High-level de novo biosynthesis of glycosylated zeaxanthin and astaxanthin in Escherichia coli

Abstract

Because of wide applications in food, feed, pharmaceutical and cosmetic industries, the carotenoid market is growing rapidly. Most carotenoids are hydrophobic, which limits their bioavailability. Glycosylation is a natural route that substantially increases the water solubility, as well as the bioavailability, photostability and biological activities of carotenoids. Here, we report metabolic engineering efforts (e.g., promoter and RBS engineering, optimization of carbon sources and supplementation of bottleneck genes) to produce glycosylated carotenoids in Escherichia coli. By fine-tuning the carotenoid-biosynthetic genes (crtX, crtZ and crtY), our strain produced up to 47.2 mg/L (~ 11,670 ppm) of zeaxanthin glucosides, ~ 78% of the total carotenoids produced. In another construct with mevalonate, astaxanthin pathway and crtX genes, the strain produced a mixture of carotenoid glucosides including astaxanthin and adonixanthin glucosides with a total yield of 8.1 mg/L (1774 ppm). Our work demonstrated a proof-of-concept study for the microbial biosynthesis of glycosylated carotenoids.

Keywords

Carotenoids / Metabolic engineering / Synthetic biology / Glycosylation / Glucosyltransferase / Zeaxanthin / Astaxanthin / UPD-glucose

References

[1] Choi SK, Osawa A, Maoka T, . 3-beta-Glucosyl-3'-beta-quinovosyl zeaxanthin, a novel carotenoid glycoside synthesized by Escherichiacoli cells expressing the Pantoeaananatis carotenoid biosynthesis gene cluster. Appl Microbiol Biotechnol, 2013, 97(19): 8479-8486.

[2] Dembitsky VM. Astonishing diversity of natural surfactants: 3 Carotenoid glycosides and isoprenoid glycolipids. Lipids, 2005, 40(6): 535-557.

[3] Elshahawi SI, Shaaban KA, Kharel MK, Thorson JS. A comprehensive review of glycosylated bacterial natural products. Chem Soc Rev, 2015, 44(21): 7591-7697.

[4] Farasat I, Kushwaha M, Collens J, . Efficient search, mapping, and optimization of multi-protein genetic systems in diverse bacteria. Mol Syst Biol, 2014, 10(6): 731-731.

[5] Hada M, Nagy V, Deli J, Agocs A. Hydrophilic carotenoids: recent progress. Molecules, 2012, 17(5): 5003-5012.

[6] Hashimoto H, Uragami C, Cogdell RJ. Stange C. Carotenoids and photosynthesis. Carotenoids in nature: biosynthesis, regulation and function, 2016, Cham: Springer International Publishing, 111-139.

[7] Hundle BS, O'Brien DA, Alberti M, . Functional expression of zeaxanthin glucosyltransferase from Erwinia herbicola and a proposed uridine diphosphate binding site. Proc Natl Acad Sci USA, 1992, 89(19): 9321-9325.

[8] Krubasik P, Takaichi S, Maoka T, . Detailed biosynthetic pathway to decaprenoxanthin diglucoside in Corynebacterium glutamicum and identification of novel intermediates. Arch Microbiol, 2001, 176(3): 217-223.

[9] Mao Z, Shin HD, Chen RR. Engineering the E. coli UDP-glucose synthesis pathway for oligosaccharide synthesis. Biotechnol Prog, 2006, 22(2): 369-374.

[10] Matsushita Y, Suzuki R, Nara E, . Antioxidant activity of polar carotenoids including astaxanthin-β-glucoside from marine bacterium on PC liposomes. Fish Sci, 2000, 66(5): 980-985.

[11] Misawa N, Nakagawa M, Kobayashi K, . Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichiacoli. J Bacteriol, 1990, 172(12): 6704-6712.

[12] Mitra K, Ubarretxena-Belandia I, Taguchi T, . Modulation of the bilayer thickness of exocytic pathway membranes by membrane proteins rather than cholesterol. Proc Natl Acad Sci USA, 2004, 101(12): 4083-4088.

[13] Mohamed HE, van de Meene AM, Roberson RW, Vermaas WF. Myxoxanthophyll is required for normal cell wall structure and thylakoid organization in the cyanobacterium Synechocystis sp. strain PCC 6803. J Bacteriol, 2005, 187(20): 6883-6892.

[14] Polyakov NE, Leshina TV, Meteleva ES, . Water soluble complexes of carotenoids with arabinogalactan. J Phys Chem, 2009, 113(1): 275-282.

[15] Richter TK, Hughes CC, Moore BS. Sioxanthin, a novel glycosylated carotenoid, reveals an unusual subclustered biosynthetic pathway. Environ Microbiol, 2015, 17(6): 2158-2171.

[16] Rivera SM, Christou P, Canela-Garayoa R. Identification of carotenoids using mass spectrometry. Mass Spectrom Rev, 2014, 33(5): 353-372.

[17] Sandmann G. Antioxidant protection from UV- and light-stress related to carotenoid structures. Antioxidants, 2019, 8(7): 219.

[18] Shrestha B, Pandey RP, Darsandhari S, . Combinatorial approach for improved cyanidin 3-O-glucoside production in Escherichiacoli. Microb Cell Fact, 2019, 18(1): 7.

[19] Wurtzel ET. Changing form and function through carotenoids and synthetic biology. Plant Physiol, 2019, 179(3): 830-843.

[20] Yabuzaki J. Carotenoids Database: structures, chemical fingerprints and distribution among organisms. Database, 2017

[21] Yokoyama A, Adachi K, Shizuri Y. New Carotenoid glucosides, astaxanthin glucoside and adonixanthin glucoside, isolated from the astaxanthin-producing marine bacterium Agrobacterium aurantiacum. J Nat Prod, 1995, 58(12): 1929-1933.

[22] Yokoyama A, Shizuri Y, Misawa N. Production of new carotenoids, astaxanthin glucosides, by Escherichia coli transformants carrying carotenoid biosynthesis genes. Tetrahedron Lett, 1998, 39(22): 3709-3712.

[23] Zha J, Wu X, Koffas MA. Making brilliant colors by microorganisms. Curr Opin Biotechnol, 2020, 61: 135-141.

[24] Zhang C (2018) Biosynthesis of Carotenoids and Apocarotenoids by Microorganisms and Their Industrial Potential. In: Progress in Carotenoid Research. IntechOpen, London

[25] Zhang C, Chen X, Zou R, . Combining genotype improvement and statistical media optimization for isoprenoid production in E coli. PLoS One, 2013, 8(10): e75164.

[26] Zhang C, Zou R, Chen X, . Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production. Appl Microbiol Biotechnol, 2015, 99(9): 3825-3837.

[27] Zhang C, Seow VY, Chen X, Too HP. Multidimensional heuristic process for high-yield production of astaxanthin and fragrance molecules in Escherichia coli. Nat Commun, 2018, 9(1): 1858.

[28] Zhang C, Chen X, Too H-P. Microbial astaxanthin biosynthesis: recent achievements, challenges, and commercialization outlook. Appl Microbiol Biotechnol, 2020, 104(13): 5725-5737.

Funding

Agency for Science, Technology and Research(A2084c0064)