Center for Synthetic Biology

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Center for Synthetic Biology

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Andersen-Ranberg J.,Center for Synthetic Biology | Kongstad K.T.,Copenhagen University | Nielsen M.T.,Center for Synthetic Biology | Jensen N.B.,Evolva AS | And 7 more authors.
Angewandte Chemie - International Edition | Year: 2016

Plant-derived diterpenoids serve as important pharmaceuticals, food additives, and fragrances, yet their low natural abundance and high structural complexity limits their broader industrial utilization. By mimicking the modularity of diterpene biosynthesis in plants, we constructed 51 functional combinations of class I and II diterpene synthases, 41 of which are "new-to-nature". Stereoselective biosynthesis of over 50 diterpene skeletons was demonstrated, including natural variants and novel enantiomeric or diastereomeric counterparts. Scalable biotechnological production for four industrially relevant targets was accomplished in engineered strains of Saccharomyces cerevisiae. © 2015 The Authors.


Liu J.-K.,CAS Institute of Plant Physiology and Ecology | Chen W.-H.,CAS Institute of Plant Physiology and Ecology | Ren S.-X.,CAS Institute of Plant Physiology and Ecology | Zhao G.-P.,CAS Institute of Plant Physiology and Ecology | And 4 more authors.
PLoS ONE | Year: 2014

The BioBricks standard has made the construction of DNA modules easier, quicker and cheaper. So far, over 100 BioBricks assembly schemes have been developed and many of them, including the original standard of BBF RFC 10, are now widely used. However, because the restriction endonucleases employed by these standards usually recognize short DNA sequences that are widely spread among natural DNA sequences, and these recognition sites must be removed before the parts construction, there is much inconvenience in dealing with large-size DNA parts (e.g., more than couple kilobases in length) with the present standards. Here, we introduce a new standard, namely iBrick, which uses two homing endonucleases of I-SceI and PI-PspI. Because both enzymes recognize long DNA sequences (>18 bps), their sites are extremely rare in natural DNA sources, thus providing additional convenience, especially in handling large pieces of DNA fragments. Using the iBrick standard, the carotenoid biosynthetic cluster (>4 kb) was successfully assembled and the actinorhodin biosynthetic cluster (>20 kb) was easily cloned and heterologously expressed. In addition, a corresponding nomenclature system has been established for the iBrick standard. Copyright: © 2014 Liu et al.


PubMed | Center for Synthetic Biology, National University of Singapore, Copenhagen University and University of Birmingham
Type: Journal Article | Journal: Science (New York, N.Y.) | Year: 2016

Metabolic highways may be orchestrated by the assembly of sequential enzymes into protein complexes, or metabolons, to facilitate efficient channeling of intermediates and to prevent undesired metabolic cross-talk while maintaining metabolic flexibility. Here we report the isolation of the dynamic metabolon that catalyzes the formation of the cyanogenic glucoside dhurrin, a defense compound produced in sorghum plants. The metabolon was reconstituted in liposomes, which demonstrated the importance of membrane surface charge and the presence of the glucosyltransferase for metabolic channeling. We used in planta fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to study functional and structural characteristics of the metabolon. Understanding the regulation of biosynthetic metabolons offers opportunities to optimize synthetic biology approaches for efficient production of high-value products in heterologous hosts.


