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Development of xanthophyll producing Mucor circinelloides strains
New Biotechnology · Volume 29S · September 2012
Keywords: First integral; Estimation; Batch alcohol fermentation; Immobilized cells http://dx.doi.org/10.1016/j.nbt.2012.08.599 Poster 5.0.160 Complete DNA sequence and analysis pCD033, a plasmid derived from a Lactobacillus plantarum silage strain S. Heinl∗ , K. Spath, S. Heiss, R. Grabherr CD-Laboratory for Genetically Engineered Lactic Acid Bacteria, Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, 1190 Vienna, Austria Lactobacillus plantarum belongs to the heterogeneous group of lactic acid bacteria (LAB). The species is frequently found in fermented food and feed products as well as in degrading plant matter. L. plantarum CD033 was isolated from a stable grass silage process in Austria. The strain was found to harbour one circular plasmid which was isolated by a modified miniprep protocol, subcloned and sequenced by Sanger technology. The 7.9 kb plasmid was designated pCD033. The plasmid revealed a very low GC-content of 36% and was predicted to contain 9 coding sequences (CDS). One CDS encodes a putative replication protein which shares 57% similarity with the RepB protein of the lactococcal plasmid pUCL22, which was shown to replicates via theta mode. This gene is flanked by a 1.7 kb untranslated region which is supposed to be involved in plasmid replication and shows similarity to an untranslated region of plasmid pLP9000 from L. plantarum strain AS1.2986. Furthermore the plasmid encodes a putative transposase, a putative tyrosine recombinase, a DGQHR domain protein, a putative pemI/pemK like plasmid addiction system as well as three proteins of unknown function. The plasmid provides a valuable resource for the generation of stably maintained shuttle and expression vectors or L. plantarum and possibly also for other LAB. http://dx.doi.org/10.1016/j.nbt.2012.08.600 Poster 5.0.161 Development of xanthophyll producing Mucor circinelloides strains Tamás Papp∗ , Árpád Csernetics, Gábor Nagy, Eszter Tóth, Csaba Vágvölgyi Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Közép fasor 52, H-6726 Szeged, Hungary Mucor circinelloides is a -carotene producing fungus. In the present study carotenoid biosynthesis of M. circinelloides was modified to construct strains able to synthesize different oxygenated and hydroxylated -carotene derivatives, such as canthaxanthin, astaxanthin, zeaxanthin and -cryptoxanthin, in considerable amount. Expression vectors based on bacterial -carotene ketolase and hydroxydase genes and the crtS gene of the astaxanthin producing basidiomycete yeast, Xanthophyllomyces dendrorhous were constructed. The genes were transformed in the recipient Mucor strain both on different autonomously replicating plasmids and
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in integrative transformation systems. In the latter case different transformation strategies, such as usage of linear DNA fragments in PEG-mediated protoplast transformation, REMI or Agrobacteriummediated transformation (AMT) were tested to obtain stable integrative transformants. Integration of the exogenous genes into the Mucor genome and the fate of the autoreplicative plasmids were analysed by Southern hybridization and inverse-PCR technique, while the copy number of the crtW gene in the different transformants was analysed with qPCR. All tested methods, except AMT, generated stable transformants. Astaxanthin production remained low in the transformants, but several strains producing canthaxanthin as the main carotenoid could be created. In these strains, canthaxanthin content achieved was comparable with those of certain wild-type xanthophyll-producing microbes. Conditions of fermentation, such as the effect of several carbon sources, chemical additives or the cultivation temperature on the carotenoid composition were also tested. In case of some transformants, special culture conditions were found, where the -carotene-canthaxanthin conversion was nearly complete. This work was supported by a KTIA-OTKA grant (OTKA A 081-2009-0049). http://dx.doi.org/10.1016/j.nbt.2012.08.601 Poster 5.0.162 Modification of the acetate-mevalonate pathway in the beta-carotene producing fungus, Mucor circinelloides Tamás Papp∗ , Árpád Csernetics, Gábor Nagy, Anita Farkas, Csaba Vágvölgyi Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary Carotenoids have several beneficial effects on the human health. Microbial sources may offer economic and environmental-friendly processes to satisfy the increasing carotenoid demand of the food, feed, pharmaceutical and cosmetic industries. Mucor circinelloides is a -carotene accumulating filamentous fungus. The availability of efficient transformation systems and the capacity to express exogenous genes make this fungus an attractive object of biotechnological developments. Our aim is to investigate the biological requirements of the application of M. circinelloides as a carotenoid producer and construct strains and methods serving as a basis for further applied research. Carotenoids are isoprenoid compounds. In