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Metab Eng. 2018 Sep;49:47-58. doi: 10.1016/j.ymben.2018.07.017. Epub 2018 Jul 26.

Characterization and engineering of a carotenoid biosynthesis operon from Bacillus megaterium.

Author information

1
Department of Biochemistry, Saarland University, Campus B 2.2, 66123 Saarbrücken, Germany.
2
Metabolomic Discoveries GmbH, Am Mühlenberg 11, 14476 Potsdam, Germany.
3
Department of Biochemistry, Saarland University, Campus B 2.2, 66123 Saarbrücken, Germany. Electronic address: f.hannemann@mx.uni-saarland.de.

Abstract

Bacillus megaterium belongs to the group of pigmented bacilli producing carotenoids that ensure self-protection from UV radiation-induced and collateral oxidative damage. Metabolite profiling of strain MS941 revealed the presence of the C30 carotenoids 4,4'-diapophytofluene and 4,4'-diaponeurosporenic acid. A gene function analysis demonstrated the presence of a corresponding C30 carotenoid biosynthetic pathway with pharmaceutical importance. We identified a gene cluster comprising putative genes for a farnesyl diphosphate synthase (IspA), a diapophytoene synthase (CrtM) and three distinct diapophytoene desaturases (CrtN1-3). Intriguingly, crtM was organized in an operon together with two of the identified crtN genes. The individual activities of the encoded enzymes were determined by heterologous expression and product analysis in the non-carotenogenic model organism Escherichia coli. Our experimental data show that the first catalytic steps of C30 carotenoid biosynthesis in B. megaterium share significant similarity to the corresponding biosynthetic pathway of Staphylococcus aureus. The biosynthesis of farnesyl diphosphates and their subsequent condensation to form 4,4'-diapophytoene are catalyzed by the identified IspA and CrtM, respectively. The following desaturation reactions to form 4,4'-diaponeurosporene, however, require the activities of multiple diapophytoene desaturases. A biosynthetic operon was engineered and successfully expressed in an E. coli whole-cell system creating a cell factory for a high-yield production of the C30 carotenoid 4,4'-diaponeurosporene which has promising potential in the treatment of various inflammatory diseases.

KEYWORDS:

Bacillus megaterium; Carotenoid biosynthesis; Diaponeurosporene; Diapophytoene desaturase; Diapophytoene synthase; Prenyl diphosphate synthase

PMID:
30055324
DOI:
10.1016/j.ymben.2018.07.017
[Indexed for MEDLINE]

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