ePathBrick: A Synthetic Biology Platform for Engineering Metabolic Pathways in E. coli Peng Xu, Amerin Vansiri, Namita Bhan, and Mattheos A. G. Koffas* Center for Biotechnology and Interdisciplinary Studies and Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, United States * S Supporting Information ABSTRACT: Harnessing cell factories for producing biofuel and pharmaceutical molecules has stimulated efforts to develop novel synthetic biology tools customized for modular pathway engineering and optimization. Here we report the develop- ment of a set of vectors compatible with BioBrick standards and its application in metabolic engineering. The engineered ePathBrick vectors comprise four compatible restriction enzyme sites allocated on strategic positions so that different regulatory control signals can be reused and manipulation of expression cassette can be streamlined. Specifically, these vectors allow for fine-tuning gene expression by integrating multiple transcriptional activation or repression signals into the operator region. At the same time, ePathBrick vectors support the modular assembly of pathway components and combinatorial generation of pathway diversities with three distinct configurations. We also demonstrated the functionality of a seven-gene pathway (∼9 Kb) assembled on one single ePathBrick vector. The ePathBrick vectors presented here provide a versatile platform for rapid design and optimization of metabolic pathways in E. coli. KEYWORDS: metabolic engineering, transcriptional fine-tuning, gene assembly, pathway configuration, T7 promoter activity A s an emerging discipline, synthetic biology is becoming increasingly important to design, construct, and optimize metabolic pathways leading to desired phenotypes such as overproduction of biofuels 1-3 and pharmaceuticals 4,5 in genetically tractable organisms. One of the major challenges for heterologous expression of multigene pathways is to orchestrate the expression level of each of the enzymes among the selected pathways and achieve optimal catalytic efficiency. Thus, delicately designed molecular control elements have been integrated into the cell chassis to enable the host strain to precisely respond to environmental stimuli or cellular intermediates and drive carbon flux toward a target pathway. For example, engineering promoter architecture has achieved tunable gene expression in both E. coli 6 and yeast 7 at transcriptional levels; engineered metabolite-responsive ribos- witches 8 and synthetic ribosome binding sites 9 can be used to precisely control protein expression at the translational level; metabolic flux channeling by spatial recruitment of desired metabolic enzymes at a stoichiometric ratio on a synthetic protein scaffold can efficiently prevent the loss of intermediates due to diffusion and has resulted in a 77-fold improvement in production titer. 10 As witnessed by these endeavors, the advances in synthetic biology have greatly speeded up our ability to design and construct cell factories for metabolic engineering application. To date, a number of synthetic biology approaches have been applied to metabolic pathway optimization including modification of plasmid copy number, 11 promoter strength, 12,13 and gene codon usage. 14 However, most of these approaches are not modular and require time-consuming work tweaking the indi- vidual pathway components until the desired performance is achieved, which greatly compromises our ability to unlock the metabolic potential of host metabolism. From an engineering perspective, we need a more efficient approach that allows us to streamline the process of pathway construction and engineering of biological systems to be much easier and faster. Despite the availability of advanced cloning tools such as the DNA assembler, 15,16 sequence and ligation-independent cloning (SLIC), 17 and Gibson isothermal assembly, 18 these tools largely rely on site-specific homologous recombination and are limited in terms of automation and the number of DNA fragments that can be assembled for pathway optimization; 19 in particular, these advanced cloning tools are not amenable to very short genetic elements such as promoter, ribosome binding site, operator, and other regulatory control elements. 20 Hence, there exists a pressing need to develop efficient molecular assembly tools customized for metabolic pathway optimization. Assembly of biological standard parts that conform to the BioBrick paradigm is being adopted rapidly and is becoming a standard practice in metabolic pathway engineering 21 and Received: March 21, 2012 Published: April 23, 2012 Research Article pubs.acs.org/synthbio © 2012 American Chemical Society 256 dx.doi.org/10.1021/sb300016b | ACS Synth. Biol. 2012, 1, 256-266