Marine Flavobacteriaceae produce zeaxanthin via the mevalonate pathway

Abstract

Zeaxanthin, an oxygenated carotenoid derivative with potent antioxidative properties, is produced by many organism taxa. Flavobacteriaceae are widely distributed in marine environments; however, the zeaxanthin biosynthesis property in this family remains incompletely explored. Here, we characterized zeaxanthin production by marine Flavobacteriaceae strains and elucidated underlying molecular mechanisms. Eight Flavobacteriaceae strains were isolated from the phycosphere of various dinoflagellates. Analyses of the zeaxanthin production in these strains revealed yields ranging from 5 to 3289 µg/g of dry cell weight. Genomic and molecular biology analyses revealed the biosynthesized zeaxanthin through the mevalonate (MVA) pathway diverging from the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway commonly observed in most Gram-negative bacteria. Furthermore, comprehensive genome analyses of 322 culturable marine Flavobacteriale strains indicated that the majority of Flavobacteriaceae members possess the potential to synthesize zeaxanthin using precursors derived from the MVA pathway. These data provide insight into the zeaxanthin biosynthesis property in marine Flavobacteriaceae strains, highlighting their ecological and biotechnological relevance.

References

[1] Alonso-Gutierrez J, Kim EM, Batth TS, Cho N, Hu Q, Chan LJG, Petzold CJ, Hillson NJ, Adams PD, Keasling JD, Garcia Martin H, Lee TS. Principal component analysis of proteomics (PCAP) as a tool to direct metabolic engineering Metab Eng, 2015, 28: 123-133.

[2] Andreassi JL, Leyh TS. Molecular functions of conserved aspects of the GHMP kinase family Biochem, 2004, 43: 14594-14601.

[3] Baltscheffsky M, Brosche M, Hultman T, Lundvik L, Nyren P, Sakai-Nore Y, Severin A, Strid A. A 3-hydroxy-3-methylglutaryl-CoA lyase gene in the photosynthetic bacterium Rhodospirillum rubrum Biochim Biophys Acta, 1997, 1337: 113-122.

[4] Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing J Comput Biol, 2012, 19: 455-477.

[5] Bauer M, Kube M, Teeling H, Richter M, Lombardot T, Allers E, Wurdemann CA, Quast C, Kuhl H, Knaust F, Woebken D, Bischof K, Mussmann M, Choudhuri JV, Meyer F, Reinhardt R, Amann RI, Glockner FO. Whole-genome analysis of the marine Bacteroidetes Gramella forsetii reveals adaptations to degradation of polymeric organic matter Environ Microbiol, 2006, 8: 2201-2213.

[6] Becker S, Scheffel A, Polz MF, Hehemann JH. Accurate quantification of laminarin in marine organic matter with enzymes from marine microbes Appl Environ Microbiol, 2017, 83: e03389-e3416.

[7] Begley M, Gahan CG, Kollas AK, Hintz M, Hill C, Jomaa H, Eberl M. The interplay between classical and alternative isoprenoid biosynthesis controls γδ T cell bioactivity of Listeria monocytogenes FEBS Lett, 2004, 561: 99-104.

[8] Bergsveinson J, Pittet V, Ziola B. RT-qPCR analysis of putative beer-spoilage gene expression during growth of Lactobacillus brevis BSO 464 and Pediococcus claussenii ATCC BAA-344^(T) in beer Appl Microbiol Biotechnol, 2012, 96: 461-470.

[9] Bhosale P, Bernstein PS. β-carotene production by Flavobacterium multivorum in the presence of inorganic salts and urea J Ind Microbiol Biotechnol, 2004, 31: 565-571.

[10] Bhosale P, Larson AJ, Bernstein PS. Factorial analysis of tricarboxylic acid cycle intermediates for optimization of zeaxanthin production from Flavobacterium multivorum J Appl Microbiol, 2004, 96: 623-629.

