Current chemical synthesis based on a petroleum based platform have led to a disruption of environmental systems and continue to be a strong contributor to the emission of greenhouse gases. The accumulation of greenhouse gases, most notably carbon dioxide, is already creating dramatic changes in the worldwide temperatures and climate . In 2016, our planet has reached the warmest global temperature ever recorded . Human activity has been shown to be the main impacting factor of the increase of greenhouse gas concentrations during the last 150 years . It has been proposed that even if the carbon dioxide emission stops immediately it would take 1000 years for the concentrations to return to normal level .
Climate change is one the most important issue affecting our modern world, and we need to consider alternative ways of life to reduce our environmental impact. Several measures are being taken by scientists and industry to reduce the emission of greenhouse gas as much as possible, with carbon dioxide as the main focus. We have let this issue be an inspiration to us and came up with the main idea of our project. Inspired by nature, the idea is to utilize carbon dioxide as a carbon source for a system that could produce useful products while reusing excess carbon dioxide.
Our project aims to use the carbon fixing ability of a photosynthetic microorganism to produce a carbon source that can be utilized by a wide range of microorganisms that are already used in industry for the production of chemical products today. For our photosynthetic microorganism we chose the cyanobacteria Synechocystis. We chose Synechocystis over, for example, microalgae because as a cyanobacteria it generally grows faster and have more tools available for genetic engineering .
The basic design
Since the carbon source is a major part of the cost for running industrial biosynthesis, utilizing carbon dioxide could be a major advantage not only from environmental perspective but also from an economical point of view. In order to achieve a stable co-culture, a dependent relationship between the two organisms is desired. This can be achieved in several ways, for example through quorum sensing  or metabolite exchange . An exchange of metabolites was chosen due to the advantages of such a system, for example by providing additional fitness benefits due to the division of metabolic labor . It has also been shown to be capable of purging cheaters from the system, i.e. cheaters do not seem to be able to outcompete the cooperating population . Furthermore, in 2015 the Amsterdam iGEM team achieved a proof of concept in which they created a symbiotic culture between the cyanobacterium Synechocystis and Escherichia coli based on auxotrophy. This provided us with great inspiration on how to achieve our production system.
The resulting system will allow for the direct conversion of atmospheric carbon dioxide into commercially used chemicals that are currently derived from fossil sources of carbon, as illustrated in Figure 1.
The chosen production organisms were Bacillus subtilis, Escherichia coli, Yarrowia lipolytica and Saccharomyces cerevisiae. They were chosen since they have all proven to be capable of industrial level production. Furthermore, all of them have been the subject of extensive research, providing a good foundation for continued development.
How will this work?
The modifications planned for each organism in the project are summarized in Figure 2. The project is divided into two systems: either with a prokaryotic or a eukaryotic production organism. For the prokaryotic organisms, arginine is overproduced and secreted. The cyanobacterium, which has its own gene coding for arginine biosynthesis knocked out, is therefore allowed to grow. The same strategy is used for the eukaryotic system, except glutamine is used instead of arginine. In both systems cyanobacteria will secrete acetate, acetate will be the only carbon source available for the production organisms thus leading to a co-dependency. This is achieved by overexpression and knock-out of several genes. For more details about the modifications, see the constructs page.
Why different production organisms?
With the use of organisms that have different strengths when it comes to product synthesis, a more efficient system can be obtained. Thus the production organism can easily be exchanged depending on the desired product. The result is a highly modular system with a wide range of production capabilities. Furthermore, there are several disadvantages when working with cyanobacteria alone. One of them is their slower growth rate than most chemotrophs , making genetic engineering of them slower. Since a future expansion of the biosynthesis field certainly will require extensive genetic engineering, working with this organism is not ideal. Leaving the product synthesis to faster growing organisms such as Saccharomyces cerevisiae and Bacillus subtilis will allow for faster implementation of new synthesis pathways.
Additionally the use of photosynthetic microorganisms allows us to avoid several of the ethical and social issues that results from the current use of plant based substrate causes. For more information on this check out the integrated human practices part of our wiki.
E. coli is one of the most frequently used gram-negative prokaryotes in research and has been a model organism since the beginning of modern microbiology. There are many advantages to use it as one of the production organisms due to the abundant research available on it. Furthermore, it has a short generation time and can be easily transformed with altered plasmids which make it very easy to clone . As a production organism E. coli was chosen for its ability to produce both heterologous and recombinant proteins .
The Bacillus group of microorganisms, where B. subtilis being the most well known, are one of the most efficient and versatile groups of microorganisms at secreting proteins . B. subtilis is mainly used on an industrial scale for production of enzymes that are used in a wide range of applications such as medical care and laundry detergents . Due to its wide usage in both industry and research, the B. subtilis genome is well annotated , facilitating further development of the organism for industrial use. Being a gram-positive bacteria, in contrast to E. coli which belongs to the gram-negative group, choosing B. subtilis will provide greater versatility to the project.
Yarrowia lipolytica is one of the most used non-conventional yeasts, both in industry and research. It has the notable trait of being able to efficiently accumulate lipids, something that only a few microorganisms are capable of. The wild-type of this microorganism is able to accumulate lipids up to 10-15% of its biomass and with genetic engineering this ratio can reach over 90% . Y. lipolytica is an obligate aerobic yeast with a wide substrate range, covering compounds such as alkanes and fatty acids . Products from Y. lipolytica are diverse and include lipids, organic acids, proteases, and lysine. Most strains of Y. lipolytica can grow on acetate as a single carbon source, which is important for our project. Sodium acetate concentration of 0.4% is well tolerated, but higher concentrations weaken the growth rate and a concentration of 1% limits the growth .
Saccharomyces cerevisiae is one of the most widely used organisms in synthetic biology today. Thus, it has already been thoroughly investigated and thus there are many molecular biology tools available for it. As a eukaryote, its post-translational modification machinery gives it potential to synthesize proteins which are not available when using prokaryotes . Because of those reasons, it was a reasonable choice for our project. On top of that, there is a lot of competence on S. cerevisiae at the synthetic biology department at Chalmers (the department where the project was performed).
To be able to create a fine-tuned co-culture dependance between the cyanobacteria and S. cerevisiae a promoter for the glutamine production with a very specific strength is needed to avoid auxotrophy of glutamate in the S. cerevisiae . A promoter study was developed as being a separate entity alongside of the project to provide aid for the choice of promoter for glutamine synthetase. In order to characterize the strength of the different promoters on BG-11 with 0.5% of acetate compared to 2% glucose we used GFP as reporter gene. The promoters characterized are shown in Table 1. The complete promoter study can be read in promoter study part of wiki.
|pAQR1||Induced by amino acids |
|pGLN1||Repressed by glutamine |
|pPCK1||Repressed by glucose and 10 times increased expression by acetate |
|pPYK2||Repressed by glucose |
|pTEF1||Strong constitutive promoter |
Characterization of a previously existing BioBrick Part
Through the promoter study, existing BioBricks of the native promoter pTEF1 in S. cerevisiae (BBa_K319003 and BBa_K563004) were characterized further and the data was added to registry pages. The study was performed in S. cerevisiae via a replicate plasmid and the expression was quantified with GFP. The expression was investigated with two different carbon sources, glucose and acetate. More detailed description of the promoter study and its results can be also viewed in promoter study part of wiki.
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