Overview

To address the topic of possible cross pollination, we are taking genes from naturally clestigamous soybean cultivars and applying them to cultivars that grow well in our country.

The Cross-Pollination Problem

Soybeans (Glycine max.) are a globally important source of livestock food, biofuels, oils, and direct food products. Soybeans comprise 50% of all bioengineered crops and of all soybeans grown worldwide, around 80% of them are bioengineered¹. However, bioengineered crops have yet to be embraced by the larger public for a few reasons. One of these reasons is the valid concern that genes from a bioengineered crop may be accidentally introduced into non-engineered crops through cross-pollination.

The Second Problem

The Yew tree family, Taxaceae, and their associated endophytes are the only naturally occurring sources of paclitaxel, an important chemotherapeutic, but produce low yields of it. Every treatment of traditionally harvested taxol requires the death of six, one-hundred year old yew trees^2,3! The miniscule quantities of taxol in the natural world have led many research groups to focus on better ways of manufacturing this chemical including complete and semi-complete synthesis. Complete chemical synthesis of paclitaxel is complex and not currently feasible for commercial production^4–6, although some pharmaceutical companies use semi-synthesis from an intermediate molecule named baccatin III, obtained by either plant cell fermentation or Yew-needle harvest^2,7. While plant cell fermentation and yew-needle harvest have both provided a more sustainable way of producing taxol, these methods are still unable to produce paclitaxel in sufficient quantities. More recently, research groups have begun to engineer microbes such as S. cerevisiae (yeast) and E. coli to contain at least some of the 19 enzymes required for Taxol production^8,9. These microbes, however, are insufficient hosts for this complicated pathway that includes many hydroxylations and organelle-specific enzymes. To date, no bioengineering attempts of microbes have successfully produced large enough quantities of intermediate Taxadiene-5α-ol.

Other attempts have been made to bioengineer this pathway into tobacco species (Nicotiana benthamiana) but these attempts have had issues with tobacco's native enzymes mis-hydroxylating the taxane skeleton.

Our Project

Our project is to test enzymes of the paclitaxel pathway in the common soybean to evaluate if the soybean will be a better chassis for this pathway. Unlike microbes, soybeans have the proper organelles and have many native enzymes like CYP450 reductases that may support the necessary chemistry. Unlike the tested tobacco species, native soybean proteins may interfere less with Taxadiene-5α-ol yield. Additionally, soybeans are cheap and easy to grow, easy to scale up in production, and could be a feasible chassis for long term biopharming of complex natural molecules such as paclitaxel.

Engineering of Enzymes

To begin implementing this pathway into soybean, we have chosen to test three different taxadiene synthases (TDS), three different Geranylgeranyl Pyrophosphate synthases (GGPPS), one T5ɑHydroxyase (T5ɑOH), and one Taxadiene-5α-ol-acetyl Transferase (TAT). In order to test these enzymes and their function in soybean, we needed to find and test a suitable reporter genes, promoter, and terminator.

All of these parts required special consideration for expression in our plant host Soybean. All coding sequences were codon optimized for soybean. We also had to consider where these parts were located in the native Yew hosts. GGPPS and TDS are both naturally located in the chloroplast, and an unknown transport mechanism moves TDS product, Taxadiene, out of the chloroplast and into the cytosol, where it is then acted upon by several endoplasmic reticulum (ER) associated enzymes. Based on previous literature, we wanted taxadiene to be assembled in the cytosol¹¹. To do this, we had to use enzyme modeling predictors to determine where the chloroplast location tag was located in the gene sequence and remove it from our fragments.

To test protein solubility in the cytosol, we also included a maltose binding protein tag, a common solubility tag in protein engineering, onto two of our taxadiene synthases. We also had to determine if our ER associated proteins contained their active site facing towards or away from the cytosol. To do this, we used Phobius modeling and determined that the active site of T5ɑOH was cytosol facing. Because of this modeling program, we decided to keep the location tag on all of our ER associated enzymes to preserve enzyme solubility and function. This is a different approach compared to present literature¹². As of the WikiFreeze, we are almost done constructing two of our final plasmids and are in the process of testing our other plasmid in soybean.

Engineering Method to Test Our Enzymes

In order to test these enzymes and their function in soybeans, we needed to first find and test suitable reporter genes, promoter, and terminator. Based on past tests in our lab, we chose to use the promoter part Gmubi and AtHSP. We built plasmids to test three different reporter genes using Gmubi and AtHSP as the promoter and terminator. The first two reporter genes tested were unsatisfactory but showed that Gmubi and AtHSP would be sufficient candidates for the rest of our genes. Finally, we found and tested an acceptable reporter gene RUBY¹³, which produces betalain. Presence of hot pink coloration on our transfected soybeans showed the RUBY was appropriately translated and transcripted with the help of Gmubi and AtHSP.