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Building enhancers from the ground up: a synthetic biology approach.

ABSTRACT: A challenge of the synthetic biology approach is to use our understanding of a system to recreate a biological function with specific properties. We have applied this framework to bacterial enhancers, combining a driver, transcription factor binding sites, and a poised polymerase to create synthetic modular enhancers. Our findings suggest that enhancer-based transcriptional control depends critically and quantitatively on DNA looping, leading to complex regulatory effects when the enhancer cassettes contain additional transcription factor binding sites for TetR, a bacterial transcription factor. We show through a systematic interplay of experiment and thermodynamic modeling that the level of gene expression can be modulated to convert a variable inducer concentration input into discrete or step-like output expression levels. Finally, using a different DNA-binding protein (TraR), we show that the regulatory output is not a particular feature of the specific DNA-binding protein used for the enhancer but a general property of synthetic bacterial enhancers.

SUBMITTER: Amit R

PROVIDER: S-EPMC3155781 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Building enhancers from the ground up: a synthetic biology approach.

Amit Roee R Garcia Hernan G HG Phillips Rob R Fraser Scott E SE

Cell 20110701 1

A challenge of the synthetic biology approach is to use our understanding of a system to recreate a biological function with specific properties. We have applied this framework to bacterial enhancers, combining a driver, transcription factor binding sites, and a poised polymerase to create synthetic modular enhancers. Our findings suggest that enhancer-based transcriptional control depends critically and quantitatively on DNA looping, leading to complex regulatory effects when the enhancer casse ...[more]

PMID: 21729783

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Project description:BackgroundMenaquinone-7 (MK-7), which is associated with complex and tightly regulated pathways and redox imbalances, is produced at low titres in Bacillus subtilis. Synthetic biology provides a rational engineering principle for the transcriptional optimisation of key enzymes and the artificial creation of cofactor regeneration systems without regulatory interference. This holds great promise for alleviating pathway bottlenecks and improving the efficiency of carbon and energy utilisation.ResultsWe used a bottom-up synthetic biology approach for the synthetic redesign of central carbon and to improve the adaptability between material and energy metabolism in MK-7 synthesis pathways. First, the rate-limiting enzymes, 1-deoxyxylulose-5-phosphate synthase (DXS), isopentenyl-diphosphate delta-isomerase (Fni), 1-deoxyxylulose-5-phosphate reductase (DXR), isochorismate synthase (MenF), and 3-deoxy-7-phosphoheptulonate synthase (AroA) in the MK-7 pathway were sequentially overexpressed. Promoter engineering and fusion tags were used to overexpress the key enzyme MenA, and the titre of MK-7 was 39.01 mg/L. Finally, after stoichiometric calculation and optimisation of the cofactor regeneration pathway, we constructed two NADPH regeneration systems, enhanced the endogenous cofactor regeneration pathway, and introduced a heterologous NADH kinase (Pos5P) to increase the availability of NADPH for MK-7 biosynthesis. The strain expressing pos5P was more efficient in converting NADH to NADPH and had excellent MK-7 synthesis ability. Following three Design-Build-Test-Learn cycles, the titre of MK-7 after flask fermentation reached 53.07 mg/L, which was 4.52 times that of B. subtilis 168. Additionally, the artificially constructed cofactor regeneration system reduced the amount of NADH-dependent by-product lactate in the fermentation broth by 9.15%. This resulted in decreased energy loss and improved carbon conversion.ConclusionsIn summary, a "high-efficiency, low-carbon, cofactor-recycling" MK-7 synthetic strain was constructed, and the strategy used in this study can be generally applied for constructing high-efficiency synthesis platforms for other terpenoids, laying the foundation for the large-scale production of high-value MK-7 as well as terpenoids.

Dataset Information

Building enhancers from the ground up: a synthetic biology approach.

Publications

Building enhancers from the ground up: a synthetic biology approach.

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