CHE.496/2008/Responses/a12

Metabolic pathway engineering

 * Discussion leader: Patrick Gildea (Discussion guide)

Eyad Lababidi's Response

 * Bioengineering novel in vitro metabolic pathways using synthetic biology
 * Okay so i read this article but i constantly had to reread sections because i was missing the point. I believe it was about how to create new enzymes in cells and the article discussed the most effective way to model the enzymes, but i really wasn't sure how it was relevant to us or how the methods they were using were relevant to us, so the article seemed a bit dense to me and to be honest i didnt really get much out of it, sorry George.
 * Synthetic biology for synthetic chemistry
 * This articles is a very good all encompassing crash course to how one would start a Igem project. Each important piece of a synthetic bio cell is defined and explained to best be setup for a cell meant to be harnessed for synthetic biology. The insight on factors that could destroy a project especially through differing metabolic rates seems useful. other explanations were also useful such as why synth bio parts have been mutated to be a bare bones chassis that will readily take up plasmid vectors and evolutionary abilities of the cell have been disabled such as to not evolve away from containing the plasmid.
 * Eyad Lababidi 01:25, 10 March 2008 (EDT)

Kevin Hershey's Response

 * Bioengineering novel in vitro metabolic pathways using synthetic biology
 * The article by Meyer et. al. discusses modeling in synthetic biology. The article begins by discussing multi-enzyme assembly, which can in itself present many challenges. Then, it discusses data generation from these enzymes. The generation can be troublesome because it needs to be obtained on the small scale in vitro experiments. Once the data is compiled, mathematical models are fit to the data. These are mostly differential equations dealing with mass balances and rate constants. With the system modeled, the network can then optimized due to the engineering principles of synthetic biology.
 * Synthetic biology for synthetic chemistry
 * The review by Keasling discusses some of the new principles and applications to synthetic biology. It begins by discussing the ideal chassis. This chassis would be very robust, hardy, and capable of surviving on minimal carbon. Information on the chassis is insightful from this author, who produced the minimal genome organism. He then discusses artemisinin, an anti-malarial drug, production. The advantage to this synthetic biology approach is that the precursor to artemisinin is squalene. Squalene is used as many other precursors, and the modular approach will allow Keasling and team to produce valuable products based on their work so far.
 * KPHershey 15:15, 12 March 2008 (CDT)

George Washington's Response

 * Bioengineering novel in vitro metabolic pathways using synthetic biology
 * This article discussed the techniques available today for development of entire metabolic pathways for use in industrial production of biological chemicals. It seemed to focus heavily on the analysis of experimental data, as there are ways to start with a (usually non-linear) model, extract parameters for the model from experimental data through various mathematical techniques, and even use those data to suggest ways to improve the model or what parameters need to be enhanced.  Thus, straightforward experimentation can pinpoint which enzymes need to be modified, and how.  Modifying the proteins can be extremely difficult, although often possible.  While all this sounds great, it still feels extremely computationally and conceptually intensive, so truly optimizing a many enzyme system may prove outside of our ability.  However, if optimization is not as important, we could probably develop a system with a reasonable model, although we would not be able to ensure our model is completely accurate without much work.  The iterative approach described in the article should suffice to permit enzymes to be made and tested one at a time, only having to do fine-tuning when there is inhibition back in the chain.
 * Synthetic Biology for Synthetic Chemistry
 * Keasling's article talked about the challenges and progress in enabling large-scale metabolic engineering processes. Specifically, induction of protein generation can be difficult to do reliably, as many inducers are all-or-none, so will result in a heterogeneous product.  However, there are becoming more useful inducers that are not all-or-none, so this problem is probably solvable, and, combined with copy-number management, should be useful in producing homogeneous product in industrial settings.  Keasling then discusses his progress with artemisinic acid production, which has been fairly successful.  He steps through the process, how each of the steps was optimized individually to produce rather large concentrations of the desired product.  This is a demonstration of the success of current metabolic engineering techniques, and an excellent example of how it works.  I doubt we would be able to anything as successful as this for our project, but something with fewer steps should be feasible, and even optimization of just two or three steps would be admirable.
 * George Washington 17:25, 12 March 2008 (CDT)

George McArthur's Response

 * Bioengineering novel in vitro metabolic pathways using synthetic biology
 * Panke et al. describe a semi-rational approach to construct systems of enzymes (i.e., metabolic pathways) that biologically produce desirable chemicals. What tools are necessary to do this kind of work?  DNA synthesis, genome engineering, high-throughput analytics, and model-based analyses are all coming together to make this possible.  What is preventing this kind of work from becoming a reality?  Simply that the economics are not worked out.  Assembling a system of enzymes is extremely difficult and time-consuming.  In addition, because our existing models of cell physiology is limited (even for E. coli), we cannot understand/predict how the enzymes will interact with the exisiting infrastructure.  An over-expressed enzyme could lead to disease and cell death.  This article illustrates the rational design approach to constructing biological systems (in this case a system of enzymes, see Figure 2) and emphasizes the necessity for data generation via high-throughput techniques (e.g., MS/MS).
 * Synthetic Biology for Synthetic Chemistry
 * Keasling presents the motivation and scientific background behind metabolic engineering, highlighting his own research on the bioproduction of artemisinin. Synthetic biology offers an engineering approach for the metabolic engineering of microorganisms.  Standardized biological parts that can be assembled into a more complex system (e.g., a metabolic pathway) coupled with a cellular chassis that is designed and suited to host the metabolic chemistry you're interested in (i.e., genome engineering) will pave the way for future metabolic engineering projects.  The difficulty lies in the fact that biological systems behave nonlinearly and are often unpredictable due to the complex interactions it experiences with the rest of the cell.  Design rules must be created to manage this complexity.  This will enable the integration of biological systems and the ability to develop computer-aided design (CAD) software for biological engineering.  The development of this software will be just as critical to the advancement of synthetic biology as the fundamentals and applications are/will be.
 * GMcArthurIV 19:14, 12 March 2008 (CDT)

Dan Tarjan's Response

 * Bioengineering novel in vitro metabolic pathways using synthetic biology
 * The authors of this article are optimistic about the advances made in metabolic engineering through synbio. Overall the message is that the tools for studying metabolic systems are getting better. The article also gives a good framework on how to conduct metabolic pathway engineering. It's become easier to achieve enzyme concentrations needed for useful systems. Cell-free systems allow the study of a potential metabolic pathway without the interference of other cellular processes. Researchers have taken advantage of increased computational power to help automate the search for an appropriate model to fit a pathway being characterized. Challenges still remain though. A main challenge that most any metabolically involved pathway will face is the interaction with a cell's carbon cycle. That and other limitations of the chassis will need to be taken into consideration for any potential projects.
 * Synthetic Biology for Synthetic Chemistry
 * Keasling and his coauthors provide a valuable reference resource and summary of pathway engineering efforts. The article deals with many specific considerations of pathway engineering. Promoters, DNA stability, gene networks, and the characterization of the pathway are discussed. A specific problem that hampers characterization efforts is need for a promoter which can be activated in a proportional as opposed to binary way. This would allow researchers to modify specific parameters of a pathway and study the response without having to grow a new cell line with new DNA that has promoters with adjusted strengths. Crosstalk between different inducible promoters is another practical issue that needs to be kept in mind. An interesting topic they touch on are starvation promoters - promoters which are activated when a certain chemical/food-source is low. They could be a useful tool.
 * Daniel R Tarjan 00:04, 17 March 2008 (CDT)