[11] Bode HB, Ring MW, Schwar G, Kroppenstedt RM, Kaiser D, Muller R. 3-Hydroxy-3-methylglutaryl-coenzyme A (CoA) synthase is involved in biosynthesis of isovaleryl-CoA in the myxobacterium Myxococcus xanthus during fruiting body formation J Bacteriol, 2006, 188: 6524-6528.

[12] Boucher Y, Doolittle WF. The role of lateral gene transfer in the evolution of isoprenoid biosynthesis pathways Mol Microbiol, 2000, 37: 703-716.

[13] Boucher Y, Huber H, L'Haridon S, Stetter KO, Doolittle WF. Bacterial origin for the isoprenoid biosynthesis enzyme HMG-CoA reductase of the archaeal orders Thermoplasmatales and Archaeoglobales Mol Biol Evol, 2001, 18: 1378-1388.

[14] Buchan A, LeCleir GR, Gulvik CA, Gonzalez JM. Master recyclers: features and functions of bacteria associated with phytoplankton blooms Nat Rev Microbiol, 2014, 12: 686-698.

[15] Chaumeil PA, Mussig AJ, Hugenholtz P, Parks DH. GTDB-Tk: a toolkit to classify genomes with the Genome Taxonomy Database Bioinformatics, 2019, 36: 1925-1927.

[16] Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor Bioinformatics, 2018, 34: i884-i890.

[17] Cui B, Smooker PM, Rouch DA, Deighton MA. Selection of suitable reference genes for gene expression studies in Staphylococcus capitis during growth under erythromycin stress Mol Genet Genom, 2016, 291: 1795-1811.

[18] Dahllof I, Baillie H, Kjelleberg S. rpoB-based microbial community analysis avoids limitations inherent in 16S rRNA gene intraspecies heterogeneity Appl Environ Microbiol, 2000, 66: 3376-3380.

[19] Flint KP. A note on a selective agar medium for the enumeration of Flavobacterium species in water J Appl Bacteriol, 1985, 59: 561-566.

[20] Gallardo Rodriguez JJ, Sanchez Miron A, Garcia Camacho F, Ceron Garcia MC, Belarbi EH, Chisti Y, Molina Grima E. Causes of shear sensitivity of the toxic dinoflagellate Protoceratium reticulatum Biotechnol Prog, 2009, 25: 792-800.

[21] Gavriilidou A, Gutleben J, Versluis D, Forgiarini F, van Passel MWJ, Ingham CJ, Smidt H, Sipkema D. Comparative genomic analysis of Flavobacteriaceae: insights into carbohydrate metabolism, gliding motility and secondary metabolite biosynthesis BMC Genom, 2020, 21: 569.

[22] Hameed A, Arun AB, Ho HP, Chang CM, Rekha PD, Lee MR, Singh S, Young CC. Supercritical carbon dioxide micronization of zeaxanthin from moderately thermophilic bacteria Muricauda lutaonensis CC-HSB-11T J Agric Food Chem, 2011, 59: 4119-4124.

[23] Hameed A, Shahina M, Lin SY, Sridhar KR, Young LS, Lee MR, Chen WM, Chou JH, Young CC. Siansivirga zeaxanthinifaciens gen. nov., sp. nov., a novel zeaxanthin-producing member of the family Flavobacteriaceae isolated from coastal seawater of Taiwan FEMS Microbiol Lett, 2012, 333: 37-45.

[24] Hamidi M, Kozani PS, Kozani PS, Pierre G, Michaud P, Delattre C. Marine bacteria versus microalgae: who is the best for biotechnological production of bioactive compounds with antioxidant properties and other biological applications? Mar Drugs, 2019, 18: 28.

[25] Huerta-Cepas J, Forslund K, Coelho LP, Szklarczyk D, Jensen LJ, von Mering C, Bork P. Fast genome-wide functional annotation through orthology assignment by eggNOG-Mapper Mol Biol Evol, 2017, 34: 2115-2122.

