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Recent advances in metabolic engineering have demonstrated the potential to exploit biological chemistry for the synthesis of complex molecules. Much of the progress to date has leveraged increasingly precise genetic tools to control the transcription and translation of enzymes for superior biosynthetic pathway performance. However, applying these approaches and principles to the synthesis of more complex natural products will require a new set of tools for enabling various classes of metabolic chemistries (i.e., cyclization, oxygenation, glycosylation, and halogenation) in vivo. Of these diverse chemistries, oxygenation is one of the most challenging and pivotal for the synthesis of complex natural products. Here, using Taxol as a model system, we use nature’s favored oxygenase, the cytochrome P450, to perform high-level oxygenation chemistry in Escherichia coli. An unexpected coupling of P450 expression and the expression of upstream pathway enzymes was discovered and identified as a key obstacle for functional oxidative chemistry. By optimizing P450 expression, reductase partner interactions, and N-terminal modifications, we achieved the highest reported titer of oxygenated taxanes (∼570 ± 45 mg/L) in E. coli. Altogether, this study establishes E. coli as a tractable host for P450 chemistry, highlights the potential magnitude of protein interdependency in the context of synthetic biology and metabolic engineering, and points to a promising future for the microbial synthesis of complex chemical entities.

To achieve globally outlined sustainability goals, multiple fields and a range of approaches will be required. Built upon the inherent advantages of biological chemistry, biological engineering is likely to play a role in broader sustainability efforts through the development of new chemical manufacturing approaches. Among the various approaches within biological engineering, the utilization of renewable biomass, specifically the upgrading (valorization) of the biomass waste component lignin, is particularly attractive. Lignin is the structural component of plants and is comprised of a complex heteropolymer of aromatic subunits. It is a common byproduct of biomass processing and is typically simply burned for process heat. Though it possesses value beyond combustion, its complexity and heterogeneity have precluded facile chemical approaches to its upgrading, and biological methods are seen as an emerging alternative. Metabolic engineering, the engineering of metabolism to generate a product of interest, appears particularly suited to unlock the potential of lignin valorization. Microbial metabolism inherently enables the assimilation of a variety and mixtures of carbon sources including those derived from lignin. Within this concept, a complex lignin mixture could be funneled to central carbon metabolism, providing tractable starting points for metabolic engineering. Focusing specifically on bacteria, efforts are underway to identify and develop chassis strains capable of assimilating lignin derivatives and synthesizing a product of interest. Although strains like Pseudomonas putida or Rhodococcus opacus are presently favored, Acinetobacter baylyi ADP1 possesses distinct advantages with respect to cloning workflows as a naturally competent host capable of native homologous recombination and is an intriguing alternative. In this dissertation, ADP1 is investigated with respect to both its engineerability (compatibility with high-throughput synthetic biology workflows) and its tractability as a metabolic engineering host. To begin, a standardized set of cloning procedures is established, including a novel one-step scarless and marker-less genomic integration technique utilizing Cas9. Second, a set of expression tools are developed including a promoter library, a set of ribosomal binding site variants, and the exploration of genomic integration sites for heterologous expression. Following, these protocols and tools are applied for the synthesis of two valuable natural products from a mock alkaline pretreatment liquor (APL), the flavoring agent vanillin-glucoside and the nutraceutical resveratrol. Collectively, this work establishes ADP1 not only as a viable but perhaps preferred host for lignin-based metabolic engineering.