E. coli Metabolic Engineering and Green Applications of Natural Products
Kamal Ahmadi, Mahmoud
MetadataShow full item record
Abstracts In this dissertation Escherichia coli has been extensively engineered to produce variety of natural products with potentials in green chemistry setting, including nonribosomal peptide-polyketide siderophore Yersiniabactin (Ybt) and aromatic compound salicylate 2-O-β-D-glucoside. Furthermore, detailed synthetic biology strategies such as gene and pathway synthesis and organization, plasmid copy number, promoter activity, ribosome binding site strength, gene knockout/in, site directed and random mutation, transporter modulation, co-culture, and precursor directed biosynthesis have been used to optimize production and to generate library of non-natural analogs and to develop green applications for these natural products. In first chapter green applications and E.coli metabolic engineering for heterologous natural products production has been reviewed through literatures. Yersiniabactin is natively produced by pathogenic bacteria “Yersinia Pestis” that causes the deadly disease bubonic plague which limits exploring applications of this valuable molecule. The Ybt pathway has been engineered for expression and biosynthesis using Escherichia coli as a heterologous host (section 2.1). The biosynthetic process for Ybt formation has been further improved 7-times through metabolic engineering and exogenous substrate provision (section 2.2). Furthermore on section 2.3 we developed a simple aqueous two phase purification method to concentrated produced Ybt from crude extract. On chapter 4 we have applied precursor-directed biosynthesis to systematically produce Ybt analogs. The new analogs show altered affinity toward precious metals such as gold. Upon doing so, resulting compounds were tested in applications that highlight the metal chelating nature of the compound (chapter 3). For instance, Ybt was immobilized on polymeric resin and then incorporated into a packed-bed column prototype to continuously remove copper from water samples with results that included: 1) 100% removal capability; 2) variation in removal across pH levels, providing an opportunity for in situ resin regeneration and metal recovery; and 3) selective removal from a copper-zinc mixture. The combined biosynthetic and recovery processes offer an alternative opportunity for selective removal of copper and other metals contaminating water samples with the goal of aiding the environmental and economic outcomes associated with processes across the electrical, plating, semiconductor, solar panel, automotive, and e-waste sectors. On last chapter, we have engineered E.coli for gram per liter production of Salicylate 2-O-β-D-glucoside (SAG). In plants, salicylic acid (SA) plays important role in biological and physiological processes, including a signaling role for systematic acquired resistance system. SA is usually modified through chemical attaching of glycosyl, methyl, or other groups to mediate its storage and utility. SAG is more water-soluble than SA, enabling easier transport and storage, and possibly preventing plants from over-exposure to active SA. To explore agricultural applications, the biosynthesis pathway of this compound was reconstituted in E.coli. Through several rounds of plasmid engineering (ribosome binding site strength), gene knockout, and co-culture the bacterial platform produces 2.5 gr/L of SAG. Although we could not test potential agricultural applications, we have explored novel application as an anti-inflammatory agent.