[26] Johnsen G, Nelson NB, Jovine RVM, Prezelin BB. Chromoprotein- and pigment-dependent modeling of spectral light absorption in two dinoflagellates, Prorocentrum minimum and Heterocapsa pygmaea Mar Ecol Prog Ser, 1994, 114: 245-258.

[27] Joshi C, Singhal RS. Modelling and optimization of zeaxanthin production by Paracoccus zeaxanthinifaciens ATCC 21588 using hybrid genetic algorithm techniques Biocatal Agric Biotechnol, 2016, 8: 228-235.

[28] Kappelmann L, Kruger K, Hehemann JH, Harder J, Markert S, Unfried F, Becher D, Shapiro N, Schweder T, Amann RI, Teeling H. Polysaccharide utilization loci of North Sea Flavobacteriia as basis for using SusC/D-protein expression for predicting major phytoplankton glycans ISME J, 2019, 13: 76-91.

[29] Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability Mol Biol Evol, 2013, 30: 772-780.

[30] Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J. Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species Int J Syst Evol Microbiol, 2012, 62: 716-721.

[31] Kuzuyama T, Seto H. Diversity of the biosynthesis of the isoprene units Nat Prod Rep, 2003, 20: 171-183.

[32] Lange BM, Rujan T, Martin W, Croteau R. Isoprenoid biosynthesis: the evolution of two ancient and distinct pathways across genomes Proc Natl Acad Sci USA, 2000, 97: 13172-13177.

[33] Laupitz R, Hecht S, Amslinger S, Zepeck F, Kaiser J, Richter G, Schramek N, Steinbacher S, Huber R, Arigoni D, Bacher A, Eisenreich W, Rohdich F. Biochemical characterization of Bacillus subtilis type II isopentenyl diphosphate isomerase, and phylogenetic distribution of isoprenoid biosynthesis pathways Eur J Biochem, 2004, 271: 2658-2669.

[34] Lee JH, Hwang YM, Baik KS, Choi KS, Ka JO, Seong CN. Mesoflavibacter aestuarii sp. nov., a zeaxanthin-producing marine bacterium isolated from seawater Int J Syst Evol Microbiol, 2014, 64: 1932-1937.

[35] Liu C-L, Fan L-H, Liu L, Tan T-W. Combinational biosynthesis of isoprene by engineering the MEP pathway in Escherichia coli Process Biochem, 2014, 49: 2078-2085.

[36] Liu C-L, Lv Q, Tan T-W. Joint antisense RNA strategies for regulating isoprene production in Escherichia coli RSC Adv, 2015, 5: 74892-74898.

[37] Liu CL, Bi HR, Bai Z, Fan LH, Tan TW. Engineering and manipulation of a mevalonate pathway in Escherichia coli for isoprene production Appl Microbiol Biotechnol, 2019, 103: 239-250.

[38] Liu X, Zhang Y, Huang K, Yin T, Li Q, Zou Q, Guo D, Zhang X. rpoB and efp are stable candidate reference genes for quantitative real-time PCR analysis in Saccharopolyspora spinosa Biotechnol Biotechnol Equip, 2021, 35: 619-632.

[39] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT Method Methods, 2001, 25: 402-408.

[40] Lombard J, Moreira D. Origins and early evolution of the mevalonate pathway of isoprenoid biosynthesis in the three domains of life Mol Biol Evol, 2011, 28: 87-99.

[41] Matthews JL, Hoch L, Raina JB, Pablo M, Hughes DJ, Camp EF, Seymour JR, Ralph PJ, Suggett DJ, Herdean A. Symbiodiniaceae photophysiology and stress resilience is enhanced by microbial associations Sci Rep, 2023, 13: 20724.

[42] McKee LS, La Rosa SL, Westereng B, Eijsink VG, Pope PB, Larsbrink J. Polysaccharide degradation by the bacteroidetes: mechanisms and nomenclature Environ Microbiol Rep, 2021, 13: 559-581.

