Synthetic promoter design for new microbial chassis

Gilman, James; Love, John

doi:10.1042/bst20160042

Cited by 50 publications

(55 citation statements)

References 79 publications

(107 reference statements)

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“…By splicing together artificial pieces of promoter elements, novel promoters that do not exist in nature can be constructed rapidly (Blazeck and Alper, 2013). These promoters have the potential to be independent of the endogenous transcription regulation and function more robustly than those naturally derived ones (Gilman and Love, 2016;Portela et al, 2017). Some synthetic promoters such as the J231 family in the Registry of Standard Biological Parts have gained wide application in gene circuit construction (Qi et al, 2012), tool development (Jervis et al, 2019), chemical production (Meng et al, 2016;Choi et al, 2017;Zhou et al, 2017) and the characterization of biological systems (Pasotti et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Context‐dependency of synthetic minimal promoters in driving gene expression: a case study

Jin

Nawab

Xia

et al. 2019

Microbial Biotechnology

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Summary Synthetic promoters are considered ideal candidates in driving robust gene expression. Most of the available synthetic promoters are minimal promoters, for which the upstream sequence of the 5′ end of the core region is usually excluded. Although the upstream sequence has been shown to mediate transcription of natural promoters, its impact on synthetic promoters has not been widely studied. Here, a library of chromosomal DNA fragments is randomly fused with the 5′ end of the J23119 synthetic promoter, and the transcriptional performance of the promoter is evaluated through β‐galactosidase assay, fluorescence intensity and chemical biosynthesis. Results show that changes in the upstream sequence can induce significant variation in the promoter strength of up to 5.8‐fold. The effect is independent of the length of the insertions and the number of potential transcription factor binding sites. Several DNA fragments that are able to enhance the transcription of both the natural and the synthetic promoters are identified. This study indicates that the synthetic minimal promoters are susceptible to the surrounding sequence context. Therefore, the upstream sequence should be treated as an indispensable component in the design and application of synthetic promoters, or as an independent genetic part for the fine‐tuning of gene expression.

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Section: Introductionmentioning

confidence: 99%

Context‐dependency of synthetic minimal promoters in driving gene expression: a case study

Jin

Nawab

Xia

et al. 2019

Microbial Biotechnology

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“…As highly active promoters have been created for use in all bioproduction hosts, a component with the required functionality can be simply selected by screening a small panel (e.g., <10) of previously characterized synthetic constructs (Brown & James, 2015;Gilman & Love, 2016;Redden, Morse, & Alper, 2014). As highly active promoters have been created for use in all bioproduction hosts, a component with the required functionality can be simply selected by screening a small panel (e.g., <10) of previously characterized synthetic constructs (Brown & James, 2015;Gilman & Love, 2016;Redden, Morse, & Alper, 2014).…”

Section: Gene Transcriptionmentioning

confidence: 99%

“…Context-specific maximization of recombinant gene transcription requires identification of an appropriate promoter part. As highly active promoters have been created for use in all bioproduction hosts, a component with the required functionality can be simply selected by screening a small panel (e.g., <10) of previously characterized synthetic constructs (Brown & James, 2015;Gilman & Love, 2016;Redden, Morse, & Alper, 2014). Alternatively, a "gold standard" part can be used directly if it has been specifically designed or shown to maximize transcriptional output in diverse expression conditions.…”

Section: Gene Transcriptionmentioning

confidence: 99%

Whole synthetic pathway engineering of recombinant protein production

Brown

Gibson

Hatton

et al. 2018

Biotech & Bioengineering

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The output from protein biomanufacturing systems is a function of total host cell biomass synthetic capacity and recombinant protein production per unit cell biomass.In this study, we describe how these two properties can be simultaneously optimized via design of a product-specific combination of synthetic DNA parts to maximize flux through the protein synthetic pathway and the use of a host cell chassis with an increased capability to synthesize both cell and product biomass. Using secreted alkaline phosphatase (SEAP) production in Chinese hamster ovary cells as our example, we demonstrate how an optimal composition of input components can be assembled from a minimal toolbox containing rationally designed promoters, untranslated regions, signal peptides, product coding sequences, cell chassis, and genetic effectors. Product titer was increased 10-fold, compared with a standard reference system by (a) identifying genetic components that acted in concert to maximize the rates of SEAP transcription, translation, and translocation, (b) selection of a cell chassis with increased biomass synthetic capacity, and (c) engineering the host cell factory's capacity for protein folding and secretion. This whole synthetic pathway engineering process to design optimal expression cassette-chassis combinations should be applicable to diverse recombinant protein and host cell-type contexts.

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“…Although natural promoters have been widely used in relatively steady laboratory application, the number of functional promoters, however, is in such a low level, considering the large potential space of base combinations. In recent years, researchers increasingly turned to extract promoter features [1][2][3] and constructed libraries of synthetic promoter sequences for specific engineering aims [4][5][6] . Many molecular approaches to generate of synthetic promoter libraries has been applied, such as methods based on mutagenesis [6][7][8] , integrating the shorter functional parts [9][10][11] and combination of biological motifs 12,13 , etc.…”

Section: Introductionmentioning

confidence: 99%

Synthetic Promoter Design in Escherichia coli based on Generative Adversarial Network

Wang

Wei

et al. 2019

Preprint

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Synthetic promoters are commonly applied elements in circuit design for fine-tuning the protein expression levels. Promoter engineering was mostly focused on the random mutation or combination of regulation elements such as transcription factor binding sites. However, the size of promoter sequence space is still overwhelming and better navigation method is required. On the other hand, the generative adversarial network (GAN) is known for its great ability to reduce the searching space by learning to generate new data on the similar manifold of original data. Here, we applied WGAN-GP model into de novo promoter sequence design to generate entirely new promoter sequences. In total, 83 of model-generated promoter sequences were tested in promoter activity screening by regulating the expression of sfGFP gene in Escherichia coli. As a result, 26 out of 83 newly designed promoters were found functional and successfully expressed with varying activities, with similarity score to natural promoters all less than 0.7. Moreover, 3 of them showed higher promoter strength than the wild type promoters and their highly expression mutants. The much higher successful rate and promoter activity with much lower similarity score in our model-designed novel promoters confirmed the effectiveness of promoter sequence learning. Our work provides insights into an area of navigation of novel functional promoter sequence space automatically, as well as speeding up evolution process of naturally existing promoters, indicating the potential ability for deep generative models to be applied into genetic element designing in the future.

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Synthetic promoter design for new microbial chassis

Cited by 50 publications

References 79 publications

Context‐dependency of synthetic minimal promoters in driving gene expression: a case study

Context‐dependency of synthetic minimal promoters in driving gene expression: a case study

Whole synthetic pathway engineering of recombinant protein production

Synthetic Promoter Design in Escherichia coli based on Generative Adversarial Network

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