Dataset Information

Genome Mining Reveals Two Missing CrtP and AldH Enzymes in the C30 Carotenoid Biosynthesis Pathway in Planococcus faecalis AJ003^T.

ABSTRACT: Planococcus faecalis AJ003^T produces glycosyl-4,4'-diaponeurosporen-4'-ol-4-oic acid as its main carotenoid. Five carotenoid pathway genes were presumed to be present in the genome of P. faecalis AJ003^T; however, 4,4-diaponeurosporene oxidase (CrtP) was non-functional, and a gene encoding aldehyde dehydrogenase (AldH) was not identified. In the present study, a genome mining approach identified two missing enzymes, CrtP2 and AldH2454, in the glycosyl-4,4'-diaponeurosporen-4'-ol-4-oic acid biosynthetic pathway. Moreover, CrtP2 and AldH enzymes were functional in heterologous Escherichia coli and generated two carotenoid aldehydes (4,4'-diapolycopene-dial and 4,4'-diaponeurosporene-4-al) and two carotenoid carboxylic acids (4,4'-diaponeurosporenoic acid and 4,4'-diapolycopenoic acid). Furthermore, the genes encoding CrtP2 and AldH2454 were located at a distance the carotenoid gene cluster of P. faecalis.

SUBMITTER: Lee JH

PROVIDER: S-EPMC7764019 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Genome Mining Reveals Two Missing CrtP and AldH Enzymes in the C30 Carotenoid Biosynthesis Pathway in Planococcus faecalis AJ003T.

Lee Jun Ho JH Kim Jin Won JW Lee Pyung Cheon PC

Molecules (Basel, Switzerland) 20201212 24

Planococcus faecalis AJ003T produces glycosyl-4,4'-diaponeurosporen-4'-ol-4-oic acid as its main carotenoid. Five carotenoid pathway genes were presumed to be present in the genome of P. faecalis AJ003T; however, 4,4-diaponeurosporene oxidase (CrtP) was non-functional, and a gene encoding aldehyde dehydrogenase (AldH) was not identified. In the present study, a genome mining approach identified two missing enzymes, CrtP2 and AldH2454, in the glycosyl-4,4'-diap ...[more]

PMID: 33322786

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Project description:BackgroundMembers of the genus Planococcus have been revealed to utilize and degrade solvents such as aromatic hydrocarbons and alkanes, and likely to acquire tolerance to solvents. A yellow marine bacterium Planococcus maritimus strain iso-3 was isolated from an intertidal sediment that looked industrially polluted, from the Clyde estuary in the UK. This bacterium was found to produce a yellow acyclic carotenoid with a basic carbon 30 (C30) structure, which was determined to be methyl 5-glucosyl-5,6-dihydro-4,4'-diapolycopenoate. In the present study, we tried to isolate and identify genes involved in carotenoid biosynthesis from this marine bacterium, and to produce novel or rare C30-carotenoids with anti-oxidative activity in Escherichia coli by combinations of the isolated genes.ResultsA carotenoid biosynthesis gene cluster was found out through sequence analysis of the P. maritimus genomic DNA. This cluster consisted of seven carotenoid biosynthesis candidate genes (orf1-7). Then, we isolated the individual genes and analyzed the functions of these genes by expressing them in E. coli. The results indicated that orf2 and orf1 encoded 4,4'-diapophytoene synthase (CrtM) and 4,4'-diapophytoene desaturase (CrtNa), respectively. Furthermore, orf4 and orf5 were revealed to code for hydroxydiaponeurosporene desaturase (CrtNb) and glucosyltransferase (GT), respectively. By utilizing these carotenoid biosynthesis genes, we produced five intermediate C30-carotenoids. Their structural determination showed that two of them were novel compounds, 5-hydroxy-5,6-dihydro-4,4'-diaponeurosporene and 5-glucosyl-5,6-dihydro-4,4'-diapolycopene, and that one rare carotenoid 5-hydroxy-5,6-dihydro-4,4'-diapolycopene is included there. Moderate singlet oxygen-quenching activities were observed in the five C30-carotenoids including the two novel and one rare compounds.ConclusionsThe carotenoid biosynthesis genes from P. maritimus strain iso-3, were isolated and functionally identified. Furthermore, we were able to produce two novel and one rare C30-carotenoids in E. coli, followed by positive evaluations of their singlet oxygen-quenching activities.

Project description:BACKGROUND: Carotenoids are a heterogeneous group of plant isoprenoids primarily involved in photosynthesis. In plants the cleavage of carotenoids leads to the formation of the phytohormones abscisic acid and strigolactone, and C13-norisoprenoids involved in the characteristic flavour and aroma compounds in flowers and fruits and are of specific importance in the varietal character of grapes and wine. This work extends the previous reports of carotenoid gene expression and photosynthetic pigment analysis by providing an up-to-date pathway analysis and an important framework for the analysis of carotenoid metabolic pathways in grapevine. RESULTS: Comparative genomics was used to identify 42 genes putatively involved in carotenoid biosynthesis/catabolism in grapevine. The genes are distributed on 16 of the 19 chromosomes and have been localised to the physical map of the heterozygous ENTAV115 grapevine sequence. Nine of the genes occur as single copies whereas the rest of the carotenoid metabolic genes have more than one paralogue. The cDNA copies of eleven corresponding genes from Vitis vinifera L. cv. Pinotage were characterised, and four where shown to be functional. Microarrays provided expression profiles of 39 accessions in the metabolic pathway during three berry developmental stages in Sauvignon blanc, whereas an optimised HPLC analysis provided the concentrations of individual carotenoids. This provides evidence of the functioning of the lutein epoxide cycle and the respective genes in grapevine. Similarly, orthologues of genes leading to the formation of strigolactone involved in shoot branching inhibition were identified: CCD7, CCD8 and MAX1. Moreover, the isoforms typically have different expression patterns, confirming the complex regulation of the pathway. Of particular interest is the expression pattern of the three VvNCEDs: Our results support previous findings that VvNCED3 is likely the isoform linked to ABA content in berries. CONCLUSIONS: The carotenoid metabolic pathway is well characterised, and the genes and enzymes have been studied in a number of plants. The study of the 42 carotenoid pathway genes of grapevine showed that they share a high degree of similarity with other eudicots. Expression and pigment profiling of developing berries provided insights into the most complete grapevine carotenoid pathway representation. This study represents an important reference study for further characterisation of carotenoid biosynthesis and catabolism in grapevine.

Dataset Information

Genome Mining Reveals Two Missing CrtP and AldH Enzymes in the C30 Carotenoid Biosynthesis Pathway in Planococcus faecalis AJ003^T.

Publications

Genome Mining Reveals Two Missing CrtP and AldH Enzymes in the C30 Carotenoid Biosynthesis Pathway in <i>Planococcus faecalis</i> AJ003<sup>T</sup>.

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