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

Abstract

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.

Keywords: P450; Taxol; metabolic engineering; natural products; oxygenated taxanes.

Fig. 1.

Taxol biosynthesis schematic. The schematic depicts an abbreviated overview of the Taxol pathway, beginning with this work's carbon source glycerol. From glycerol, the basic metabolic building blocks of glyceraldehyde-3-phosphate (GAP) and pyruvate (PYR) are made. These building blocks are then carried through the methylerythritol phosphate (MEP) pathway to create the ubiquitous isoprenoid precursors IPP and DMAPP. IPP and DMAPP then are polymerized and cyclized by GGPPS and TxS to form the 20-carbon backbone taxa-4 (5),11 (12)-diene (taxadiene). The blue star indicates the focus of this study, which is the putative next pathway the fifth carbon hydroxylation as performed by the cytochrome P450 CYP725A4 and its reductase partner. Further decoration leads to the advanced intermediate Baccatin III, which combined with group transfer produces the final Taxol compound.

Fig. 2.

P450 module optimization screen. The control references the taxadiene producing chromosomally integrated strain that contains no P450 or CPR. The first graph depicts the physically linked chimera strains in order of increasing expression strength: single-copy chromosomal integration with Trc (1) and T7 (2), Trc on a five-copy (3) and 10-copy plasmid (4), and T7 on a five- (5) and 10-copy plasmid (6). Above each strain are given qualitative, relative expression values for the construct collected by GFP experiments and normalized to (1) (SI Appendix, Fig. S2). The second graph depicts the operon construction mimicking the expression strength of strains 3–6 (strains 7–10). The third graph depicts the strains with varying CPR expression, with the P450 held constant at Trc with a five-copy plasmid. Strain 11 corresponds to the CPR expressed from a single chromosomal integration with a T7 promoter. Strain 12 corresponds to the CPR expression with the P450 on the five copy plasmid, but with its own separate T7 promoter. The fourth graph depicts the strains with various N-terminal modifications to the P450 and CPR, ordered in increasing hydrophilicity.

Fig. 3.

Targeted transcriptomic and proteomic data. Upper indicates the targeted transcriptomics results for the 24-h time point. Genes depicted include the first gene in each module’s operon. DOXP synthase (dxs) accounts for the MEP module, TxS for the synthase module, and taxadiene 5-α hydroxylase (T5aOH) for the P450 module. Lower indicates the targeted proteomics results. The insoluble fraction is depicted on top, whereas the soluble fraction is on the bottom. For both transcriptomic and proteomic figures, Left indicates strains with relative productivity, whereas Right indicates strains with poor productivity of terpene molecules.

Fig. 4.

Bioreactor performance of strain 7. Left depicts the growth and productivity curves, represented in OD₆₀₀ and oxygenated taxanes (mg/L) values. Right depicts glycerol and acetate concentrations throughout the duration of the fermentation.