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.

Metabolic engineering of Saccharomyces cerevisiae for de novo production of dihydrochalcones with known antioxidant, antidiabetic, and sweet tasting properties

“…by higher copy numbers of HvCHS2 . A similar strategy of using enzymes as filters for selective production of the compound of interest was recently used in E. coli for unnatural C50 carotenoids (Furubayashi et al, 2015). …”

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…For instance, Furubayashi et al designed an unnatural biosynthetic route toward C 50 -astaxanthin which has stronger antioxidant property than the natural C 40 -astaxanthin (Figure 5a). [46] For C 50 -astaxanthin biosynthesis, farnesyl diphosphate (FPP) should be first converted to a C 25 precursor. The authors brought FPP synthase from Geobacillus stearothermophilus and performed site-directed mutagenesis to synthesize C 25 PP.…”

“…As a result, C 50 -astaxanthin could be successfully produced in E.coli. [46] For the production of taxol intermediates, many papers have used a biosynthetic route through taxa-4(5)-11(12)-diene, usually known as taxadiene (Figure 4). However, Edgar et al found out that taxadiene is easily degraded after first epoxidation.…”

Directed evolution and expression tuning of geraniol synthase for efficient geraniol production in <i>Escherichia coli</i>

“…pUCara-GES M53 was constructed by PCR-amplifying the reading frame of GES M53 from pUC-GES M53 (Furubayashi et al, 2014) and subcloning it into the EcoRI-ApaI site of the pUCara vector (Furubayashi et al, 2015).…”