Team:UCopenhagen/Interdependency
Introduction
Why interdependency? In order to have a stable relationship where the host-endosymbiont relationship are maintained through generations, the host and endosymbionts must depend on each other for their continued survival.
Within the endosymbiotic relationship, we envision a resource based cross-network between host and symbiont. The exchange of vital metabolites necessary for proliferation would ensure the co-existence of the symbiotic pair while also ensuring mutual demise should either host or symbiont perish. Our goal would be that the pair would survive only if the condition of endosymbiosis is met. This is important, as our project is about laying the foundation for stable modular endosymbiosis.
In this subproject, we will use yeast as a substitute for the host, and E.coli as a substitute for an endosymbiont. This choice was done due to both organisms being readily available and since metabolic pathways in E.coli are well studied. Endosymbiosis between fungi and bacteria are occurring naturally in mycorrhiza (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC92331/), showing that endosymbiotic relationships between these two groups of organisms can occur.
Our aim is to make yeast depend on a metabolite produced by E.coli - an auxotrophic dependence. This will be achieved by engineering an E. coli strain to produce sufficient L-tryptophan to complement the lack of tryptophan synthesis in the yeast strain when grown in a media depleted for L-tryptophan.
We do not attempt to make E.coli depend on yeast at this stage, but in an endosymbiotic relationship the yeast can be grown in a media the E.coli could not survive on its own, the symbiont is thus dependent on the host for survival in these growth conditions.
Final Design
Goal: In synthetic yeast media there are 76 mg tryptophane pr liter (Sigma-Aldrich, 2017), so the goal is to produce and export similar amounts from E.coli. A strain by Gu et al. (2012) had few modifications and accumulated 1.7 g tryptophane per liter: Sufficient for yeast growth.
In addition to this, we have included tryptophane use and production in yeast and E.coli in the modelling to check how many endosymbionts would be necessary pr host.
Genes: Based on the papers by Gu et al., (2012) and Wang et al., (2013) three genes are overexpressed. Tryptophane production is regulated by feedback mechanisms, that we try to overcome.
trpE belongs to the tryptophan operon and has been over-expressed frequently in L-tryptophan producing E. coli strains.
aroG: the starting enzyme of the shikimate pathway, leading to synthesis of tryptophan. Being the first enzyme in the pathway, it determines the carbon flow towards tryptophan synthesis and thus the production.
Both aroG and trpE (figure) are regulated by the concentration of the tryptophane they produce. This feedback signalling reduces the tryptophane concentration we can achieve in a WT E.coli. We have made use of known (Gu et al., 2012) mutant feedback resistant alleles for trpE and aroG to overcome this regulation. For trpE, mutation in a methionine to threonine at position 293 is required, and for aroG the proline at 150 is changed to leucine.
yddG: an aromatic amino acid exporter. yddG is responsible for the secretion of L-tryptophan, and the over-expression of this increase the accumulation (Gu et al 2012), likely due to bypassing the feedback sensitive regulatory steps in tryptophane biosynthesis by decreasing the intracellular concentration.
Codon-optimised, synthetically produced.
Experiments
Why interdependency? In order to have a stable relationship where the host-endosymbiont relationship are maintained through generations, the host and endosymbionts must depend on each other for their continued survival.
Within the endosymbiotic relationship, we envision a resource based cross-network between host and symbiont. The exchange of vital metabolites necessary for proliferation would ensure the co-existence of the symbiotic pair while also ensuring mutual demise should either host or symbiont perish. Our goal would be that the pair would survive only if the condition of endosymbiosis is met. This is important, as our project is about laying the foundation for stable modular endosymbiosis.
In this subproject, we will use yeast as a substitute for the host, and E.coli as a substitute for an endosymbiont. This choice was done due to both organisms being readily available and since metabolic pathways in E.coli are well studied. Endosymbiosis between fungi and bacteria are occurring naturally in mycorrhiza (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC92331/), showing that endosymbiotic relationships between these two groups of organisms can occur.
Our aim is to make yeast depend on a metabolite produced by E.coli - an auxotrophic dependence. This will be achieved by engineering an E. coli strain to produce sufficient L-tryptophan to complement the lack of tryptophan synthesis in the yeast strain when grown in a media depleted for L-tryptophan.
We do not attempt to make E.coli depend on yeast at this stage, but in an endosymbiotic relationship the yeast can be grown in a media the E.coli could not survive on its own, the symbiont is thus dependent on the host for survival in these growth conditions.
Design process
Natural endosymbiosis (our model) Shortly: what is endosymbiosis?
Synthetic endosymbiosis will have innumerable applications if developed to be the orthogonal system we envision. We have chosen to take a bottom-up approach to it, through the investigation of the following three essential mechanisms.
Add something about how our idea started?
Interdependence between host and endosymbiont is necessary in order for the endosymbiotic relationship to be stable and maintained throughout evolution or, more relevant for synthetic biology, through generations. The relationship should be beneficial and crucial for the host and the endosymbiont alike. Interdependency would thus decrease possible safety concerns: without the dependency relationship, the endosymbiotic relationship will not spread and be maintained in a wild population.
Number control addresses our concern that the endosymbiont will thrive too well in the host, and replicate uncontrollably - thus overwhelming the host system - or be lost in host replications. Thus we strive for a way to maintain a stable number of endosymbionts in the system. A control system that repress endosymbiont reproduction in high concentration/numbers, and allow replication when the concentration is decreased, would address this concern.
Protein import is a common trait observed in naturally occurring endosymbiotic relationships, such as mitochondria and chloroplasts. In these cases a down regulation of protein expression in the symbiont is observed, and the protein expression is instead moved to the cell. Proteins expressed in mitochondria and chloroplasts are transcribed in the nucleus, translated in the cytoplasm and subsequently transported across the cell membranes into the endosymbionts. This mechanism would tighten the relationship between host and symbiont, and would allow endosymbiotic manipulation of precursors produced in the host.