[43] Misic AM, Cain CL, Morris DO, Rankin SC, Beiting DP. Divergent isoprenoid biosynthesis pathways in Staphylococcus Species constitute a drug target for treating infections in companion animals Msphere, 2016, 1: e00258-e316.

[44] Motone K, Takagi T, Aburaya S, Miura N, Aoki W, Ueda M (2020) A zeaxanthin-producing bacterium isolated from the algal phycosphere protects coral endosymbionts from environmental stress. mBio 11:e01019-19

[45] Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies Mol Biol Evol, 2015, 32: 268-274.

[46] Olsen JV, de Godoy LM, Li G, Macek B, Mortensen P, Pesch R, Makarov A, Lange O, Horning S, Mann M. Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap Mol Cell Proteom, 2005, 4: 2010-2021.

[47] Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, Edwards RA, Gerdes S, Parrello B, Shukla M, Vonstein V, Wattam AR, Xia F, Stevens R. The SEED and the rapid Annotation of microbial genomes using Subsystems Technology (RAST) Nucl Acids Res, 2014, 42: D206-D214.

[48] Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes Genome Res, 2015, 25: 1043-1055.

[49] Perez-Gil J, Rodriguez-Concepcion M. Metabolic plasticity for isoprenoid biosynthesis in bacteria Biochem J, 2013, 452: 19-25.

[50] Pitera DJ, Paddon CJ, Newman JD, Keasling JD. Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli Metab Eng, 2007, 9: 193-207.

[51] Prabhu S, Rekha PD, Young CC, Hameed A, Lin SY, Arun AB. Zeaxanthin production by novel marine isolates from coastal sand of India and its antioxidant properties Appl Biochem Biotechnol, 2013, 171: 817-831.

[52] Rodríguez-Concepción M, Boronat A (2012) Isoprenoid biosynthesis in prokaryotic organisms. In: Isoprenoid synthesis in plants and microorganisms, pp 1–16

[53] Ruivo M, Amorim A, Cartaxana P. Effects of growth phase and irradiance on phytoplankton pigment ratios: implications for chemotaxonomy in coastal waters J Plankton Res, 2011, 33: 1012-1022.

[54] Sacristan-Soriano O, Winkler M, Erwin P, Weisz J, Harriott O, Heussler G, Bauer E, West Marsden B, Hill A, Hill M. Ontogeny of symbiont community structure in two carotenoid-rich, viviparous marine sponges: comparison of microbiomes and analysis of culturable pigmented heterotrophic bacteria Environ Microbiol Rep, 2019, 11: 249-261.

[55] Seemann T. Prokka: rapid prokaryotic genome annotation Bioinformatics, 2014, 30: 2068-2069.

[56] Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes Nat Protoc, 2006, 1: 2856-2860.

[57] Silva SG, Blom J, Keller-Costa T, Costa R. Comparative genomics reveals complex natural product biosynthesis capacities and carbon metabolism across host-associated and free-living Aquimarina (Bacteroidetes, Flavobacteriaceae) species Environ Microbiol, 2019, 21: 4002-4019.

[58] Singh D, Puri M, Wilkens S, Mathur AS, Tuli DK, Barrow CJ. Characterization of a new zeaxanthin producing strain of Chlorella saccharophila isolated from New Zealand marine waters Bioresour Technol, 2013, 143: 308-314.

[59] Song GH, Kim SH, Choi BH, Han SJ, Lee PC. Heterologous carotenoid-biosynthetic enzymes: functional complementation and effects on carotenoid profiles in Escherichia coli Appl Environ Microbiol, 2013, 79: 610-618.

[60] Sowmya R, Sachindra NM. Carotenoid production by Formosa sp. KMW, a marine bacteria of Flavobacteriaceae family: Influence of culture conditions and nutrient composition Biocatal Agric Biotechnol, 2015, 4: 559-567.

[61] Sudharshan P, Rekha PD, Arun A. Zeaxanthin biosynthesis by members of the genus Muricauda Pol J Microbiol, 2014, 63: 115-119.

