A synthetic biology approach to metabolic pathway engineering

Merrick, Christine (2015) A synthetic biology approach to metabolic pathway engineering. PhD thesis, University of Glasgow.

Abstract

Microbial biosynthesis of commodity compounds offers a cheaper, greener and more reliable method of production than does chemical synthesis. However, engineering metabolic pathways within a microbe for biosynthesis of a target compound is a complicated process: levels of gene expression, protein stability, enzyme activity, and metabolic flux must be balanced for high productivity without compromising host cell viability. A major rate-limiting step in engineering microbes for optimum biosynthesis of a target compound is DNA assembly, as current methods can be cumbersome and costly.
This study aimed to develop a new, synthetic biology tool for rapid DNA assembly that can be applied to engineering and optimizing metabolic pathways for the microbial biosynthesis of commodity compounds. The potential of using serine site-specific recombinases as synthetic biology tools to assemble DNA was investigated and a new DNA assembly method, Serine Integrase Recombinational Assembly (SIRA), using PhiC31 integrase was established. It was demonstrated that SIRA can clone DNA parts ranging in size from 71 bp to 12.7 kb, assemble as many as five DNA parts in a one-pot reaction, facilitate targeted post-assembly modification of an assembled construct and generate variation between DNA constructs in a single assembly reaction.
SIRA was used to generate variation between constructs containing genes of the violacein biosynthesis pathway, the lycopene biosynthesis pathway, or the DXP pathway for isoprenoid biosynthesis in E. coli. By studying the phenotypes and genotypes of the constructs generated, it was possible to identify rate-limiting steps within these pathways.
Finally, a lycopene-producing in vivo biosensor screen was developed in E. coli to screen DNA assemblies, made with SIRA, encoding genes from the DXP pathway, for enhanced isoprenoid production. By optimizing the expression conditions for assemblies of DXP pathway genes that enhanced isoprenoid production and genes for lycopene biosynthesis in E. coli, 35.78 mg lycopene per gram dry cell weight was obtained - the highest recorded level of lycopene produced from engineering of the DXP pathway alone in E. coli.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Synthetic biology, DNA assembly, isoprenoid biosynthesis, site-specific recombination, carotenoid biosynthesis.
• Subjects: Q Science > QH Natural history > QH301 Biology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Rosser, Professor Susan
• Date of Award: 2015
• Embargo Date: 18 May 2021
• Depositing User: Miss Christine Merrick
• Unique ID: glathesis:2015-6383
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 15 Jun 2015 13:39
• Last Modified: 18 May 2020 05:51
• URI: https://theses.gla.ac.uk/id/eprint/6383