Team:ICT-Mumbai/Description

The days where kilograms of animal and plant tissues were needed for the purification of small amounts of a given protein are almost gone. From the initial establishment of recombinant protein production in the pharmaceutical industry in the 1980s, systems and technologies have evolved in step with developments in other areas to enable rapid production of many different target proteins, and their variants, specifically designed for their end use.

In the past century, the idea of improving desirable characteristics in the living bodies by controlling the expression of target genes or engineering organisms to behave in a certain manner was just an imagination. However, in the recent era, by virtue of recombinant DNA technology, crucial proteins required for health problems and dietary purposes can be produced safely, affordably, and sufficiently. Whether it is the creation of proteins like erythropoietin (epoetin alfa) which can be effectively used in curing of anemia or cyanobacteria mediating hydrogen production ^[1], this field has demonstrated unique impacts in bringing advancement to human life.

In efforts to identify an optimal approach for the production and purification of proteins, many different technologies and strategies have been explored. A revolution has been observed in the traditional approaches of protein production owing to the blooming popularity of recombinant protein technology. It has been a common objective to balance success rates with ease and breadth of use, speed, cost and versatility. The widespread impact and future implications of this technology is highly exciting for us. The opportunity to work for the betterment of this technology rather than being silent bystanders is what drives and inspires our team.

So, what's the matter?

The expression of recombinant proteins is commonly accomplished by inserting the gene of interest into a multicopy plasmid and hence transforming plasmid into the host. Plasmids pose a metabolic load for the host organism as genes on the plasmid are expressed simultaneously with other chromosomal genes. This leads to competition between chromosomal genes and plasmid-borne genes for cellular metabolic resources and transcription-translation machinery leading to plasmid loss in several cases. The negative effects of the presence of multicopy plasmid on growth rate and cell viability are also well known ^[2]. Along with this, it is a known fact that even identical cells in the same environment may produce different amounts of metabolically relevant proteins ^[3]. Such different protein expression implies a possibility of metabolic heterogeneity in bacterial cells used in fermenters ^[4] which lead to heterologous growth phenotypes reducing protein titer. This has increased the desirability of uncoupling biomass production phase and protein production phase ^[5].

Quiescenar Dohaeris- All quiescent cells must serve!

We are creating an industrially employable segregation system in model organism Escherichia coli. We aim to engineer E. coli to enable facile separation of cells based on their metabolic state. This will enable us to separate the protein production phase spatially and temporally from the biomass production phase. We plan to effect this separation by creation of a stationary phase induced flagellar genetic circuit which will allow automated concentration gradient induced movement of the cells from one fermentor to the next. Now, as the protein production phase will be in a homologous growth phenotype, we can expect higher titers of the target protein.

In one embodiment of this innovation, cells can be grown in an upstream of the two fermenters to high cell densities, and cells that have achieved a specific metabolic state (for example, have become quiescent), will be separated from the cell culture into the downstream fermenter and used for carrying out a biotransformation. This strategy will allow a fermenter to be maintained in a continuous steady state without the need for extensive manual reactor control.

In another embodiment, in a batch or fed-batch process, cells that have achieved stationary phase and have stopped production of the product of interest can be separated from cells that are producing the protein. Such a strategy has the potential to increase titres of the recombinant protein, as nutrients will be available for growing cells and not be diverted towards cellular maintenance by cells in the stationary phase.

References:

Khan, Suliman et al. “Role of Recombinant DNA Technology to Improve Life.” International journal of genomics vol. 2016 (2016): 2405954. doi:10.1155/2016/2405954

Kurland CG and Dong H. “Bacterial growth inhibition by overproduction of protein.” The Journal of Molecular Microbiology vol. 21,1 (1996): 1-4.

Şimşek, Emrah, and Minsu Kim. “The emergence of metabolic heterogeneity and diverse growth responses in isogenic bacterial cells.” The ISME journal vol. 12,5 (2018): 1199-1209. doi:10.1038/s41396-017-0036-2

Labhsetwar, Piyush et al. “Heterogeneity in protein expression induces metabolic variability in a modeled Escherichia coli population.” Proceedings of the National Academy of Sciences of the United States of America vol. 110,34 (2013): 14006-11. doi:10.1073/pnas.1222569110

Rowe, D C, and D K Summers. “The quiescent-cell expression system for protein synthesis in Escherichia coli.” Applied and environmental microbiology vol. 65,6 (1999): 2710-5.