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Butanol in Plants Project

The butanol project’s goal is to synthetically produce the enzymes that make up the University of California’s cyanobacteria pathway to produce n-butanol within physcomitrella. Thereby making a plant directly produce n-butanol, an industrially relevant compound that can serve as a more efficient biofuel than ethanol. The project took on another goal when it was realized that an intermediary compound in the pathway could be used to produce (R)-Polyhydroxybutyrate, a biodegradable plastic.

Introduction

Butanol is a four carbon alcohol with several different industrial uses such as the creation of polymers and pharmaceuticals. However, its most notable use is as a fuel for internal combustion engines. Butanol is fairly non-polar due to its longer hydrocarbon chain, and is more similar to gasoline than other small alcohols such as ethanol. The fact that it is similar in function to gasoline means that butanol can be used in automobiles without any changes. This is significant because butanol can be synthesized from biomass. Organisms such as cyanobacteria, diatoms, and the bacterium Clostridium acetobutylicum have already been shown to produce butanol using various methods.

Our other potential product is polyhydroxybutyrate or PHB. PHB is a polymer in the polyesters class that has many uses similar to other plastics of the same variety. The reason this one is an interesting option is the fact that it is nontoxic and biodegradable. This fact makes PHB an excellent choice for potential use in the medical industry as well as others. Biosynthesis of the polymer is currently achieved using microorganisms. Our goal was to extend the production capabilities to plants; specifically Physcomitrella patens.

Background

Our work focuses on the ATP dependent pathway described by the University of California. The image below gives an outline of the pathway used. Refer to the red line.

http://www.pnas.org/content/109/16/6018/F1.large.jpg

We designed three constructs based on the pathways for the synthesis of butanol and PHB. The first of the three (Castor) is the same for both pathways and brings the reaction to the intermediate 3-(R)-hydroxybutyryl-CoA. After that the reaction can proceed as shown above or with the enzyme Polyhydroxyalkanoate synthase or PhaC the intermediate can be used to make PHB. Castor can be ligated together with either of the other two constructs to create a useful pathway for one of our products.

Method

The amino acid sequences used for the enzymes were converted back into nucleotide sequences and then optimized for use in plants, or more specifically Physcomitrella patens. The sequences were then optimized into ~500 base pair gBlocks final constructs were then built using Gibson Assembly or CBAR. Then the constructs were ligated into our vectors; which were Ti-plasmids. TM which were synthesized. The

The entire pathway was broken into two distinct pieces, each of which gives us one of our two final desired products. The first half of both constructs brings us to 3-(R)- hydroxybutyryl-CoA and is referred to as Castor. This can then be converted into either butanol or PHB depending on the following enzyme(s) in the pathway. The first construct consists of the preliminary pathway and an additional four enzymes in a construct referred to as Pollux which converts 3-(R)-hydroxybutyryl-CoA into butanol. The other is a single enzyme after the first half that gives us PHB. The construct containing the single enzyme is named after the enzyme: PhaC.

Results

Pending...

After transfection with Agrobacterium of the Castor and PhaC constructs into Nicotiana, the transiently transformed leaf segments were excised and stained. The leaf segments and control leaf cuttings were examined with spinning disk confocal microscopy for the presence of Polyhydroxybutyrate (PHB) granules. The preliminary staining results were inconclusive.

Expected Results:

Given ideal staining and PHB production in the cell cytoplasm, we expect to see granules of PHB within the cells. These are expected to be well defined against the background fluorescence of the stomata, tricomes, and cell walls. It is also expected that GC-MS should be able to detect the PHB from the leaf cuttings.

Discussion

The preliminary results were inconclusive primarily due to issues with staining. The dye used was not very soluble in water and produced a colloidal suspension. This resulted in some of the dye binding to the surface of the leaf indiscriminately giving us false positives. A better dye that can more readily penetrate the leaf and have better solubility may be required to obtain a clear result.

Further Study

The future goals are to continue testing with the Castor and PhaC constructs in Nicotiana, and expand to testing in Physcomitrella. We are going to work on getting clearer microscopy results and GCMS results.

Another goal for the Butanol project is to assemble and test the Pollux construct for the production of n-butanol. At this time we could also complete a protein extraction on samples using the His-tags and categorize the 2A sequence utilized in the project. This could help us to ascertain the efficiency of the 2A sequence in the respected test organisms.

Further categorization of the submitted constructs and their products is an ongoing task.

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