News Article | December 12, 2016
Site: phys.org

"Right now, one of the most promising frontiers in cancer treatment is immunotherapy—harnessing the immune system to combat a wide range of cancers," said Joshua N. Leonard, the senior author of the study. "The simple cell rewiring we've done ultimately could help overcome immunosuppression at the tumor site, one of the most intransigent barriers to making progress in this field." When cancer is present, molecules secreted at tumor sites render many immune cells inactive. The Northwestern researchers genetically engineered human immune cells to sense the tumor-derived molecules in the immediate environment and to respond by becoming more active, not less. This customized function, which is not observed in nature, is clinically attractive and relevant to cancer immunotherapy. The general approach for rewiring cellular input and output functions should be useful in fighting other diseases, not just cancer. "This work is motivated by clinical observations, in which we may know why something goes wrong in the body, and how this may be corrected, but we lack the tools to translate those insights into a therapy," Leonard said. "With the technology we have developed, we can first imagine a cell function we wish existed, and then our approach enables us to build—by design—a cell that carries out that function." Currently, scientists and engineers lack the ability to program cells to exhibit all the functions that, from a clinical standpoint, physicians might wish them to exhibit, such as becoming active only when next to a tumor. This study addresses that gap, Leonard said. Leonard, who focuses on integrating synthetic biology into medicine, is an associate professor of chemical and biological engineering at the McCormick School of Engineering. He is a member of the Robert H. Lurie Comprehensive Cancer Center of Northwestern University. The research comes out of a rich collaboration that Leonard's team has with clinical oncologists, immunologists and basic cancer researchers at Northwestern University Feinberg School of Medicine as well as other synthetic biologists. The study, to be published Dec. 12 by the journal Nature Chemical Biology, provides details of the first synthetic biology technology enabling researchers to rewire how mammalian cells sense and respond to a broad class of physiologically relevant cues. Kelly A. Schwarz, a graduate student in Leonard's research group, is the study's first author. "This work is exciting because it addresses a key technical gap in the field," Schwarz said. "There is great promise for using engineered cells as programmable therapies, and it is going to take technologies such as this to truly realize that goal." Starting with human T cells in culture, the research team genetically engineered changes in the cells' input and output, including adding a sensing mode, and built a cell that is relevant to cancer immunotherapy. Specifically, the engineered cells sense vascular endothelial growth factor (VEGF), a protein found in tumors that directly manipulates and in some ways suppresses the immune response. When the rewired cells sense VEGF in their environment, these cells, instead of being suppressed, respond by secreting interleukin 2 (IL-2), a protein that stimulates nearby immune cells to become activated specifically at that site. Normal unmodified T cells do not produce IL-2 when exposed to VEGF, so the engineered behavior is both useful and novel. This work was carried out in cells in culture, and the technology next will be tested in animal studies. While Leonard's team has initially focused on the application of this cell programming technology to enabling cancer immunotherapy, it can be readily extended to distinct cellular engineering goals and therapeutic applications. Leonard's "parts" are also intentionally modular, such that they can be combined with other synthetic biology innovations to write more sophisticated cellular programs. "To truly accelerate the rate at which we can translate scientific insights into treatments, we need technologies that let us rapidly try out new ideas, in this case by building living cells that manifest a desired biological function," said Leonard, who also is a founding member of the Center for Synthetic Biology and a member of the Chemistry of Life Processes Institute. "Our technology also provides a powerful new tool for fundamental research, enabling biologists to test otherwise untestable theories about how cells coordinate their functions in complex, multicellular organisms," he said. Related to this research, Leonard was an invited conferee at a special meeting held in October, "Systems and Synthetic Biology for Designing Rational Cancer Immunotherapies," as part of President Obama and Vice President Biden's Cancer Moonshot Initiative.