[62] Takagi T, Aoyama K, Motone K, Aburaya S, Yamashiro H, Miura N, Inoue K. Mutualistic interactions between dinoflagellates and pigmented bacteria mitigate environmental stress Microbiol Spectrosc, 2023, 11. e0246422

[63] Tanaka RD, Lee LY, Schafer BL, Kratunis VJ, Mohler WA, Robinson GW, Mosley ST. Molecular cloning of mevalonate kinase and regulation of its mRNA levels in rat liver Proc Natl Acad Sci USA, 1990, 87: 2872-2876.

[64] Thawornwiriyanun P, Tanasupawat S, Dechsakulwatana C, Techkarnjanaruk S, Suntornsuk W. Identification of newly zeaxanthin-producing bacteria isolated from sponges in the Gulf of Thailand and their zeaxanthin production Appl Biochem Biotechnol, 2012, 167: 2357-2368.

[65] Tuli HS, Chaudhary P, Beniwal V, Sharma AK. Microbial pigments as natural color sources: current trends and future perspectives J Food Sci Technol, 2015, 52: 4669-4678.

[66] Volke DC, Rohwer J, Fischer R, Jennewein S. Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis Microb Cell Fact, 2019, 18: 192.

[67] Vranova E, Coman D, Gruissem W. Network analysis of the MVA and MEP pathways for isoprenoid synthesis Annu Rev Plant Biol, 2013, 64: 665-700.

[68] Wakahama T, Laza-Martinez A, Bin Haji Mohd Taha AI, Okuyama H, Yoshida K, Kogame K, Awai K, Kawachi M, Maoka T, Takaichi S (2012) Structural confirmation of a unique carotenoid lactoside, P457, in Symbiodinium sp. strain nbrc 104787 isolated from a sea anemone and its distribution in dinoflagellates and various marine organisms. J Phycol 48:1392–402

[69] Walsh PS, Metzger DA, Higushi R. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material Biotechniques, 2013, 54: 134-139.

[70] Wang CH, Hou J, Deng HK, Wang LJ. Microbial production of mevalonate J Biotechnol, 2023, 370: 1-11.

[71] Wang H, Laughinghouse HDt, Anderson MA, Chen F, Willliams E, Place AR, Zmora O, Zohar Y, Zheng T, Hill RT (2012) Novel bacterial isolate from Permian groundwater, capable of aggregating potential biofuel-producing microalga Nannochloropsis oceanica IMET1. Appl Environ Microbiol 78:1445–1453

[72] Wilding EI, Brown JR, Bryant AP, Chalker AF, Holmes DJ, Ingraham KA, Iordanescu S, So CY, Rosenberg M, Gwynn MN. Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in gram-positive cocci J Bacteriol, 2000, 182: 4319-4327.

[73] Wisniewski JR, Zougman A, Nagaraj N, Mann M. Universal sample preparation method for proteome analysis Nat Methods, 2009, 6: 359-362.

[74] Xie J, Chen Y, Cai G, Cai R, Hu Z, Wang H. Tree Visualization By One Table (tvBOT): a web application for visualizing, modifying and annotating phylogenetic trees Nucl Acids Res, 2023, 51: W587-W592.

[75] Yang D, Shipman LW, Roessner CA, Scott AI, Sacchettini JC. Structure of the Methanococcus jannaschii mevalonate kinase, a member of the GHMP kinase superfamily J Biol Chem, 2002, 277: 9462-9467.

[76] Zhang H, Yoshizawa S, Sun Y, Huang Y, Chu X, Gonzalez JM, Pinhassi J, Luo H. Repeated evolutionary transitions of flavobacteria from marine to non-marine habitats Environ Microbiol, 2019, 21: 648-666.

[77] Zhang Y, Liu Z, Sun J, Xue C, Mao X. Biotechnological production of zeaxanthin by microorganisms Trends Food Sci Technol, 2018, 71: 225-234.

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