Bioretrosynthetic construction of a didanosine biosynthetic pathway

Abstract

Concatenation of engineered biocatalysts into multistep pathways markedly increases their utility, but the development of generalizable assembly methods remains a major challenge. Herein we evaluate 'bioretrosynthesis', which is an application of the retrograde evolution hypothesis, for biosynthetic pathway construction. To test bioretrosynthesis, we engineered a pathway for synthesis of the antiretroviral nucleoside analog didanosine (2',3'-dideoxyinosine). Applying both directed evolution- and structure-based approaches, we began pathway construction with a retro-extension from an engineered purine nucleoside phosphorylase and evolved 1,5-phosphopentomutase to accept the substrate 2,3-dideoxyribose 5-phosphate with a 700-fold change in substrate selectivity and threefold increased turnover in cell lysate. A subsequent retrograde pathway extension, via ribokinase engineering, resulted in a didanosine pathway with a 9,500-fold change in nucleoside production selectivity and 50-fold increase in didanosine production. Unexpectedly, the result of this bioretrosynthetic step was not a retro-extension from phosphopentomutase but rather the discovery of a fortuitous pathway-shortening bypass via the engineered ribokinase.

Figure 1. Model inosine biosynthetic pathway and proposed bioretrosynthesis of didanosine

(a) The three enzyme metabolic pathway for inosine used as a model to construct a didanosine pathway. (b) Comparison of the forward and retro-evolution strategies of pathway construction. Enzymes evolved in the forward direction proceed in the order of biosynthesis (ribokinase, RK; phosphopentomutase, PPM; then purine nucleoside phosphorylase, PNP), requiring individual screening assays for each enzymatic step (gray boxes). Retro-evolution requires one screening assay for terminal enzyme activity (orange boxes) for evolution of each enzyme in the reverse order of biosynthesis (PNP, PPM then RK) in increasingly tandem assays.

Figure 2. First shell residues targeted for PPM saturation mutagenesis

Electron density maps for substrate and Ile195 are |F_o|-|F_c| omit maps calculated in Refmac5^, after the removal of relevant atoms from each PDB file and contoured at 2σ. Electron density around Ile195 in panels a-c allows comparison of the electron density quality between structures. Hydrogen bonds are shown as dashed lines. Active site residues Ser154, Val158 and Ile195 are highlighted (blue) in the costructures of wild-type PPM with (a) ribose 5-phosphate (gold, PDB entry 3M8Z) and (b) dideoxyribose 5-phosphate (green). Due to the close proximity (<4.5 Å) of each residue to the bound substrates, all three positions were targeted for saturation mutagenesis. (c) Costructure of Ser154Gly variant with dideoxyribose 5-phosphate (green). The glycine Cα atom shown as a sphere. (d) Overlay of Val158Leu structure (green) with wild-type PPM (gray) with ribose 5-phosphate bound (gold, PDB entry 3M8Z). A favorable hydrogen bond in the wild-type structure between Arg193 and ribose 5-phosphate induced upon substrate binding is indicated. Putative unfavorable interactions in the Val158Leu variant between the Leu158 side chain and both Arg193 (2.3 Å) and the overlaid ribose 5-phosphate (2.5 Å) prevent ribose 5-phosphate from binding in an orientation optimal for catalysis.

Figure 3. Lineage and characterization of PPM variants through generations of evolution

(a) Lineage tree of PPM variants. Clone name is underlined, with mutations listed below. New mutations accumulated through the indicated method of mutagenesis are shown in red. (b) Comparison of changes in ribose 5-phosphate and dideoxyribose 5-phosphate turnover rate by PPM variants in cell lysate normalized to wild-type PPM, presented as changes in nucleoside production through the two enzyme product-screening assay. Assays were performed in tandem with wild-type PNP or PNP-46D6 using 1 mM ribose 5-phosphate or dideoxyribose 5-phosphate, respectively. Data are mean ± s.d. (n=3). Wild-type PPM turnover is 3.18 ± 0.52 μM min⁻¹ for ribose 5-phosphate and 0.486 ± 0.083 μM min⁻¹ for dideoxyribose 5-phosphate. (c) Location of amino acid substitutions mapped on the structure of wild-type PPM with ribose 5-phosphate bound (PDB entry 3M8Z). Positions 81, 238 and 361 are located in the core domain, 154 and 158 are located in the cap domain and 101 and 190 are in the hinge region connecting the two domains. (d) Domain movement observed in 4H11 structure. The core and cap domains of the 4H11 variant (green) are related by a different interdomain angle than wild-type PPM in both its active, phosphorylated form (gray, PDB entry 3TWZ) and the unphosphorylated and unactive form (blue, PDB entry 3TX0). One consequence of the cap domain movement is highlighted in the inset. Asp156 has previously been shown to coordinate an active site Mn²⁺. However, in the 4H11 structure, the location of this coordinating residue is shifted 4.6 Å away from the Mn²⁺ and the coordination sphere is instead completed by water molecules. Water molecules are not shown.

Figure 4. Optimization of RK via didanosine production assay

(a) The interaction between the active site residue Asp16 and bound ribose is highlighted in wild-type E. coli RK (PDB entry 1GQT). This interaction was targeted for removal in preliminary site directed mutagenesis studies by mutation to leucine, asparagine and alanine (water molecules not shown). (b) RK variants were tested for production of inosine and didanosine from ribose and dideoxyribose, respectively, using the three enzyme engineered pathway and ATP regeneration cycle. Data are mean ± s.d. (n=2).

Figure 5. Selectivity and activity changes in selected variant enzymes as assessed using coupled assays

Nucleoside production was measured via HPLC/MS analysis (a) from in situ generated sugar 1-phosphates in one step biocatalysis (PNP only), (b) from sugar 5-phosphates in the two-step tandem pathway (PPM and PNP) or (c) from ribose or dideoxyribose in the full biosynthetic pathway (RK, PPM and PNP) or the pathway without PPM. Enzyme variants used in each reaction are listed under bars (dashed line means no variant included). Direct phosphorylation of the sugar C1 position by wild-type RK in panel (c, second from right) was tested in tandem with wild-type PNP for inosine production and PNP-46D6 for didanosine production to allow the best detection of activity. Turnover in panel (a) was normalized to PNP variant concentration and assay length. Production in panel (b) was normalized to incubation time. The full inosine pathway in (c) was incubated for 5 min, while didanosine production was for 10 h. Reactions in (c) without PPM were incubated for 10 h, however inosine production was normalized to 5 min to be directly comparable to production by the full pathway with PPM. Data are mean ± s.d. (n=2).

Figure 6. Progression of pathway evolution throughout enzyme engineering stages

(a) The original proposed three enzyme biosynthetic pathway. (b) The full pathway including the ATP regeneration cycle after identifying the kinase progenitor for phosphorylation of dideoxyribose and recognizing inhibition of PPM by nucleotide substrates and products. (c) The two step didanosine biosynthetic pathway as a result of the discovered bypass activity of the engineered RK. Dashed arrows indicate expected degradation products.