Chin Giaw Lim

Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli

Significance Metabolic engineering is an economically feasible and sustainable alternative for the production of natural products, pharmaceuticals, nutraceuticals, flavors, and fragrances. Of the model systems used to demonstrate and develop this approach, the anticancer agent Taxol stands out for its structural complexity and therapeutic value. A major challenge for the biosynthesis of Taxol and many other natural products is the involvement of cytochrome P450-mediated oxygenation. P450 enzymes are intransigent to functional heterologous expression, especially in Escherichia coli, leading many laboratories to abandon this organism when engineering P450-containing pathways. Here, through a series of optimizations, we demonstrate E. coli as a viable host for P450-mediated oxidative chemistry, advancing Taxol’s biosynthesis through a fivefold increase in oxygenated terpene titers. 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.

Efficient utilization of pentoses for bioproduction of the renewable two-carbon compounds ethylene glycol and glycolate.

The development of lignocellulose as a sustainable resource for the production of fuels and chemicals will rely on technology capable of converting the raw materials into useful compounds; some such transformations can be achieved by biological processes employing engineered microorganisms. Towards the goal of valorizing the hemicellulose fraction of lignocellulose, we designed and validated a set of pathways that enable efficient utilization of pentoses for the biosynthesis of notable two-carbon products. These pathways were incorporated into Escherichia coli, and engineered strains produced ethylene glycol from various pentoses, including simultaneously from D-xylose and L-arabinose; one strain achieved the greatest reported titer of ethylene glycol, 40 g/L, from D-xylose at a yield of 0.35 g/g. The strategy was then extended to another compound, glycolate. Using D-xylose as the substrate, an engineered strain produced 40 g/L glycolate at a yield of 0.63 g/g, which is the greatest reported yield to date.

Engineering a novel biosynthetic pathway in Escherichia coli for production of renewable ethylene glycol

Ethylene glycol (EG) is an important commodity chemical with broad industrial applications. It is presently produced from petroleum or natural gas feedstocks in processes requiring consumption of significant quantities of non‐renewable resources. Here, we report a novel pathway for biosynthesis of EG from the renewable sugar glucose in metabolically engineered Escherichia coli. Serine‐to‐EG conversion was first achieved through a pathway comprising serine decarboxylase, ethanolamine oxidase, and glycolaldehyde reductase. Serine provision in E. coli was then enhanced by overexpression of the serine‐biosynthesis pathway. The integration of these two parts into the complete EG‐biosynthesis pathway in E. coli allowed for production of 4.1 g/L EG at a cumulative yield of 0.14 g‐EG/g‐glucose, establishing a foundation for a promising biotechnology. Biotechnol. Bioeng. 2016;113: 376–383.

Orthogonal Assays Clarify the Oxidative Biochemistry of Taxol P450 CYP725A4

Natural product metabolic engineering potentially offers sustainable and affordable access to numerous valuable molecules. However, challenges in characterizing and assembling complex biosynthetic pathways have prevented more rapid progress in this field. The anticancer agent Taxol represents an excellent case study. Assembly of a biosynthetic pathway for Taxol has long been stalled at its first functionalization, putatively an oxygenation performed by the cytochrome P450 CYP725A4, due to confounding characterizations. Here, through combined in vivo (Escherichia coli), in vitro (lipid nanodisc), and metabolite stability assays, we verify the presence and likely cause of this enzymes inherent promiscuity. Thereby, we remove the possibility that promiscuity simply existed as an artifact of previous metabolic engineering approaches. Further, spontaneous rearrangement and the stabilizing effect of a hydrophobic overlay suggest a potential role for nonenzymatic chemistry in Taxols biosynthesis. Taken together, this work confirms taxadiene-5α-ol as a primary enzymatic product of CYP725A4 and provides direction for future Taxol metabolic and protein engineering efforts.