Structure and biosynthesis of carotenoids produced by a novel Planococcus sp. isolated from South Africa

Abstract

Background The genus Planococcus is comprised of halophilic bacteria generally reported for the production of carotenoid pigments and biosurfactants. In previous work, we showed that the culturing of the orange-pigmented Planococcus sp. CP5-4 isolate increased the evaporation rate of industrial wastewater brine effluent, which we attributed to the orange pigment. This demonstrated the potential application of this bacterium for industrial brine effluent management in evaporation ponds for inland desalination plants. Here we identified a C30-carotenoid biosynthetic gene cluster responsible for pigment biosynthesis in Planococcus sp. CP5-4 through isolation of mutants and genome sequencing. We further compare the core genes of the carotenoid biosynthetic gene clusters identified from different Planococcus species’ genomes which grouped into gene cluster families containing BGCs linked to different carotenoid product chemotypes. Lastly, LC–MS analysis of saponified and unsaponified pigment extracts obtained from cultures of Planococcus sp. CP5-4, revealed the structure of the main (predominant) glucosylated C30-carotenoid fatty acid ester produced by Planococcus sp. CP5-4. Results Genome sequence comparisons of isolated mutant strains of Planococcus sp. CP5-4 showed deletions of 146 Kb and 3 Kb for the non-pigmented and “yellow” mutants respectively. Eight candidate genes, likely responsible for C30-carotenoid biosynthesis, were identified on the wild-type genome region corresponding to the deleted segment in the non-pigmented mutant. Six of the eight candidate genes formed a biosynthetic gene cluster. A truncation of crtP was responsible for the “yellow” mutant phenotype. Genome annotation revealed that the genes encoded 4,4′-diapolycopene oxygenase (CrtNb), 4,4′- diapolycopen-4-al dehydrogenase (CrtNc), 4,4′-diapophytoene desaturase (CrtN), 4,4′- diaponeurosporene oxygenase (CrtP), glycerol acyltransferase (Agpat), family 2 glucosyl transferase 2 (Gtf2), phytoene/squalene synthase (CrtM), and cytochrome P450 hydroxylase enzymes. Carotenoid analysis showed that a glucosylated C30-carotenoid fatty acid ester, methyl 5-(6-C17:3)-glucosyl-5, 6′-dihydro-apo-4, 4′-lycopenoate was the main carotenoid compound produced by Planococcus sp. CP5-4. Conclusion We identified and characterized the carotenoid biosynthetic gene cluster and the C30-carotenoid compound produced by Planococcus sp. CP5-4. Mass-spectrometry guided analysis of the saponified and unsaponified pigment extracts showed that methyl 5-glucosyl-5, 6-dihydro-apo-4, 4′-lycopenoate esterified to heptadecatrienoic acid (C17:3). Furthermore, through phylogenetic analysis of the core carotenoid BGCs of Planococcus species we show that various C30-carotenoid product chemotypes, apart from methyl 5-glucosyl-5, 6-dihydro-apo-4, 4′-lycopenoate and 5-glucosyl-4, 4-diaponeurosporen-4′-ol-4-oic acid, may be produced that could offer opportunities for a variety of applications.

Carotenoides

“…GGPP, geranilgeranil difosfato; PSY, fitoeno sintasa; PDS, fitoeno desaturasa; Z-ISO, 15-cis-δ-caroteno isomerasa; ZDS, ζ-caroteno desaturasa; CRTISO, carotenoide isomerasa; LCYB, licopeno β-ciclasa; LCYE, licopeno ε-ciclasa; BCH1/2, β-caroteno hidroxilasa 1/2; VDE, violaxantina deepoxidasa; ZEP, zeaxantina epoxidasa; NSY, neoxantina sintasa (Makhneva et al, 2021). (Moyo et al, 2022). Sin embargo, la presencia de ésteres de carotenoides glucosilados en bacterias a menudo se pasa por alto debido al uso rutinario de saponifcación para hidrolizar los ésteres y eliminar los ácidos (Moyo et al, 2022).…”

“…(Moyo et al, 2022). Sin embargo, la presencia de ésteres de carotenoides glucosilados en bacterias a menudo se pasa por alto debido al uso rutinario de saponifcación para hidrolizar los ésteres y eliminar los ácidos (Moyo et al, 2022).…”

“…De los pigmentos producidos por las bacterias, los carotenoides tienen diversas funciones biológicas que incluyen, coloración, fotoprotección, captación de luz y regulación de la fluidez de la membrana de la bicapa de fosfolípidos bacterianos (Moyo et al, 2022).…”

“…El crecimiento de la bacteria Planococcus y otros microorganismos halófilos pigmentados en estanques de agua salada contribuye a la coloración rojo anaranjada y aumenta las tasas de evaporación de la salmuera durante la producción de sal (Fig. 83) (Moyo et al, 2022). (Moyo et al, 2022).…”

Characterization of C30 carotenoid and identification of its biosynthetic gene cluster in Methylobacterium extorquens AM1

“…strains [ 14 ], and Planococcus spp. strains [ [15] , [16] , [17] , [18] ], the unique C 30 based carotenoids were identified.…”