A highly selective biosynthetic pathway to non-natural C50 carotenoids assembled from moderately selective enzymes

Abstract

Synthetic biology aspires to construct natural and non-natural pathways to useful compounds. However, pathways that rely on multiple promiscuous enzymes may branch, which might preclude selective production of the target compound. Here, we describe the assembly of a six-enzyme pathway in Escherichia coli for the synthesis of C50-astaxanthin, a non-natural purple carotenoid. We show that by judicious matching of engineered size-selectivity variants of the first two enzymes in the pathway, farnesyl diphosphate synthase (FDS) and carotenoid synthase (CrtM), branching and the production of non-target compounds can be suppressed, enriching the proportion of C50 backbones produced. We then further extend the C50 pathway using evolved or wild-type downstream enzymes. Despite not containing any substrate- or product-specific enzymes, the resulting pathway detectably produces only C50 carotenoids, including ∼ 90% C50-astaxanthin. Using this approach, highly selective pathways can be engineered without developing absolutely specific enzymes.

Figure 1. Design of a C₅₀-astaxanthin biosynthetic pathway in E. coli.

Non-natural steps are indicated with red arrows. Starting from endogenous C₁₅PP, the pathway includes 15 biochemical steps catalysed by six enzymes (see Supplementary Fig. 1 to see all pathway intermediates). Bottom insets: colonies and cell pellets of E. coli expressing pathways to astaxanthin (left) and C₅₀-astaxanthin (right) constructed in this study. DMAPP, dimethylallyl diphosphate; IPP, isopentenyl diphosphate; OPP, diphosphate unit.

Figure 2. Co-expression of broad-specificity enzymes results in the complex mixture of undesired carotenoid byproducts.

(a) HPLC trace showing the backbone distribution resulting from co-expression of two enzyme variants, FDS_Y81A and CrtM_F26A,W38A, previously reported to have C₂₅PP synthase activity and C₅₀ backbone synthase activity, respectively. See c for the pathways generated by these enzymes. (b) Co-expression of the six broad-specificity enzymes (FDS_Y81A, CrtM_F26A,W38A, CrtI, CrtY, CrtW, CrtZ) encoding all of the biochemical functions required in theory to synthesize C₅₀-astaxanthin results in a complex mixture of undesired carotenoids. (c) Biosynthetic pathways to natural (C₃₀, C₄₀) and non-natural (C₃₅, C₄₅ and C₅₀) carotenoid backbones by FDS_Y81A and CrtM_F26A,W38A.

Figure 3. Directed evolution of C₂₅PP synthase and C₅₀ backbone synthase.

(a) Directed evolution of a more efficient C₂₅PP synthase. The Y81A mutant of FDS was subjected to random mutagenesis and screening for improved C₂₀PP consumption. The library was visually assayed in colonies co-expressing crtE, crtB and crtI; hits were colonies with reduced red (lycopene) pigmentation. (b) In vitro activity of FDS variants provided with DMAPP and [1-¹⁴C]IPP as substrates. Note that the products have been dephosphorylated to the corresponding alcohol. See Supplementary Note 3 for details. (c) Directed evolution of improved C₅₀ backbone synthases. Random mutants of CrtM_W38A were screened for maintenance of C₄₀ synthase function in the presence of crtE and crtI, followed by selection for reduction of C₃₀ synthase function in the presence of crtN. (d) Effect of selectivity-altering mutations on the cellular activity of CrtM mutants. Carotenoid backbones produced by E. coli harbouring pUC-crtM mutants and pAC-fds_Y81A. FDS_Y81A provides C₁₅PP, C₂₀PP and C₂₅PP for the CrtM variants. DNR, diaponeurosporene (C₃₀ carotenoid); LYC, lycopene (C₄₀ carotenoid).

Figure 4. Combinatorial co-expression of enzyme variants for selective production of carotenoid backbones.

(a) Size distributions of carotenoids produced by combinatorial pairing of FDS and CrtM variants. Bars with stars represent asymmetric C₄₀ backbones, produced from C₁₅PP+C₂₅PP⁹. (b) Illustration of metabolic filtering. A modest shift in the precursor distribution effected by the choice of FDS variant, combined with a modest shift in backbone synthase substrate selectivity driven by the choice of CrtM variant can result in substantial focusing of pathway flux to a target product (for example, C₃₅ or C₅₀ carotenoids). Thicker arrows represent preferred enzyme selectivities, while larger dots represent greater metabolite concentrations. The boxes in a labelled i, ii and iii, correspond to the labelled diagrams in b. See Supplementary Note 6 and Supplementary Fig. 7 for a more extensive and quantitative illustration.

Figure 5. Selective production of C₃₀, C₃₅, C₄₀ and C₅₀ backbones by selected combinations of FDS and CrtM variants.

Peaks labelled with asterisks correspond to unidentified non-carotenoid compounds. Percentages refer to the mole fraction of total carotenoid backbones.

Figure 6. Directed evolution of a C₅₀ carotenoid desaturase.

(a) CrtI was subjected to PCR mutagenesis and colonies were screened for increased pigmentation in cells predominantly producing the C₅₀ backbone. Abolition of phytoene was prerequisite for assaying desired C₅₀ (versus undesired C₄₀) function. (b) C₅₀ desaturase activity of CrtI and CrtI_N304P in E. coli expressing FDS_Y81A,V157A and CrtM_{F26A,W38A,F233S}. Insets show the colony hue of each transformant. CrtM*, CrtM_{F26A,W38A,F233S}; FDS*, FDS_Y81A,V157A.

Figure 7. Selective formation of cyclic C₅₀ carotenoids.

E. coli harbouring FDS-CrtM pairs for selective production of C₅₀ (FDS_{Y81A,T121A,V157A} and CrtM_F26A,F233S) or C₄₀ (FDS_Y81M and CrtM_F26A,W38A) backbones were additionally transformed with the indicated genes to produce (a) C₅₀-β-carotene or C₄₀ (natural) β-carotene, (b) C₅₀ oxo-cyclic carotenoids or C₄₀ (natural) counterparts. Shown from left to right are the pathway constructs, cell pellets, HPLC chromatograms, absorption spectra and the corresponding carotenoid structure. As illustrated, the plasmid constructs and expression contexts were identical for all wild-type and variants of each gene under comparison (see Supplementary Table 10).