The Metabolic Link between E. coli and C. hutchinsonii
When C. hutchinsonii is grown on pure crystalline cellulose, soluble sugars (including glucose, cellobiose, cellotriose, and cellotetraose) accumulate extracellularly. It is hypothesised that C. hutchinsonii is not able to efficiently use all of the soluble sugars it generates, such as xylose (Xie et al. 2007) (see the C. hutchinsonii page for more information).

E. Coli is capable of using some of these sugars, such as glucose and xylose (Desai and Rao 2010), for its own growth. Thus, there is a hypothesized metabolic link between C. hutchinsonii and E. Coli. We engineered E. Coli to produce fatty acid ethyl esters from these sugars alone, enabling direct conversion of lignocellulose to biodiesel by the C. hutchinsonii and modified E. Coli co-culture.

Co-culture Instability
Zuroff et al. (2013) predicted that “synthetic syntrophic interactions may be unstable” since at least one organism does not necessarily rely on the other for survival. Without an additional level of control the community may breakdown. Obligate mutualisms such as those described by You et al. 2004 and Shou et al. 2007 may be a more stable approach for consortia-mediated lignocellulosic ethanol production” (Zuroff 2013).

Our naive co-culture experiment showed that the C. hutchinsonii and E. Coli co-culture is neither stable nor efficient without the addition of a population control system - C. hutchinsonii vastly dominates the community, not allowing E. Coli enough access to sugars. Our task was then to design a population control circuit to address this problem.
Introduction to Microbial Consortia Engineering for Consolidated Bioprocessing
Microbial consortia engineering has the potential to more effectively generate useful products, ranging from biofuels to specialty chemicals, than current technology based on mono-cultures of bacteria (Shong 2012). Communities of microbes can better handle the complex process of the conversion of substrates to products by dividing the metabolic load among multiple species. In addition, communities of microbes exhibit increased production rates, metabolic efficiency, and robustness to changes in environmental conditions relative to mono-cultures due to synergistic interactions between species. Currently, there are many challenges in creating synthetic microbial consortia. For instance, natural microbial communities have evolved to be capable of maintaining homeostasis, but synthetic communities are not. When creating synthetic microbial consortia, one must ensure that the members do not out-compete each other, do not exhaust the resources in their environments, and do not have unstable genetic compositions. Thus, engineering microbial consortia requires the establishment of population control systems. The use of synthetic microbial consortia for consolidated bio-processing also faces the same challenges as the use of mono-cultures, including economic feasibility relative to current methods of production.
Our Approach - Designing a Co-Culture for Conversion of Cellulosic Waste to Biodiesel
We aim to create a stable and robust synthetic microbial consortia that converts agricultural waste, lignocellulose, into a useful product, biodiesel. Our system consists of a co-culture of Cytophaga hutchinsonii, an aerobic bacteria that rapidly digests crystalline cellulose, and Escherichia coli, which can grow on the sugars produced from cellulose degradation and is genetically modified to produce the fatty acid esters that comprise biodiesel. In addition to this existing metabolic link, we introduce a synthetic communication pathway to ensure a synergistic relationship between them. Our main focus is thus to ensure stable and efficient ratios of the populations of the bacteria through synthetic biology. In order to predict the interactions between the bacteria and design the communication network, we model the dynamics of our co-culture using whole-genome scale metabolic models with an approach called dynamic flux balance analysis. Our co-culture has many characteristics that make it better than current methods of generation of biodiesel and other products. It is a stable co-culture as opposed to a mono-culture, so it is capable of performing the complex task of conversion of the cellulosic waste into high-value products in one reactor. It does not require additional pre-processing steps of cellulosic substrate, reducing production costs. The ability of our co-culture to use cellulosic waste to produce biodiesel also makes it environmentally friendly, compared to other methods of fuel production. Also, our culture is resilient to environmental changes, which reduces operating costs because optimal operating conditions do not have to be maintained. In addition, our co-culture can be grown aerobically at room temperature, which also cuts down on operating costs.
General Applicability of Our Approach
Here, we demonstrate a robust, environmentally friendly, and economically effective system for production of biodiesel, but our system can be applied to the production of many different products. One could replace the biodiesel genes we have chosen for E. coli with genes of their choice to generate a desired product. In addition, our method of creating synthetic communication pathways to stabilize our synthetic microbial consortia is an extremely important contribution to the field of synthetic biology. One could use this approach to stabilize different co-cultures with bacteria of varying phenotypes and metabolisms, or be utilized modularly so that population ratio can be modified via inducible signal.
Desai and Rao, Appl Environ Microbiol., 76(5):1524-32 (2010)

Shou et al., Proc Natl Acad Sci USA, 104: 1877–1882 (2007)

Xie et al., J Exp Bot., 58(11):2799-810 (2007)

You et al., Nature, 428(6985):868-71 (2004)

Zuroff et al., Biotechnology for Biofuels, 6:59 (2013)