Evolutionary synthetic biology enabled the construction of a pathway for the biosynthesis of “supernatural” purple carotenoids

The construction of a 6-enzyme pathway to a literally supernatural, purple 50-carbon analogue of the orange carotenoid astaxanthin in Escherichia coli. More than a decade ago, chemists have synthesized carotenoids with C₅₀ backbones and found that they are more potent antioxidants than natural carotenoids, but biosynthetic pathways for these carotenoids requiring at least 14 non-existing biochemical steps, and therefore has not been accessible until the present work published in Nature Communications.

The heterologous installation of biosynthetic pathways for small molecules found in nature has progressed substantially over the last 15 years. However, engineering pathways for the biosynthesis of products not found in nature, such as natural product analogues, has lagged. A major reason has been reliance on so-called “promiscuous” enzymes, which accept non-standard precursors or produce alternative reaction products, to effect such pathways. Use of these promiscuous enzymes often leads to highly-branched, byproduct-laden synthetic pathways with very poor selectivity for the desired unnatural product.

Daisuke Umeno and his co-workers describe the methods they employed to coordinate six enzymes carrying out 15 chemical transformations to produce C₅₀-astaxanthin. The initial assembly of promiscuous enzymes resulted in a complex mixture of non-target carotenoids. However, through directed evolution and judicious matching of evolved enzyme variants with appropriately-overlapping specificity ranges (a strategy the authors term, “metabolic filtering”), they achieved an impressively selective pathway to C₅₀-astaxanthin from only moderately-specific enzyme “parts.” The authors believe this work provides a plausible model for how plants and microorganisms routinely synthesize particular metabolites with high selectivity using enzymes that are, in fact, often rather unspecific.

Having established a robust and selective biosynthetic route to C₅₀ carotenoids, the authors are interested in systematically exploring this new carotenoid “space” by mixing and matching hundreds of carotenoid-modifying enzymes found in nature.