Team:Macquarie Australia/Description
Nanocompartment production within common prokaryotic expression vectors is highly desirable, and an active area of research. These compartments can be used for the sequestration of cellular products such as proteins and small molecules, aiding the recombinant production of pharmaceuticals and fine chemicals. Additionally, confining cytotoxic compounds extends the utility of recombinant production by ensuring the survival of cells. As well as this, bulk isolation of compartments potentially simplifies purification procedures, while confining enzymes and metabolic pathways within enclosed compartments also aids biosynthetic efficiency, by bringing enzymes and substrates closer in proximity. Additionally, nanocompartments have also been investigated for use in slow release or targeted drug delivery, as well as improving drug bioavailability. Similarly, biological nanocompartments have applications in materials production, cosmetics, and agriculture. Presented herein, we engineer synthetic vesicles into Escherichia coli based upon the known spontaneous vesicle formation in plants and algae associated with the chlorophyll biosynthesis pathway. By mimicking this natural process within E. coli, we extend the aforementioned benefits of biological nanocompartments to a ubiquitous expression vector, thereby enhancing industrial and academic molecular biology and biotechnology.
Ultimately, in order to produce vesicles within E. coli, we need to mimic chlorophyll biosynthesis. The metabolites and enzymes associated with this pathway spontaneously yield vesicles. It has long been known that E. coli produces protoporphyrin as a natural metabolite (Cox and Charles, 1973) and we will be using this as a starting point for chlorophyll biosynthesis in E. coli. To do this, we will be using the genes from the chlorophyll biosynthesis pathway in Chlamydomonas reinhardtii, a well studied alga used widely as a model organism. In support of this, we have carried out literature searches to identify the expression level necessary for each gene and optimised this through computer modelling. Based on this research, several operons have been designed, each with the optimal expression levels necessary. Once complete, each of these operons will be assembled into one plasmid designed around a standardised biobrick design. This allows for vesicle formation to be introduced into any E. coli culture via a single transformation.
These cells, when grown in the absence of light, cause protochlorophyllide (pchlide) to accumulate into semi-crystalline aggregates with phospholipids and key enzymes. These aggregates, termed prolamellar bodies, are the foundation of our vesicles. The cells are subsequently exposed to light, activating the POR (protochlorophyllide oxidoreductase) enzyme, resulting in the production of chlorophyll 𝛼 from pchlide (Bradbeer et al. 1974). This conversion has been experimentally demonstrated to result in the spontaneous formation of phospholipid vesicles from the prolamellar bodies. Thus, we will enable E. coli to produce vesicles, generating a tool that can be used in research and industry with profound implications.
Cox, R. and Charles, H.P., 1973. Porphyrin-accumulating mutants of Escherichia coli. Journal of bacteriology, 113(1), pp.122-132.
Bradbeer, J.W., Gyldenholm, A.O., Ireland, H.M.M., Smith, J.W., Rest, J. and Edge, H.J.W., 1974. Plastid development in primary leaves of Phaseolus vulgaris. New phytologist, 73(2), pp.271-279.