News Article | December 12, 2016
Site: www.eurekalert.org

A major challenge in truly targeted cancer therapy is cancer's suppression of the immune system. Northwestern University synthetic biologists now have developed a general method for "rewiring" immune cells to flip this action around. "Right now, one of the most promising frontiers in cancer treatment is immunotherapy -- harnessing the immune system to combat a wide range of cancers," said Joshua N. Leonard, the senior author of the study. "The simple cell rewiring we've done ultimately could help overcome immunosuppression at the tumor site, one of the most intransigent barriers to making progress in this field." When cancer is present, molecules secreted at tumor sites render many immune cells inactive. The Northwestern researchers genetically engineered human immune cells to sense the tumor-derived molecules in the immediate environment and to respond by becoming more active, not less. This customized function, which is not observed in nature, is clinically attractive and relevant to cancer immunotherapy. The general approach for rewiring cellular input and output functions should be useful in fighting other diseases, not just cancer. "This work is motivated by clinical observations, in which we may know why something goes wrong in the body, and how this may be corrected, but we lack the tools to translate those insights into a therapy," Leonard said. "With the technology we have developed, we can first imagine a cell function we wish existed, and then our approach enables us to build -- by design -- a cell that carries out that function." Currently, scientists and engineers lack the ability to program cells to exhibit all the functions that, from a clinical standpoint, physicians might wish them to exhibit, such as becoming active only when next to a tumor. This study addresses that gap, Leonard said. Leonard, who focuses on integrating synthetic biology into medicine, is an associate professor of chemical and biological engineering at the McCormick School of Engineering. He is a member of the Robert H. Lurie Comprehensive Cancer Center of Northwestern University. The research comes out of a rich collaboration that Leonard's team has with clinical oncologists, immunologists and basic cancer researchers at Northwestern University Feinberg School of Medicine as well as other synthetic biologists. The study, to be published Dec. 12 by the journal Nature Chemical Biology, provides details of the first synthetic biology technology enabling researchers to rewire how mammalian cells sense and respond to a broad class of physiologically relevant cues. Kelly A. Schwarz, a graduate student in Leonard's research group, is the study's first author. "This work is exciting because it addresses a key technical gap in the field," Schwarz said. "There is great promise for using engineered cells as programmable therapies, and it is going to take technologies such as this to truly realize that goal." Starting with human T cells in culture, the research team genetically engineered changes in the cells' input and output, including adding a sensing mode, and built a cell that is relevant to cancer immunotherapy. Specifically, the engineered cells sense vascular endothelial growth factor (VEGF), a protein found in tumors that directly manipulates and in some ways suppresses the immune response. When the rewired cells sense VEGF in their environment, these cells, instead of being suppressed, respond by secreting interleukin 2 (IL-2), a protein that stimulates nearby immune cells to become activated specifically at that site. Normal unmodified T cells do not produce IL-2 when exposed to VEGF, so the engineered behavior is both useful and novel. This work was carried out in cells in culture, and the technology next will be tested in animal studies. While Leonard's team has initially focused on the application of this cell programming technology to enabling cancer immunotherapy, it can be readily extended to distinct cellular engineering goals and therapeutic applications. Leonard's "parts" are also intentionally modular, such that they can be combined with other synthetic biology innovations to write more sophisticated cellular programs. "To truly accelerate the rate at which we can translate scientific insights into treatments, we need technologies that let us rapidly try out new ideas, in this case by building living cells that manifest a desired biological function," said Leonard, who also is a founding member of the Center for Synthetic Biology and a member of the Chemistry of Life Processes Institute. "Our technology also provides a powerful new tool for fundamental research, enabling biologists to test otherwise untestable theories about how cells coordinate their functions in complex, multicellular organisms," he said. Related to this research, Leonard was an invited conferee at a special meeting held in October, "Systems and Synthetic Biology for Designing Rational Cancer Immunotherapies," as part of President Obama and Vice President Biden's Cancer Moonshot Initiative. The paper is titled "Rewiring Human Cellular Input-Output Using Modular Extracellular Sensors." In addition to Leonard and Schwarz, other authors are Nichole M. Daringer and Taylor B. Dolberg, both of Northwestern.


