Abstract:Lysate-based
cell-free expression (CFE) systems are accessible
platforms for expressing proteins that are difficult to synthesize in vivo, such as nonribosomal peptide synthetases (NRPSs).
NRPSs are large (>100 kDa), modular enzyme complexes that synthesize
bioactive peptide natural products. This synthetic process is analogous
to transcription/translation (TX/TL) in lysates, resulting in potential
resource competition between NRPS expression and NRPS activity in
cell-free environments. Moreover, CFE condition… Show more
“…S7 ). Thus, these results also confirm that different proteins have varying optimal CFE expression conditions 54 . Figure 4 Flaviolin measurement in CFE reactions initiated with plasmids carrying different promoter and coding sequence combinations.…”
Section: Resultssupporting
confidence: 72%
“…The formation of the red-brown flaviolin pigment can thus be monitored as it readily absorbs light at 340 nm above cellular background 24 , 53 . As metabolite production in an E. coli lysate-based cell-free system is correlated with the amount of protein that is expressed 54 , we used the amount of flaviolin produced as a proxy for estimating catalytically functional RppA production. To establish assay conditions, we first sought to determine the threshold for pigment production against a cell lysate background.…”
Section: Resultsmentioning
confidence: 99%
“…To define temperature conditions for conducting CFE experiments, we used the pET28b expression plasmid and codon-optimized rppA (O- rppA ) in a lysate-based system using E. coli BL21 Star(DE3), a BL21(DE3) strain that has the DE3 lysogen under control of a lac- UV5 promoter 55 . We have previously had success using this lysate for the production of pigments from Streptomyces 54 . Our rationale for this initial choice of coding sequence and promoter was twofold: (1) prior studies from our laboratory suggest that heterologous expression of enzymes originating from Streptomyces that form pigments show improved expression and solubility when using E. coli optimized sequences 33 and (2) the T7 promoters (and specifically pET vectors) have been widely used in CFE 54 , 56 – 60 .…”
Section: Resultsmentioning
confidence: 99%
“…We have previously had success using this lysate for the production of pigments from Streptomyces 54 . Our rationale for this initial choice of coding sequence and promoter was twofold: (1) prior studies from our laboratory suggest that heterologous expression of enzymes originating from Streptomyces that form pigments show improved expression and solubility when using E. coli optimized sequences 33 and (2) the T7 promoters (and specifically pET vectors) have been widely used in CFE 54 , 56 – 60 . These initial experiments revealed superior pigment production at 30 °C, so we proceeded with 30 °C for all subsequent experiments (Fig.…”
Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have distinct protein folding environments compared to tractable expression hosts like Escherichia coli. Consequentially, expressing biosynthetic gene clusters (BGCs) from these bacteria in E. coli often results in a myriad of unpredictable issues with regard to protein expression and folding, delaying the biochemical characterization of new natural products. Current strategies to achieve soluble, active expression of these enzymes in tractable hosts can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as a valuable expression platform as a testbed for rapid prototyping expression parameters. Here, we use a type III polyketide synthase from Streptomyces griseus, RppA, which catalyzes the formation of the red pigment flaviolin, as a reporter to investigate BGC refactoring techniques. We applied a library of constructs with different combinations of promoters and rppA coding sequences to investigate the synergies between promoter and codon usage. Subsequently, we assess the utility of cell-free systems for prototyping these refactoring tactics prior to their implementation in cells. Overall, codon harmonization improves natural product synthesis more than traditional codon optimization across cell-free and cellular environments. More importantly, the choice of coding sequences and promoters impact protein expression synergistically, which should be considered for future efforts to use CFE for high-yield protein expression. The promoter strategy when applied to RppA was not completely correlated with that observed with GFP, indicating that different promoter strategies should be applied for different proteins. In vivo experiments suggest that there is correlation, but not complete alignment between expressing in cell free and in vivo. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs, which advances CFE as a tool for natural product research.
