicholas J. Morse, Kelly A. Markham, Allison M. Wagman, Akash Gupta, Hal S. AlperAbstract:Terminators play an important role both in completing the transcription process and impacting mRNA half-life. As such, terminators are an important synthetic component considered in applications such as Heterologous Gene expression and metabolic engineering. Here, we describe a panel of short (35-70 bp) synthetic terminators that can be used for modulating Gene expression in yeast. The best of these synthetic terminator resulted in 3.7-fold more fluorescent protein output and 4.4-fold increase in transcript level compared to that with the commonly used CYC1 terminator. These synthetic terminators offer several advantages over native sequences, including an easily synthesized short length, minimal sequence homology to native sequences, and similar or better performance characteristics than those of commonly used longer terminators. Furthermore, the synthetic terminators are highly functional in both Saccharomyces cerevisiae and an alternative yeast, Yarrowia lipolytica, demonstrating that these synthetic designs are transferrable between diverse yeast species.
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A condition-specific codon optimization approach for improved Heterologous Gene expression in Saccharomyces cerevisiae
BMC systems biology, 2014Co-Authors: Amanda M. Lanza, Kathleen A. Curran, Lindsey G Rey, Hal S. AlperAbstract:Background: Heterologous Gene expression is an important tool for synthetic biology that enables metabolic engineering and the production of non-natural biologics in a variety of host organisms. The translational efficiency of Heterologous Genes can often be improved by optimizing synonymous codon usage to better match the host organism. However, traditional approaches for optimization neglect to take into account many factors known to influence synonymous codon distributions. Results: Here we define an alternative approach for codon optimization that utilizes systems level information and codon context for the condition under which Heterologous Genes are being expressed. Furthermore, we utilize a probabilistic algorithm to Generate multiple variants of a given Gene. We demonstrate improved translational efficiency using this condition-specific codon optimization approach with two Heterologous Genes, the fluorescent protein-encoding eGFP and the catechol 1,2-dioxygenase Gene CatA, expressed in S. cerevisiae. For the latter case, optimization for stationary phase production resulted in nearly 2.9-fold improvements over commercial Gene optimization algorithms. Conclusions: Codon optimization is now often a standard tool for protein expression, and while a variety of tools and approaches have been developed, they do not guarantee improved performance for all hosts of applications. Here, we suggest an alternative method for condition-specific codon optimization and demonstrate its utility in Saccharomyces cerevisiae as a proof of concept. However, this technique should be applicable to any organism for which Gene expression data can be Generated and is thus of potential interest for a variety of applications in metabolic and cellular engineering.
Lars M Blank - One of the best experts on this subject based on the ideXlab platform.
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tn7 based device for calibrated Heterologous Gene expression in pseudomonas putida
ACS Synthetic Biology, 2015Co-Authors: Sebastian Zobel, Victor De Lorenzo, Ilaria Benedetti, Lara Eisenbach, Nick Wierckx, Lars M BlankAbstract:The soil bacterium Pseudomonas putida is increasingly attracting considerable interest as a platform for advanced metabolic engineering through synthetic biology approaches. However, genomic context, Gene copy number, and transcription/translation interplay often introduce considerable uncertainty to the design of reliable Genetic constructs. In this work, we have established a standardized Heterologous expression device in which the promoter strength is the only variable; the remaining parameters of the flow have stable default values. To this end, we tailored a mini-Tn7 delivery transposon vector that inserts the constructs in a single genomic locus of P. putida’s chromosome. This was then merged with a promoter insertion site, an unvarying translational coupler, and a downstream location for placing the Gene(s) of interest under fixed assembly rules. This arrangement was exploited to benchmark a collection of synthetic promoters with low transcriptional noise in this bacterial host. Growth experiments ...
Ralf Takors - One of the best experts on this subject based on the ideXlab platform.
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genome reduction boosts Heterologous Gene expression in pseudomonas putida
Microbial Cell Factories, 2015Co-Authors: Sarah Lieder, Pablo I Nikel, Victor De Lorenzo, Ralf TakorsAbstract:Background The implementation of novel platform organisms to be used as microbial cell factories in industrial applications is currently the subject of intense research. Ongoing efforts include the adoption of Pseudomonas putida KT2440 variants with a reduced genome as the functional chassis for biotechnological purposes. In these strains, dispensable functions removed include flagellar motility (1.1% of the genome) and a number of open reading frames expected to improve genotypic and phenotypic stability of the cells upon deletion (3.2% of the genome).