Abstract
Most Mucor species accumulate β-carotene as the main carotenoid. The crtW and crtZ astaxanthin biosynthesis genes from Agrobacterium aurantiacum were placed under the control of Mucor circinelloides expression signals. Expression vectors containing the bacterial genes were constructed, and PEG-mediated transformations were performed on a selected M. circinelloides strain. Transformants that exhibited altered carotene production were isolated and analyzed. Southern analysis showed that all plasmids behave as autoreplicative elements. Northern analysis detected the actual heterologous transcription products, whereas thin layer chromatography and high-performance liquid chromatography studies revealed the presence of new carotenoid compounds and intermediates among the transformants.


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References
Appel KF, Wolff AM, Arnau J (2004) A multicopy vector system for genetic studies in Mucor circinelloides and other zygomycetes. Mol Genet Genomics 271:595–602
Arnau J, Strøman P (1993) Gene replacement and ectopic integration in the zygomycete Mucor circinelloides. Curr Genet 23:542–546
Benito EP, Díaz-Mínguez JM, Iturriaga EA, Campuzano EA, Eslava AP (1992) Cloning and sequence analysis of the Mucor circinelloides pyrG gene encoding orotidine-5′-monophosphate decarboxylase: use of pyrG for homologous transformation. Gene 116:59–67
Bhosale P (2004) Environmental and cultural stimulants in the production of carotenoids from microorganisms. Appl Microbiol Biotechnol 63:351–361
Chew BP, Park JS, Wong MW, Wong TS (1999) A comparison of the anticancer activities of dietary beta-carotene, canthaxanthin and astaxanthin in mice in vivo. Anticancer Res 19:1849–1853
Choi HT, Revuelta JL, Sadhu C, Jayaram M (1988) Structural organization of the TRP1 gene of Phycomyces blakesleeanus: implications for evolutionary gene fusions in fungi. Gene 71:85–95
Fraser PD, Miura Y, Misawa N (1997) In vitro characterization of astaxanthin biosynthetic enzymes. J Biol Chem 272:6128–6135
Hughes DA (1999) Effects of carotenoids on human immune function. Proc Nutr Soc 58:713–718
Iturriaga EA, Díaz-Mínguez JM, Benito EP, Álvarez MI, Eslava AP (1992) Heterologous transformation of Mucor circinelloides with the Phycomyces blakesleeanus leu1 gene. Curr Genet 21:215–223
Iturriaga EA, Velayos A, Eslava AP (2000) The structure and function of the genes involved in the biosynthesis of carotenoids in the Mucorales. Biotechnol Bioprocess Eng 5:263–274
Iturriaga EA, Velayos A, Eslava AP, Alvarez MI (2001) The genetics and molecular biology of carotenoid biosynthesis in Mucor. Recent Res Dev Genet 1:79–92
Iturriaga EA, Papp T, Breum J, Arnau J, Eslava AP (2005) Strain and culture conditions improvement for β-carotene production with Mucor. In: Barredo JL (ed) Methods in biotechnology, vol 18. Microbial Processes and Products, Humana Press Inc., Totowa, NJ, USA, pp 239–256
Lee PC, Momen AZR, Mijts BN, Schmidt-Dannert C (2003) Biosynthesis of structurally novel carotenoids in Escherichia coli. Chem Biol 10:453–462
Misawa N, Shimada H (1998) Metabolic engineering for the production of carotenoids in non-carotenogenic bacteria and yeasts. J Biotechnol 59:169
Misawa N, Satomi Y, Kondo K, Yokoyama A, Kajiwara S, Saito T, Ohtani T, Miki W (1995) Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level. J Bacteriol 177:6575–6584
Naguib YM (2000) Antioxidant activities of astaxanthin and related carotenoids. J Agric Food Chem 48:1150–1154
Navarro E, Ruiz-Pérez VL, Torres-Martínez S (2000) Overexpression of the crgA gene abolishes light requirement for carotenoid biosynthesis in Mucor circinelloides. Eur J Biochem 267:800–807
Nishino H, Murakosh M, Ii T, Takemura M, Kuchide M, Kanazawa M, Mou XY, Wada S, Masuda M, Ohsaka Y, Yogosawa S, Satomi Y, Jinno K (2002) Carotenoids in cancer prevention. Cancer Metastasis Rev 21:257–264
Paiva SAR, Russel RM (1999) β-carotene and other carotenoids as antioxidants. J Am Coll Nutr 5:426–433
Roncero MIG, Jepsen LP, Stroman P, van Heeswijck R (1989) Characterization of a leuA gene and an ARS element from Mucor circinelloides. Gene 84:335–343
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor, New York
Sandmann G (2001) Genetic manipulation of carotenoid biosynthesis: strategies, problems and achievements. Trends Plant Sci 6:14–17
Schmidt-Dannert C, Umeno D, Arnold FH (2000) Molecular breeding of carotenoid biosynthetic pathways. Nat Biotechnol 18:750–753
Steiger S, Sandmann G (2004) Cloning of two carotenoid ketolase genes from Nostoc punctiforme for the heterologous production of canthaxanthin and astaxanthin. Biotechnol Lett 26:813–817
Takaichi S, Sandmann G, Schnurr G, Satomi Y, Suzuki A, Misawa N (1996) The carotenoid 7,8-dihydro-ψ and group can be cyclized by the lycopene cyclase from the bacterium Erwinia uredovora and the higher plant Capsicum annuum. Eur J Biochem 241:291–296
van Heeswijck R, Roncero MIG (1984) High frequency transformation of Mucor with recombinant plasmid DNA. Carlsberg Res Commun 49:691–702
Velayos A, Blasco JL, Alvarez MI, Iturriaga EA, Eslava AP (2000a) Blue-light regulation of the phytoene dehydrogenase (carB) gene expression in Mucor circinelloides. Planta 210:938–946
Velayos A, Eslava AP, Iturriaga EA (2000b) A bifunctional enzyme with lycopene cyclase and phytoene synthase activities is encoded by the carRP gene of Mucor circinelloides. Eur J Biochem 267:1–12
Velayos A, Papp T, Aguilar-Elena R, Fuentes-Vicente M, Eslava AP, Iturriga EA, Álvarez MI (2003) Expression of the carG gene, encoding geranylgeranyl pyrophosphate synthase, is up-regulated by blue light in Mucor circinelloides. Curr Genet 43:112–120
Verdoes JC, Sandmann G, Visser H, Diaz M, van Mossel M, van Ooyen AJJ (2003) Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma). Appl Environ Microbiol 69:3728–3738
Wang C-W, Oh M-K, Liao JC (1999) Engineered isoprenoid pathway enhances astaxanthin production in Escherichia coli. Biotechnol Bioeng 62:235–241
Wolff AM, Arnau J (2002) Cloning of glyceraldehyde-3-phosphate dehydrogenase-encoding genes in Mucor circinelloides (Syn. racemosus) and use of the gpd1 promoter for recombinant protein production. Fungal Genet Biol 35:21–29
Acknowledgements
This research was supported by grants BIO2001-2040 (Spanish Ministerio de Ciencia y Tecnología); SA067/01 (Junta de Castilla y León, Spain); and the Hungarian Scientific Research Fund (OTKA) grants T037471, F046658, and D048537. Dr. N. Misawa is acknowledged for the plasmid pAK96K.
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Papp, T., Velayos, A., Bartók, T. et al. Heterologous expression of astaxanthin biosynthesis genes in Mucor circinelloides . Appl Microbiol Biotechnol 69, 526–531 (2006). https://doi.org/10.1007/s00253-005-0026-6
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DOI: https://doi.org/10.1007/s00253-005-0026-6