title: Study of E. coli metabolic pathways for efficient production of commodity chemicals using synthetic biology and genome engineering
Creators: name: Goyal, Garima
Publisher: name: UNSPECIFIED
Year: 2022

description: Although bio-based fuels and chemicals offer an appealing and more sustainable alternative to traditional petroleum-based fuels and chemicals, their widespread acceptance has been partially obstructed by their limited industry-level titer and yield improvements. Historically, E. coli developed for chemical production has been modified with intuition-based biochemical changes of genes and their expression at the plasmid level on a trial-and-error basis. This type of work can be high cost and labor-intensive, and very unstable for industrial applications. Here, we present our work on the employment of advanced synthetic biology and gene editing tools that can accelerate the understanding and engineering of the microbial metabolic pathways directed towards the systematic and stable improvement of process, yields, and rates for biofuels and bio-products production. The overall objective of the current study is focused on studying and optimizing E. coli metabolic pathways for the efficient production of target bio-based compounds. We performed engineering of two E. coli metabolic pathways for optimizing the titers of two target compounds through a shared systematic approach of utilizing gene editing, strain development and optimization, and synthetic biology tools. Chapter three of this thesis involves the parallel integration and chromosomal expansion of the isoprenoid pathway in the E. coli genome for improved isopentenol titers. In this study, we enabled integration and independent expansions of three pathway components across multiple loci. Suicide vectors were used to achieve high-efficiency site-specific integration of sequence-validated multigene components and a heat-curable plasmid was introduced to obviate recA deletion post pathway expansion. Through 3-dimensional expansion, we generated libraries of pathway component copy number variants to screen for improved titers. Machine learning studies were conducted to predict the gene expression for the isopentenol titers. Polynomial regressor statistical modeling of the production screening data suggested that increasing copy numbers of all isopentenol pathway components would further improve titers. From the library of engineered strains with isoprenoid pathway components integrated and expanded in the genome, the best strain produced 344 mg/mL of isopentenol in the absence of selection pressure. Chapter four focuses on the metabolic engineering of fatty acid biosynthesis pathway for efficient production of fatty alcohols in E. coli. Due to tight regulation of endogenous fatty acid biosynthesis pathway, growth essential pathway genes were removed from the genome via CRISPR-Cas9 and placed on the replicable and repressible bacterial artificial chromosome (BAC). Another BAC module that consisted of a heterologous pathway for fatty alcohols production was introduced. Both these modules in the defragged genome were confirmed to be orthogonal and independent of each other. BAC-A module was introduced to support the cell growth in the absence of native genes and BAC-B module's main function was to express heterologous pathway genes for producing high titers of fatty alcohols. CRISPR-Cas9 was developed for simultaneous and consequential multigene deletions of twenty-seven growth essential native pathway genes. A resulting engineered strain with defragged genome harboring BAC-A and BAC-B was tested for fatty alcohols under different express/repress conditions and performed 2.5 times better than an engineered non-FABIO strain (consists of BAC-B without repressing the growth essential genes).