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=Biosynthetic Metabolism= Proteins created from an organism's encoded DNA produce function to that organism; from its phenotypic expression to its metabolic pathways. Synthetic metabolisms are man-made biological systems are created from altering the encoded DNA to serve a specific purpose such as bioremediation for oil spills or for pharmaceutical chemical production. The common platform to use for biosynthetic metabolism includes the use of microorganisms due to their fast reproduction time. There are two ways that these synthetic pathways can be studied in microorganisms: In vitro (in a test tube) and In vivo (in a cell). Both have their own strengths and weaknesses.

In Vitro Metabolic enzyme experiments:
This process involves using exclusively enzymes, the proteins needed for catalyzing biological processes. The isolation of enzymes without the cell comes with some perks. In vitro systems have more predictable product generation rates, experimental design pliability, and they are almost fully capable of full stoichiometric conversion. As great as in vitro biosynthetic pathways are in theory, it takes a long time to procure the enzymes needed and/or their sequences and is regarded as too costly to utilize broadly. It is also notable that, though in vitro enzyme experiments don’t have to deal with the biological regulations that a cell would, scientists haven’t found a way to completely eliminate problems that enzymes face outside the cell. The interior of the cell is far more concentrated in substrates, enzymes, etc. compared to a test tube, and doesn’t give any insight into possible side-reactions that could occur in vivo. This has led to a lack of interest and sporadic research from scientists in the utilization of this process.

In Vivo Metabolic enzyme experiments:
In vivo (in a cell) testing requires the usage of whole cells and is by far the more advisable way to quantify the efficiency of a metabolic pathway. For one, the need for enzyme purification is surpassed. Second, it is easy to design experiments by slightly tweaking the amounts of substrate in an experiment to test a microbe’s metabolism. To easily map out a microbe's metabolism, scientists can simply control the amount of limiting substrate in the biological reaction and record the results (for example, a chemostat experiment). Studying metabolism with in vivo experiments can also allow scientists so observe several enzyme catalyzed pathways can be studied in at the same time. However, there are certain restraints using this method. It is, unfortunately, not currently possible to control the way cell(s) express the required concentration of enzymes; in contrast to the in vitro approach that allows for the concentration of enzymes to be constant. This places restrictions upon how scientists employ and design experiments. The data received from in vivo experiments, covers how an organism(s) can metabolize a substrate. However, the accuracy of the results depends on how exact the measurements of the metabolite concentrations in the metabolic area of interest are. For example, let’s say there is a target reaction a scientist wishes to study. That reaction is part of a large network with many steps in between the starting substrate and the product. The more steps in the pathway, the more reaction intermediates that will be obtained, hence making it more difficult for the total pathway’s metabolites to be properly measured. This fact sometimes makes it difficult to draw conclusions about the pathway if the data received isn’t correct.

De Novo Biosynthetic Pathway creation:
De Novo biosynthetic metabolic pathways are inserted pathways not found in nature that are created by scientists to optimize a metabolic pathway by diverting some of the substrate/chemical intermediates from the original pathway to the newly inserted one. Metabolic bottlenecks are a problem faced by both in vivo and in vitro testing of metabolic pathways and have become a hindrance to scientists in research and industry alike because they cause limits in chemical production. Because all enzymes have a different Vmax, an enzyme further along in a pathway with a lower Vmax than one of its predecessors can become saturated with a substrate and limit the amount of product being created. To surpass this, scientists can introduce a de novo pathway to the organism by adding or deleting genes that can then allow the organism to express new enzymes that take the pressure off of the enzyme causing the bottleneck by diverting the pathway to the new pathway of enzymes. Encoding a new enzyme is a versatile and beneficial process that has become a massive accelerator to the research of synthetic biology. There exist massive databases, such as Phyre2 and RefSeq, that can suggest similar enzymes and their DNA sequences that can be possible replacements for the old one. Scientists simply have to search the database for replacements, make the genetic addition to the organism to add the metabolic pathway, and run experiments to test the efficiency. If the process isn't optimal, the process can be repeated until favorable results are obtained.

Existing Metabolic pathway manipulation:
Another way that problems with metabolism can be surpassed with is by altering the existing metabolic pathway. This can be performed by deleting and/or inserting a gene(s) encoding a more efficient pathway. The enzymes can be from different organisms or by altering the existing enzymes into a more competent form. After experimentation, it can be quantified if the alteration has performed as expected and correct the bottleneck to the scientist's preferred chemical production rates.