Text

theses

ETD doctoral

The synthesis of aromatic compounds has received increasing interest in recent years due to its value for a wide range of industries. Over the last few decades, the rapid development of metabolic engineering and synthetic biology tools has enabled high-efficiency and low waste-emission microbial biosynthesis of value-added products from renewable carbon substrates. In microbes, aromatic compounds can be biosynthesized via the shikimate pathway, a ubiquitous pathway for making aromatic amino acids and associated aromatics. This dissertation focuses on the biosynthesis of industrially important aromatic compounds by engineering the shikimate pathway in Escherichia coli.

First, L-tryptophan and L-phenylalanine overproducing E. coli strains were developed by a variety of engineering methodologies, such as over-expression of desired pathway enzymes, deletion of competing pathway genes, and optimization of cultivation conditions. In addition, to address the metabolic heterogeneity caused by non-genetic variation, toxin/antitoxin-based cell selection systems were constructed to select for high-producing cells to improve biosynthetic performance. Specifically, a biosensor-based mechanism was utilized to monitor the intracellular concentrations of L-tryptophan/L-phenylalanine and accordingly control the growth of individual cells by regulating the expression level of toxin/antitoxin genes. This strategy was demonstrated to enhance the L-tryptophan and L-phenylalanine bioproduction significantly.

On the other hand, engineered E. coli strains were constructed for the biosynthesis of several other aromatic compounds, including tryptamine, flavonoids (naringenin, pinocembrin, sakuranetin, and acacetin), and salicylic acid. To this end, selected heterologous enzymes were functionally expressed in E. coli to establish the desired biosynthesis pathways using simple carbon substrates as starting materials. Since biosynthesis of these representative natural products involves characteristically long and complicated pathways, a novel modular co-culture engineering approach was adapted. Specifically, each pathway was divided into serial modules, each of which was accommodated in an independent E. coli strain, to address the challenge of metabolic stress reduction and biosynthetic pathway balancing. The co-cultivation of the resulting strains in one culture was utilized for the biosynthesis of the target products. By utilizing the modular co-culture approach, the heterologous biosynthesis performance was improved remarkably compared to the conventional mono-culture approaches. Moreover, rationally designed biosensor-assisted growth regulation systems were integrated into the co-cultures to sense different types of pathway metabolites (substrate, intermediate, and end product, respectively) and accordingly regulate the growth and biosynthetic behaviors during the cultivation process. In all tested cases, the production of the desired compound was significantly improved.

This thesis explores the utilization of advanced metabolic engineering approaches, including co-culture engineering, biosensing, and their combination, for microbial biosynthesis. The findings not only establish robust E. coli platforms for the production of value-added aromatic compounds but also expand the scope of metabolic engineering for cutting-edge research in the future.

ETD_11364

Ph.D.

Includes bibliographical references

doi:10.7282/t3-0q64-w552

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