Genetically engineered biosynthetic pathways for nonnatural C60 carotenoids using C5-elongases and C50-cyclases in Escherichia coli

Ling Li,Kyoichi Saito,Maiko Furubayashi,Shigeko Kawai-Noma,Takashi Maoka,Daisuke Umeno,Shifei Wang

doi:10.1038/s41598-019-39289-w

Ling Li, Kyoichi Saito + Show 5 more

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https://doi.org/10.1038/s41598-019-39289-w

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Abstract

While the majority of the natural carotenoid pigments are based on 40-carbon (C40) skeleton, some carotenoids from bacteria have larger C50 skeleton, biosynthesized by attaching two isoprene units (C5) to both sides of the C40 carotenoid pigment lycopene. Subsequent cyclization reactions result in the production of C50 carotenoids with diverse and unique skeletal structures. To produce even larger nonnatural novel carotenoids with C50 + C5 + C5 = C60 skeletons, we systematically coexpressed natural C50 carotenoid biosynthetic enzymes (lycopene C5-elongases and C50-cyclases) from various bacterial sources together with the laboratory-engineered nonnatural C50-lycopene pathway in Escherichia coli. Among the tested enzymes, the elongases and cyclases from Micrococcus luteus exhibited significant activity toward C50-lycopene, and yielded the novel carotenoids C60-flavuxanthin and C60-sarcinaxanthin. Moreover, coexpression of M. luteus elongase with Corynebacterium cyclase resulted in the production of C60-sarcinaxanthin, C60-sarprenoxanthin, and C60-decaprenoxanthin.

Highlights

Carotenoids are a class of natural pigments covering yellow, orange and red colors
Since C45 and C50 carotenoids were first reported in the 1960s8,9, many of these carotenoids have been identified in prokaryotes, including gram-positive bacteria such as Micrococcus[10], Corynebacterium[11], Dietzia[12], and extreme halophilic archaea such as Halobacterium or Haloarcula[13]
C50 carotenoid synthesis in Corynebacterium proceeds via the flavuxanthin intermediate, but the expressed cyclase (CrtYeYf) is less specific and yields a mixture of decaprenoxanthin[10,11] (ε,ε-ring), sarprenoxanthin[10] (ε,γ-ring) and sarcinaxanthin, when expressed in E. coli

Summary

Introduction

Carotenoids are a class of natural pigments covering yellow, orange and red colors. More than 750 carotenoids have been identified in various plants, fungi, and microorganisms[1], and a wide range of essential biological functions have been described, with light-harvesting, photoprotection, antioxidatant, and pro-vitamin activities, and roles in the control of membrane fluidity[2]. Carotenoid cleavage enzymes can be used to produce a range of carotenoids with different-backbone sizes (C14, C20, or C28 carbons)[7] These include precursors of bixin (C24+1), crocetin (C20), retinals (C20), and other hormonal compounds such as strigolactones (C13) and abscisic acid (C15), as reviewed previously[3]. Another source of structure and size diversity comes from the attachment of additional isoprene units (C5) to C40 carotenoids. We constructed a nonnatural C50 backbone carotenoid pathway in which two geranylfarnesyl diphosphates (C25PP) are condensed to produce C50-phytoene (Fig. 1b) This compound is subsequently subjected to a six-step desaturation reaction resulting in the synthesis of C50-lycopene. We systematically expressed elongase and cyclase in engineered E. coli strains harboring enzymes of the C50-lycopene synthetic pathway and established pathways for the synthesis of the novel carotenoids C60-flavuxanthin, C60-sarcinaxanthin, C60-sarprenoxanthin, and C60-decaprenoxanthin (Fig. 1b)

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