Tuning genetic control through promoter engineering

Alper, Hal S.; Fischer, Curt R.; Nevoigt, Elke; Stephanopoulos, Gregory

doi:10.1073/pnas.0504604102

Cited by 780 publications

(700 citation statements)

References 40 publications

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“…Library approaches can provide a direct route for obtaining and refining functional in vivo systems (39)(40)(41). In the field of metabolic engineering, for example, researchers have repeatedly improved natural product yields and synthesized analogs by searching collections of isozymes (42,43), mutant biosynthetic enzymes (3,44), or promoters and regulatory regions that modulate the expression levels of genes that alter pathway flux (45,46). Testing these multiple variables in the context of a pathway causes library sizes to rapidly swell (e.g., testing 100 mutants of enzyme A against 100 mutants of enzyme B is already Fig.…”

Section: Discussionmentioning

confidence: 99%

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

Wingler

Cornish

2011

Proc. Natl. Acad. Sci. U.S.A.

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The increasing sophistication of synthetic biology is creating a demand for robust, broadly accessible methodology for constructing multigene pathways inside of the cell. Due to the difficulty of rationally designing pathways that function as desired in vivo, there is a further need to assemble libraries of pathways in parallel, in order to facilitate the combinatorial optimization of performance. While some in vitro DNA assembly methods can theoretically make libraries of pathways, these techniques are resource intensive and inherently require additional techniques to move the DNA back into cells. All previously reported in vivo assembly techniques have been low yielding, generating only tens to hundreds of constructs at a time. Here, we develop “Reiterative Recombination,” a robust method for building multigene pathways directly in the yeast chromosome. Due to its use of endonuclease-induced homologous recombination in conjunction with recyclable markers, Reiterative Recombination provides a highly efficient, technically simple strategy for sequentially assembling an indefinite number of DNA constructs at a defined locus. In this work, we describe the design and construction of the first Reiterative Recombination system in Saccharomyces cerevisiae , and we show that it can be used to assemble multigene constructs. We further demonstrate that Reiterative Recombination can construct large mock libraries of at least 10 4 biosynthetic pathways. We anticipate that our system’s simplicity and high efficiency will make it a broadly accessible technology for pathway construction and render it a valuable tool for optimizing pathways in vivo.

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Section: Discussionmentioning

confidence: 99%

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

Wingler

Cornish

2011

Proc. Natl. Acad. Sci. U.S.A.

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“…The second approach uses combinations of individually characterized elements to attain desired expression without directly considering their DNA sequences (17)(18)(19)(20)(21)(22)(23)(24)(25). Current efforts have focused on approaches to limit the number of time-consuming steps required to characterize potential interactions and on identifying existing or engineered elements that act predictably when used in combination (26)(27)(28).…”

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confidence: 99%

Composability of regulatory sequences controlling transcription and translation in Escherichia coli

Kosuri

Goodman

Cambray

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

398

447

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The inability to predict heterologous gene expression levels precisely hinders our ability to engineer biological systems. Using well-characterized regulatory elements offers a potential solution only if such elements behave predictably when combined. We synthesized 12,563 combinations of common promoters and ribosome binding sites and simultaneously measured DNA, RNA, and protein levels from the entire library. Using a simple model, we found that RNA and protein expression were within twofold of expected levels 80% and 64% of the time, respectively. The large dataset allowed quantitation of global effects, such as translation rate on mRNA stability and mRNA secondary structure on translation rate. However, the worst 5% of constructs deviated from prediction by 13-fold on average, which could hinder large-scale genetic engineering projects. The ease and scale this of approach indicates that rather than relying on prediction or standardization, we can screen synthetic libraries for desired behavior.next-generation sequencing | synthetic biology | systems biology O rganisms can be engineered to produce chemical, material, fuel, and medical products that are often superior to nonbiological alternatives (1). Biotechnologists have sought to discover, improve, and industrialize such products through the use of recombinant DNA technologies (2, 3). In recent years, these efforts have increased in complexity from expressing a few genes at once to optimizing multicomponent circuits and pathways (4-7). To attain desired systems-level function reliably, careful and time-consuming optimization of individual components is required (8-11).To mitigate this slow trial-and-error optimization, two dominant approaches have taken hold. The first approach seeks to predict expression levels by elucidating the biophysical relationships between sequence and function. For example, several groups have modified promoters (12, 13) and ribosome binding sites (RBSs) (14-16) to see how small sequence changes affect transcription or translation. Such studies are fundamentally challenging due to the vastness of sequence space. In addition, because these approaches mostly look at either transcription or translation individually, they are rarely able to investigate interactions between these processes.The second approach uses combinations of individually characterized elements to attain desired expression without directly considering their DNA sequences (17-25). Current efforts have focused on approaches to limit the number of time-consuming steps required to characterize potential interactions and on identifying existing or engineered elements that act predictably when used in combination (26-28). However, these studies still suggest there are enough idiosyncratic interactions and context effects that it will be necessary to construct and measure many variants of a circuit to achieve desired function (29). For larger circuits, such approaches are necessarily limited in scope due to the difficulty in measuring large numbers of combinations (26, 27...

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“…In synthetic biology, most ventures proceed through four phases of strain engineering-design, construction, implementation (booting) and troubleshooting (debugging) 7 . The design of increasingly more complex biological systems has been greatly enhanced by the wide array of tools now available for controlling heterologous protein and pathway expression at both the transcriptional (promoter and messenger RNA) and translational (RBS) levels [9][10][11] . Similarly, the construction of large and complex designs has become an almost routine process through the use of cheap, automated and sophisticated techniques for both de novo DNA synthesis 12,13 and genetic parts assembly [14][15][16][17] .…”

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confidence: 99%

Implementation of stable and complex biological systems through recombinase-assisted genome engineering

Santos

Regitsky²,

Yoshikuni³

2013

Nat Commun

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Evaluating the performance of engineered biological systems with high accuracy and precision is nearly impossible with the use of plasmids due to phenotypic noise generated by genetic instability and natural population dynamics. Minimizing this uncertainty therefore requires a paradigm shift towards engineering at the genomic level. Here, we introduce an advanced design principle for the stable instalment and implementation of complex biological systems through recombinase-assisted genome engineering (RAGE). We apply this concept to the development of a robust strain of Escherichia coli capable of producing ethanol directly from brown macroalgae. RAGE significantly expedites the optimal implementation of a 34 kb heterologous pathway for alginate metabolism based on genetic background, integration locus, copy number and compatibility with two other pathway modules (alginate degradation and ethanol production). The resulting strain achieves a B40% higher titre than its plasmidbased counterpart and enables substantial improvements in titre (B330%) and productivity (B1,200%) after 50 generations.

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Tuning genetic control through promoter engineering

Cited by 780 publications

References 40 publications

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

Composability of regulatory sequences controlling transcription and translation in Escherichia coli

Implementation of stable and complex biological systems through recombinase-assisted genome engineering

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