Project description

Current commercial production of many chemicals is based on fossil sources of carbon. This inevitably results in staggering amounts of carbon dioxide being released into the atmosphere of the Earth (in excess of 35 billion tonnes annually¹). Though carbon dioxide itself is not very toxic, the current increasing atmospheric concentrations of this greenhouse gas has a massive effect on the ecosystems of the planet as well as human quality of life. Examples of this is the acidification of oceans and an increasing mean temperature of the earth.

These problems can be mediated by replacing petrochemical-based production with a sustainable production method based on biosynthesis in microorganisms. This is currently applied in the production of bioethanol, which uses a process very similar to fermenting alcoholic beverages. However, this requires large amounts of sugars used as the carbon source, which is currently taken from plant-based materials such as corn and sugarcane². This has a significant impact on the quality of life in developing parts of the world, where crops are grown and sold for fuel production instead of feeding the local population, leading to a substantial increase in food prices^3,4.

How can we solve the problem of increasing levels of carbon dioxide and the the social issues caused by current substrate production?

Our solution to this problem is to use photosynthetic bacteria to produce the carbon substrate needed for industrial fermentation. The carbon fixation ability of cyanobacteria allows for the direct conversion of carbon dioxide into a usable carbon source that the cyanobacterium then secretes. The secreted carbon source can be converted into a useful product using biosynthesis. Biosynthesis refers to the production of complex molecules within living organisms or cells. A variety of microorganisms, such as Saccharomyces cerevisiae, Bacillus subtilis and many more, are already used in the industry today for this purpose.

There are several approaches that can be used for this kind of system. The cyanobacterium could be grown separately and its supernatant could be used as medium for the separate fermentation for the production organism. This would mean that the fermentations would still be done in separate batches, similarly to how industrial fermentation is performed today. However, this method requires several purification steps: the cyanobacteria medium has to be separated from the bacteria, and the product of interest has to be separated from the production batch.

How can we decrease the number of purification steps?

To solve this issue, we will establish a continuous production system. The proposed system is a co-culture where both organisms are kept in the same container. To prevent one organism from outcompeting the other in the system, the organisms are modified to be metabolically dependent on each other, resulting in a symbiotic system. The modified cyanobacterium (Synechocystis) produces the carbon source that the production organism requires. In return, the production organism produces a specific amino acid that the auxotrophic cyanobacterium requires for survival. In this way we hope to create a self-regulated system that will maintain equilibrium between the two organisms. Furthermore, the auxotrophy of the cyanobacterium makes it non-viable outside the lab environment, preventing unwanted spread of the bacteria.

Why not produce chemicals directly from Synechocystis?

Converting carbon dioxide into useful molecules is an ability that makes cyanobacteria interesting as a production system for the biotech industry. However, with this ability a few disadvantages arise. One of them is a slower growth rate, and therefore a longer recovery time, after performing genetic modifications. Furthermore, the genetic tools needed for genetic modification are not as developed as for industrially used organisms like E. coli and S. cerevisiae. This makes introduction of new synthesis pathways slower in cyanobacteria than it is in other well-studied species. Therefore, we leave the production of chemicals to the organisms that have already proven themselves well suited for this task.

The carbon source that the cyanobacterium is modified to overproduce is acetate. It was chosen since it is naturally present in the metabolism of cyanobacteria. Furthermore, acetate production by Synechocystis has been shown to be possible both in previous publications⁵ and iGEM projects⁶. Overproduction of acetate in Synechocystis is achieved by knock-outs and knock-ins of genes involved in acetate metabolism. The cyanobacterium is also made auxotrophic for arginine or glutamine to make it reliant on the production organism and prevent it from surviving outside the lab. In this case, the arginine auxotrophic bacteria will be co-cultured with prokaryotic production organisms and the glutamine auxotrophs with eukaryotic organisms.

What production organisms will we use in this project?

Four different industrially relevant organisms are modified to be compatible with the symbiotic production system. These are Saccharomyces cerevisiae, Escherichia coli, Yarrowia lipolytica and Bacillus subtilis. As described earlier, all production organisms will have to produce an amino acid that the cyanobacterium requires. Due to the difference in metabolism and regulation of amino acid synthesis, it proved difficult to make all organisms secrete the same amino acid. Therefore the eukaryotes (S. cerevisiae and Y. lipolytica) are modified to secrete glutamine and the prokaryotes (E. coli and B. subtilis) are engineered to secrete arginine.

All of the production organisms except for Bacillus subtilis are capable of growing on acetate in their wild type state. An operon from its cousin Bacillus licheniformis is therefore incorporated into the Bacillus subtilis strain. We have chosen to work with B. subtilis over B. licheniformis due to the expertise available at our lab and the biobrick library available for this organism.

With the four production organisms we will create a library of organisms compatible with the symbiotic production system. Our solution will provide a platform for a versatile system that converts carbon dioxide into everyday products in an environmentally friendly and sustainable way.

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<sup>1. "Trends in global CO2 emissions - 2015 report - EDGAR - Europa.eu." 2015. 28 Jun. 2016 (http://edgar.jrc.ec.europa.eu/news_docs/jrc-2015-trends-in-global-co2-emissions-2015-report-98184.pdf)↩
2. Ranalli, Paolo. Improvement of crop plants for industrial end uses. Paolo Ranalli. The Netherlands: springer, 2007.↩
3. Babcock, Bruce A. "The impact of US biofuel policies on agricultural price levels and volatility." China Agricultural Economic Review 4.4 (2012): 407-426.↩
4. Gao, Zhengxu et al. "Photosynthetic production of ethanol from carbon dioxide in genetically engineered cyanobacteria." Energy & Environmental Science 5.12 (2012): 9857-9865.↩
5. Anfelt, Josefine et al. "Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production." Microbial cell factories 14.1 (2015): 1.↩
6. "Team:Amsterdam - 2015.igem.org." 2015. 30 Jun. 2016 (https://2015.igem.org/Team:Amsterdam)↩</sup>