News Article | December 1, 2015
Site: phys.org

Cytochrome P450s are enzymes that oxygenate organic compounds stereo-specifically and are involved in numerous metabolic routes. Their reaction mechanism requires electrons, usually obtained from redox cofactors such as NADPH. This availability of reducing power is often the bottleneck in heterologous expression plant pathways in microbes: in the aremisinin (1) and taxol (2) production in yeast and E. coli respectively, the biosynthetic steps catalyzed by P450s required laborious optimization. There might be however another way to overcome this hurdle, the answer could lie in photosynthesis. The subject of the latest research article from my research group is around harnessing sunlight and redirecting it towards desired metabolic compounds. The paper, titled "Metabolic engineering of light-driven cytochrome P450 dependent pathways into Synechocystis sp. PCC 6803" (3), was recently published in Metabolic Engineering. In this work, my colleagues engineer Synechocystis sp. PCC6803, a popular cyanobacterium model, to produce dhurrin (a cyanogenic glucoside from Sorghum bicolor). Sorghum utilizes dhurrin as a defense compound: when the leaf tissues are mechanically disrupted (chewed), dhurrin degrades and hydrogen cyanide releases, poisoning the unfortunate herbivores. Its biosynthesis commences from tyrosine, and involves two distinct cytochrome P450s and a glycosyltransferase. Although dhurrin has no commercial use, its biosynthesis has evolved into a model pathway for understanding plant P450 functionality (I refer the curious reader to the work of Professor Birger Møller 's research and the University of Copenhagen Center for Synthetic Biology). More interestingly, the dhurrin P450s were used to demonstrate that it is possible for P450s to obtain electrons directly from the photosynthetic apparatus. Eukaryotic P450s are normally located in the endoplasmic reticulum and rely on a NADPH-dependent dedicated oxidoreductase as a redox partner. However, in vitro and in vivo experiments have shown that if the P450s localize in the proximity of the photosystem I, they can retain activity by gaining electrons from photoreduced ferredoxin, thus bypassing the specialized reductase requirement (4,5). Since this concept worked quite well in plant chloroplasts, our group saw no reason that this principle is not transferable to cyanobacteria. Coming back to our recent paper, the authors introduced the three enzyme sequences of the dhurrin pathway into a self-replicating vector as an operon, their expression controlled by a strong inducible promoter. Under theophyline induction, cyanobacteria produce dhurrin and excrete most of it to the growth medium. The productivity was also tested in 8 Liter bioreactors, where dhurrin accumulation reached 3.2 mg dhurrin L-1OD-1 after 7 days of cultivation. The effect of dhurrin production on cell fitness was also tested. When the whole pathway was expressed, there was a small delay in cell growth. But the strains expressing only the two first enzymes (the p450s without the glycosyltransferase) displayed a severe poisoning phenotype. Electron microscopy revealed rearrangements of the thylacoid structures and the lack of glycogen granules. These side-effects were not observed in the full pathway-expressing strain, suggesting that some intermediate may have toxic effect and that the glycosilation is crucial for the detoxification and secretion of this potential poison. Hijacking electrons from photosynthesis is a promising bioengineering alternative, especially in the cases where reducing power and co-factor availability are limiting. This study shows that Synechocystis is receptive to this practice, and paves the way for further metabolic engineering work, aiming to produce more and commercially interesting compounds. Cyanobacteria are prominent vessels for synthetic biology approaches, recently receiving attention from NASA as polymer construction hosts (see the hangout with Dr. F. Zhang). Even though it might be some time before seeing photosynthetic organisms doing large scale production in space (or in Mars, according to our 2015 iGEM SpaceMoss team), the principle remains the same: light, CO2 and water—feedstock plentifully available in a resource-limited planet—are captured by photosynthetic microbial cell factories to produce any fuel, nutrient, or pharmaceutical. Explore further: New forage plant prepares farmers for climate changes More information: Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, et al. High-level semi-synthetic production of the potent antimalarial artemisinin. Nature. 2013 Apr 25;496(7446):528–32. Ajikumar PK, Xiao W-H, Tyo KEJ, Wang Y, Simeon F, Leonard E, et al. Isoprenoid Pathway Optimization for Taxol Precursor Overproduction in Escherichia coli. Science. 2010 Oct 1;330(6000):70–4. Wlodarczyk A, Gnanasekaran T, Nielsen AZ, Zulu NN, Mellor SB, Luckner M, et al. Metabolic engineering of light-driven cytochrome P450 dependent pathways into Synechocystis sp. PCC 6803. Metab Eng. 2016 Jan;33:1–11.

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