Engineering bacterial hosts cells for robust growth at high external osmolarities

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Biological Sciences

Abstract

Due to their relative simplicity and rapid growth rates bacteria have been engineered to produce a variety of bulk chemicals, from organic acids and diols to fuels such as ethanol and butanol. In line with their use as platforms for biotechnology there has been an increased interest in gaining control over bacterial growth rates. A very common, yet poorly understood, modulation of bacterial growth during bio production occurs in response to changes in external osmolarities. High-level excretion of metabolites and substrate feed during biotechnological applications constantly increases medium concentration and can result in limited cell growth and low volumetric productivity. Similarly, some of the less expensive media, used to decrease fabrication costs particularly in the areas where clean water is limited, are high in osmolarity.

Increases in the external osmolarity were previously found to effect bacterial osmotic pressure. Based on these findings it was long thought that the change in osmotic pressure leads to decreased growth rates. We recently combined cutting-edge microscopy techniques for single cell studies to show that this is not the case. Upon an increase in external osmolarity osmotic pressure of Escherichia coli recovers to the initial pre-shock value, while the cells continue to grow slower immediately after (Biophys J, under review). Therefore the osmotic pressure does not explain the slowdown in growth. Instead, our preliminary measurements of cellular energetics indicate that the reduction in growth is due to the sustained energetic burden needed to maintain higher cytoplasmic concentration. The finding opens several novel possibilities of controlling cellular growth and production yields.

In this project the student will be trained in a versatile set of interdisciplinary skills needed to explore a range of approaches to achieve more desirable responses to changes in external concentrations for biotechnology applications. The project has been structured to offer several approaches, and thus contingency options, with varying degrees of risk to ensure project success. The project aims are:

(1) Determine the energetic costs of employing different osmoregulatory components during recovery and subsequent growth.
The student will genetically engineer E.coli strains with a given combination of osmoregulatory components and use advanced microscopy techniques to measure the extent of changes in cellular energetics during subsequent growth. Our encouraging preliminary data indicated that this will generate simplified strains with a characteristic response to growth at higher external osmolarity.

(2) Design synthetic pathways that enable E. coli to use accumulated osmolites as alternative sugar sources for production.

The student will utilize increased osmolyte production during growth at higher external osmolarities, such as treahlose, to design synthetic circuit that allow E.coli to use accumulated treahlose for compound production.

(3) Light power the proton motive force of E. coli to elevate available energy levels.

The student will express a light-powered pump, proteorhodopsin, and test if artificially boosting cellular energetics with green light can enhance growth and production yields at high external osmolarity.

(4) Apply the approaches from Aim 1-3 to INEOS proprietary bacteria.

Publications

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Buda R (2016) Dynamics of Escherichia coli's passive response to a sudden decrease in external osmolarity. in Proceedings of the National Academy of Sciences of the United States of America

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Stevenson K (2016) General calibration of microbial growth in microplate readers. in Scientific reports

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M015777/1 01/07/2015 30/06/2019
1602789 Studentship BB/M015777/1 01/07/2015 30/06/2019 Keiran Stevenson
 