“…S7 ). Thus, these results also confirm that different proteins have varying optimal CFE expression conditions 54 . Figure 4 Flaviolin measurement in CFE reactions initiated with plasmids carrying different promoter and coding sequence combinations.…”
Section: Resultssupporting
confidence: 72%
“…The formation of the red-brown flaviolin pigment can thus be monitored as it readily absorbs light at 340 nm above cellular background 24 , 53 . As metabolite production in an E. coli lysate-based cell-free system is correlated with the amount of protein that is expressed 54 , we used the amount of flaviolin produced as a proxy for estimating catalytically functional RppA production. To establish assay conditions, we first sought to determine the threshold for pigment production against a cell lysate background.…”
Section: Resultsmentioning
confidence: 99%
“…To define temperature conditions for conducting CFE experiments, we used the pET28b expression plasmid and codon-optimized rppA (O- rppA ) in a lysate-based system using E. coli BL21 Star(DE3), a BL21(DE3) strain that has the DE3 lysogen under control of a lac- UV5 promoter 55 . We have previously had success using this lysate for the production of pigments from Streptomyces 54 . Our rationale for this initial choice of coding sequence and promoter was twofold: (1) prior studies from our laboratory suggest that heterologous expression of enzymes originating from Streptomyces that form pigments show improved expression and solubility when using E. coli optimized sequences 33 and (2) the T7 promoters (and specifically pET vectors) have been widely used in CFE 54 , 56 – 60 .…”
Section: Resultsmentioning
confidence: 99%
“…We have previously had success using this lysate for the production of pigments from Streptomyces 54 . Our rationale for this initial choice of coding sequence and promoter was twofold: (1) prior studies from our laboratory suggest that heterologous expression of enzymes originating from Streptomyces that form pigments show improved expression and solubility when using E. coli optimized sequences 33 and (2) the T7 promoters (and specifically pET vectors) have been widely used in CFE 54 , 56 – 60 . These initial experiments revealed superior pigment production at 30 °C, so we proceeded with 30 °C for all subsequent experiments (Fig.…”
Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have distinct protein folding environments compared to tractable expression hosts like Escherichia coli. Consequentially, expressing biosynthetic gene clusters (BGCs) from these bacteria in E. coli often results in a myriad of unpredictable issues with regard to protein expression and folding, delaying the biochemical characterization of new natural products. Current strategies to achieve soluble, active expression of these enzymes in tractable hosts can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as a valuable expression platform as a testbed for rapid prototyping expression parameters. Here, we use a type III polyketide synthase from Streptomyces griseus, RppA, which catalyzes the formation of the red pigment flaviolin, as a reporter to investigate BGC refactoring techniques. We applied a library of constructs with different combinations of promoters and rppA coding sequences to investigate the synergies between promoter and codon usage. Subsequently, we assess the utility of cell-free systems for prototyping these refactoring tactics prior to their implementation in cells. Overall, codon harmonization improves natural product synthesis more than traditional codon optimization across cell-free and cellular environments. More importantly, the choice of coding sequences and promoters impact protein expression synergistically, which should be considered for future efforts to use CFE for high-yield protein expression. The promoter strategy when applied to RppA was not completely correlated with that observed with GFP, indicating that different promoter strategies should be applied for different proteins. In vivo experiments suggest that there is correlation, but not complete alignment between expressing in cell free and in vivo. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs, which advances CFE as a tool for natural product research.
“…Thus, this is additionally appealing for NRPS characterization and engineering because it can accelerate designbuild-test-learn (DBTL) cycles. To these ends, NRPSs have recently been expressed in both lysate-based (Goering et al, 2017;Zhuang et al, 2020;Moore et al, 2021;Dinglasan et al, 2023) and PURExpress (Siebels et al, 2020a) cell-free systems. This review highlights examples of NRPS natural products that are expressed in CFE systems in synergy with different transcriptional and translational machineries.…”
Peptide natural products have a wide range of useful applications as pesticides, veterinary agents, pharmaceuticals, and bioproducts. To discover new natural products, manipulate them for analog generation, and to harness the potential of these bioactive compounds for synthetic biology, it is necessary to develop robust methods for the expression of biosynthetic genes. Cell-free synthetic biology is emerging as an important complementary approach because it is highly desirable to express protein on a more rapid timescale and does not rely upon the genetic tractability of a strain thus improving the throughput of design-build-test-learn cycles. Additionally, generating metabolites outside the cell can overcome issues such as cellular toxicity which can hamper applications like antibiotic development. In this review, we focus on the cell-free production of peptide natural products generated by non-ribosomal peptide synthetase. Nonribsomal peptides are biosynthesized by non-ribosomal peptide synthetases which are large “mega” enzymes that provide specific challenges to heterologous expression. First, we summarize NRPSs and their corresponding peptide metabolites that are expressed in cell-free systems. With that, we discuss the requirements and challenges to express such large proteins in cell-free protein synthesis as well as host machineries that have been developed for cell-free protein synthesis that could be particularly relevant to generating non-ribosomal peptide metabolites in the future. The development of cell-free systems can then be used for prototyping to accelerate efforts towards engineered biosynthesis of these complex pathways.
Cell-free protein synthesis (CFPS) has emerged as an attractive platform for biotechnology and synthetic biology due to its numerous advantages to cell-based technologies for specific applications. CFPS can be faster, less sensitive to metabolite toxicity, and amenable to systems that are not easily genetically manipulated. Due to these advantages, a promising application of CFPS is to characterize biosynthetic gene clusters, particularly those harbored within the genomes of microorganisms that generate secondary metabolites, otherwise known as natural products. In the postgenomic era, genome sequencing has revealed an incredible wealth of metabolic diversity. However, far more of these pathways are termed “cryptic,” i.e., unable to be produced under standard laboratory conditions than have been characterized. A major barrier to characterizing these cryptic natural products using CFPS is that many of these pathways require utilization of complex cofactors, many of which to date are not recycled efficiently or in an economically viable fashion. In this perspective, we outline strategies to regenerate cofactors relevant to secondary metabolite production in CFPS. This includes adenosine 5′-triphosphate, coenzyme A, redox cofactors (iron-sulfur clusters, nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide), all of which play a crucial role in important biosynthetic enzymes. Such advances in cofactor recycling enable continuous production of complex metabolites in CFPS and expand the utility of this emergent platform.
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