Applying omics and synthetic biology tools for improving the production of sustainable industrial biochemicals

Priyadharshini Chandrasekaran*

*Corresponding author for this work

Research output: Book/ReportPh.D. thesis

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Abstract

Addressing the growing concern of climate change involves various strategies and one prominent approach involves investigating sustainable alternatives to fossil fuels for the synthesis of chemicals. Within this context, microbial cell factories have emerged as key players, leveraging advanced synthetic biology tools. However, the commercial production is only feasible when they are cost competitive with respect to chemical production process. For maintaining economic viability, optimization of production in terms of titres, yields, and productivity is required. While higher titres and yields are generally achieved through genetic manipulations of the production and degradation pathways of target biochemical; productivity is primarily based on the fitness of the production strain, which in turn is the combination of fitness of the parent strain and the effects of the genetic manipulations on the strain. Escherichia coli has been one of the most popular bacteria used for production of biochemicals. Though many E. coli strains have been characterized; accessibility to genome data and genetic manipulation tools decide choice of the strain over other fitness parameters such as growth rate and reduce overflow metabolism. Such physiological advantages if included in a production strain could enhance the fitness and therefore productivity of the strain.

This thesis adopts a systems-based approach with the primary objective of identifying genetic modification targets to amplify yields, titres, and productivity for the target biochemical in the host organism E. coli. To achieve this, a comparative proteomic analysis was conducted on two E. coli strains: the preferred industrial host E. coli K-12 MG1655 and E. coli W, characterized by favourable attributes such as rapid growth, minimal overflow metabolism, and robust stress tolerance. Key differences among the two strains that contribute to the higher growth rate of E. coli W are highlighted. These are potential targets for genetic modification in the MG1655 strain to further enhance its fitness.

The omics tools developed in the above study were employed to decipher any metabolic stress in production strains and the key variants during fed-batch fermentation conditions. Proteome analyses at different growth phases were carried out. Through this study, limitations in the media components as well as the potential metabolic stress of the under-performing variants was identified. Subsequent fermentation supplemented with the limiting media component, resulted in a 46% and 31% increase in the biochemical yield and titer, respectively.

Increased production of the target biochemical is achieved by upregulating the expression of biosynthetic pathway genes typically using the plasmids. However, regulations are being imposed on both addition of antibiotic to media (for maintaining the plasmid) and the presence of antibiotic marker in the plasmid. Plasmid complementation system developed in many published studies still carry the antibiotic marker in the final strain. In this project we developed a simple synthetic biology tool for in vivo removal of antibiotic marker after initial selection of the strains using the antibiotic selection. The tool was used in other projects at Cysbio and therefore has wide application for all cell factories at large.
Original languageEnglish
Place of PublicationKgs. Lyngby, Denmark
PublisherDTU Bioengineering
Number of pages138
Publication statusPublished - 2023

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