Description We have identified the limits of optical density measurements of bacterial suspensions in microplate readers that arise from the complex relationship of size, refractive index and number of bacteria. From this study we have developed a method for the calibration of cell number in these readers that takes into account these limits allowing for continued use of microplate readers as a high throughput data collection method. We have also developed an automated cell counting protocol to improve throughput of calibration as well as collect data on cell size distributions.
Using a microplatereader and our calibration methodology we have devised a protocol for the rapid scanning of osmolarities, medium and solvent concentrations to identify optimal conditions for growth and production. In our studies we have used exposure to ethanol as an example of a toxic product of an industrial bioreactor and have looked to identify conditions that improve the resistance of our E.coli. We have so far concluded that the introduction of high osmolarity in combination with exposure to ethanol causes a reduction in the ethanol tolerance of the bacteria, likely due to the significant simultaneous stress imposed by both conditions. In addition, we have however identified that a reduction in temperature significantly improves solvent tolerance, with a 2% increase in resistance by reducing the incubation temperature 10C.
Exploitation Route We hope that the calibration technique for microplate readers we have developed will be used as a standard protocol for future researchers to help provide accurate measurements of cell number when using optical density measurements.
We also intend to continue with our research into the use of osmoregulation systems for the improvement of bacteria for industrial production of compounds, including synthetic gene constructs for over expression of various components of the osmoregulation pathways. In addition we are now beginning to measure the diffusion within the cytoplasm of the bacteria at different osmolarties in order to better understand the reduction of growth rate that occurs with the intention of finding novel ways to mitigate this.
In addition, reduction of growth temperature may be useful in certain processes for improving solvent tolerances of the bacteria, however, the reduced growth rate that results may offset that advantage and thus some further research is needed.
Sectors Manufacturing, including Industrial Biotechology

URL https://www.nature.com/articles/srep38828
 
Description HFSP program grant
Amount $1,000,000 (USD)
Funding ID RGP0041/2015 
Organisation Human Frontier Science Program (HFSP) 
Sector Charity/Non Profit
Country France
Start 06/2015 
End 12/2018
 
Description Collaboration with Matt Scott at University of Waterloo 
Organisation University of Waterloo
Country Canada 
Sector Academic/University 
PI Contribution This partnership was lead by our team in Edinburgh where we performed microscopy to measure the rates of diffusion in the E.coli cytoplasm and contributed to the development of a model describing E.coli growth at high osmolarity, building on the collaborators previous work.
Collaborator Contribution Our partners provided training and expertise in experimental protocols for the analysis of proteome fractions within bacterial cells and useful guidance in developing the model.
Impact No output yet
Start Year 2018
 
Description Collaboration with Peking University, group of Prof. Fan Bai 
Organisation Peking University
Department Biodynamics Optical Imaging Center
Country China 
Sector Academic/University 
PI Contribution This partnership was lead by our team in Edinburgh where the team at Peking University contributed to the mathematical modeling of bacterial response to osmotic downshocks. The collaboration is ongoing. The two three undergraduate students who helped with the worked secured funding for PhD positions (two) and a Msc scholarship (one).
Collaborator Contribution This partnership was lead by our team in Edinburgh where the team at Peking University contributed to the mathematical modeling of bacterial response to osmotic downshocks. The collaboration is ongoing. The two three undergraduate students who helped with the worked secured funding for PhD positions (two) and a Msc scholarship (one).
Impact Buda R*, Liu Y*, Yang J*, Hegde S*, Stevenson K, Bai F** and Pilizota T**. Dynamics of Escherichia coli's passive response to a sudden decreases in external osmolarity. PNAS September 2016, doi:10.1073/pnas.1522185113 This collaboration is interdisciplinary, it is work that falls under biological physics.
Start Year 2016
 
Title Cell counter 
Description The software automatically counts numbers of cells in a given sequence of images. 
Type Of Technology Software 
Year Produced 2018 
Impact The software will likely be a part of a spin-out company and a device in development. We are currently looking into protecting it. 
 
Title Fitderiv 
Description The software is written to provide a tool for estimating growth rates from optical density data and uses that language (although it can process any other type of data too). It has been described in more detail in the following publication Swain P S**, Stevenson K, Leary A, Montano-Gutierrez L F, Clark I B N, Vogel J and Pilizota T. Inferring time-derivatives, including cell growth rates, using Gaussian processes. Nature Communications 2016;7:13766 and made openly available at http://swainlab.bio.ed.ac.uk/software/fitderiv/ 
Type Of Technology Software 
Year Produced 2016 
Open Source License? Yes  
Impact The software is currently used by several research groups and expected to be adopted wider (publication date is December 2016) 
URL http://swainlab.bio.ed.ac.uk/software/fitderiv/