I have heard of the cellulosome for quite some time. It discribes a extracellular ‘factory’ of enzymes some bacteria (or fungi) are equipped with to degrade the components of the plant cell wall. These enzymes are held by a scaffold structure projecting out of the cell. Several bacterial species to date are known to encode some sort of cellulosome. Here, I will focus on a model species, Clostridium thermocellum. I never really thought twice about cellulosomes until recently when researching for an upcoming project. Now, however, I have a great appreciation and respect for this massive, impressive apparatus.
The backbone of sorts for the cellulosome is the scaffoldin CipA. CipA is a monstrous protein with many domains, most of which necessary to attach the enzymes needed to break down plant cell walls. CipA contains 9 cohesins, domains used to securely allow different proteins to interact. The enzymes ( I will describe soon) contain dockerin domains that interact with cohesins. CipA also contains a carbohydrate binding module (CBM) which allows it to directly interact with cell walls.
Many of the cohesins are used to attach carbohydrate degrading enzymes, usually of two classes: endoglucanases and exoglucanases. These work in concert to breakdown cellulose and other carbohydrate polymers. Apart from the catalytic portions of these proteins are dockerin, needed to bind to CipA, and other domains like the Ig domain or X domain.
These enzymes ‘fit’ onto the scaffold protein like Legos. This makes them very modular. Now consider other scaffold proteins have a different type of cohesin (cohesin II) that can be used to attach other scaffold proteins thus making polycellulosomes. For example, Cthe_0736 is a scaffold protein with 7 type II cohesins. This means Cthe_0736 can have 6 other scaffold proteins attached to it meaning this polycellulosome could contain up to 63 individual enzymes which is potentially common considering isolated cellulosomes vary in molecular weight considerably.
In a later post, I will go into a little detail on how these cellulosomal enzymes actually are able to degrade anhydrous polymers of carbohydrate.
Thanks to the decreasing costs of sequencing genomic DNA, finding novel microorganisms that add to our understanding of metabolism in myriad environments is becoming common place. Not only are we learning about the diversity of life in extreme environments, like heat, cold, pressure, and altitude, but we are also learning what life on other planets may be like. With each additional genome added into ‘the cloud’, our synthetic biology toolbox gets a little bit bigger and our ability to manipulate tiny organisms to produce novel compounds is possible. Enter the “rushing fireball”.
Pyrococcus furiosus is an archeal species that thrives near deep-sea thermal vents where temperatures are between 90 and 100 degrees Celsius (or 194 to 212 degrees F). P. furiosus can grow at temperatures as low as 70 degrees C (158 deg F). To live in such conditions, this organism’s proteins must be tolerant to what we would consider harsh conditions. This organism’s ambient conditions makes wild-type proteins well-suited for industrial processes where temperatures are near boiling.
So far, P. furiosus has been utilized to produce 3-hydroxypropionic acid, a common industrial chemical used to make various products including acrylics. The kicker is that these cells were wired to make this chemical from atmospheric carbon dioxide. It is not crazy to think of what other useful products can be produced by P. furiosus with small modifications within the genome; products like ethanol or butanol as biofuels.
Last week, I presented illustrations for yeast and a microalgal species of Chlamydomonas. Today I will expound on part of this. Ongoing research is working to identify ways to circumvent the need for fresh water, a precious commodity, and costly fertilizer to cultivate microalgae for biofuel production. These microorganisms are a rich source of oils that can be integrated into our national fuel infrastructure. However, growing the amount of microalgae necessary to decrease our need for petroleum based fuel relies on a precious and ever deminishing resource, fresh water. Also needed are nutrients like nitrogen and phosphorous, usually in the form of fertilizer.
Microalgae are adaptable to environmental changes. Recent research shows several microalgal species that can be cultivated with no need for freshwater. Instead, these species, Chlamydomonas globosa, Chlorella minutissima and Scenedesmus bijuga, are grown in something we have plenty of; wastewater.
These microalgae are able to generate energy and grow with no input of fresh, potable water or fertilizers. The ‘nutrients’ needed are all available within the wastewater. By wastewater I am referring to myriad kinds of used water predominantly from industry like the production of carpets or other products and from livestock litter. Usually, microalgae can be viewed similar to plants with respect to their carbon utilization. Algae can breathe in carbon dioxide from the atmosphere and convert it into carbohydrates and fatty acids (oils), thus releasing oxygen back into the atmosphere. They have been doing this for over 3.5 billion years and are the primary reason other life, including humans, are alive today. However, microalgae can also use different metabolic strategies to incorporate carbon. Also, these organisms can utilize both carbon dioxide and organic carbon compounds simultaneously (called mixotrophy).
This research is only the beginning. As these investigations progress, many other organisms can be identified that can lower our dependence on fossil fuels.
When it comes to synthetic biology, two species of microorganisms should automatically come to mind; E. coli and the yeast Saccharomyces cerevisiae.Both have been used extensively for proof of principle research. Thanks to these investigations, both are able to synthesize a drop-in biodiesel.
First, there was biology which began in earnest in the 19th century. Then came molecular biology in the 1920s and the foundation of mutagenesis set forth by Herman Muller in 1927. Then, genetic engineering was first applied in 1972 the lab of Paul Berg. Finally, humans had the ability to manipulate living organisms in a specific, directed way. Fast forward 38 years to the announcement by J. Craig Venter that the first synthetic organism was created with a completely synthetic genome. However, Mother Nature is very particular about what exactly humans can do with respect to organismal manipulation. The naive thought that simple addition of genes from one organism into a more suitable organism would lead to theoretical, effective production of desired chemicals was soon the way of the albatros.
This is when scientists had to take a step back and rethink their strategy. They had to consider gene regulation (positive and negative feedback), build-up of secondary metabolites, toxicity of produced end products, etc. It wasn’t enough to add genes coding for enzymes necessary for desired chemical production. Through the advancements of bioinformatics, computation biology, and a nascent field called systems biology, scientists are just now starting to see the fruits of their labor.
Humor me; type in “engineering bacteria” into Google News. Take a look at the headlines that pop up in your browser. Look at the amazing advancements that are happening currently and imagine what is to come…
Sorry it has been so long since my last post. Lots of ‘fun’ at Disney World and a big pickup in stuff to do at work. From what I have been working on, I am seeing lots of great things coming out of the Department of Energy’s Bioenergy Research Centers (BRCs). I wasn’t aware the level of pioneering work in synthetic biology coming down the pipeline and ready for industry partners. Be on the lookout for upcoming posts about this topic and others.