fungi, their biosynthesis branches from the general acetate-mevalonate pathway, of which HMG-CoA reductase is a key enzyme catalysing the formation of mevalonate. In this study, expression of the three HMG-CoA reductase genes (hmgR1, hmgR2 and hmgR3) and their effects on the carotenoid content were examined. Transcription levels during the life cycle of the fungus and under various cultivation conditions were analysed by quantitative real-time PCR. Results suggest that the three genes have different functions. HmgR1 showed a constitutively low transcription in these studies, while hmgR2 and hmgR3 seems to be involved in the general metabolism and the mycelial development, respectively. The genes were also used in transformation experiments to over-express them by elevating their copy numbers in the recipient strains. Gene dose effect led to increased carotene content in
Keywords: First integral; Estimation; Batch alcohol fermentation; Immobilized cells http://dx.doi.org/10.1016/j.nbt.2012.08.599 Poster 5.0.160 Complete DNA sequence and analysis pCD033, a plasmid derived from a Lactobacillus plantarum silage strain S. Heinl∗ , K. Spath, S. Heiss, R. Grabherr CD-Laboratory for Genetically Engineered Lactic Acid Bacteria, Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 11, 1190 Vienna, Austria Lactobacillus plantarum belongs to the heterogeneous group of lactic acid bacteria (LAB). The species is frequently found in fermented food and feed products as well as in degrading plant matter. L. plantarum CD033 was isolated from a stable grass silage process in Austria. The strain was found to harbour one circular plasmid which was isolated by a modified miniprep protocol, subcloned and sequenced by Sanger technology. The 7.9 kb plasmid was designated pCD033. The plasmid revealed a very low GC-content of 36% and was predicted to contain 9 coding sequences (CDS). One CDS encodes a putative replication protein which shares 57% similarity with the RepB protein of the lactococcal plasmid pUCL22, which was shown to replicates via theta mode. This gene is flanked by a 1.7 kb untranslated region which is supposed to be involved in plasmid replication and shows similarity to an untranslated region of plasmid pLP9000 from L. plantarum strain AS1.2986. Furthermore the plasmid encodes a putative transposase, a putative tyrosine recombinase, a DGQHR domain protein, a putative pemI/pemK like plasmid addiction system as well as three proteins of unknown function. The plasmid provides a valuable resource for the generation of stably maintained shuttle and expression vectors or L. plantarum and possibly also for other LAB. http://dx.doi.org/10.1016/j.nbt.2012.08.600 Poster 5.0.161 Development of xanthophyll producing Mucor circinelloides strains Tamás Papp∗ , Árpád Csernetics, Gábor Nagy, Eszter Tóth, Csaba Vágvölgyi Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Közép fasor 52, H-6726 Szeged, Hungary Mucor circinelloides is a -carotene producing fungus. In the present study carotenoid biosynthesis of M. circinelloides was modified to construct strains able to synthesize different oxygenated and hydroxylated -carotene derivatives, such as canthaxanthin, astaxanthin, zeaxanthin and -cryptoxanthin, in considerable amount. Expression vectors based on bacterial -carotene ketolase and hydroxydase genes and the crtS gene of the astaxanthin producing basidiomycete yeast, Xanthophyllomyces dendrorhous were constructed. The genes were transformed in the recipient Mucor strain both on different autonomously replicating plasmids and
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in integrative transformation systems. In the latter case different transformation strategies, such as usage of linear DNA fragments in PEG-mediated protoplast transformation, REMI or Agrobacteriummediated transformation (AMT) were tested to obtain stable integrative transformants. Integration of the exogenous genes into the Mucor genome and the fate of the autoreplicative plasmids were analysed by Southern hybridization and inverse-PCR technique, while the copy number of the crtW gene in the different transformants was analysed with qPCR. All tested methods, except AMT, generated stable transformants. Astaxanthin production remained low in the transformants, but several strains producing canthaxanthin as the main carotenoid could be created. In these strains, canthaxanthin content achieved was comparable with those of certain wild-type xanthophyll-producing microbes. Conditions of fermentation, such as the effect of several carbon sources, chemical additives or the cultivation temperature on the carotenoid composition were also tested. In case of some transformants, special culture conditions were found, where the -carotene-canthaxanthin conversion was nearly complete. This work was supported by a KTIA-OTKA grant (OTKA A 081-2009-0049). http://dx.doi.org/10.1016/j.nbt.2012.08.601 Poster 5.0.162 Modification of the acetate-mevalonate pathway in the beta-carotene producing fungus, Mucor circinelloides Tamás Papp∗ , Árpád Csernetics, Gábor Nagy, Anita Farkas, Csaba Vágvölgyi Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary Carotenoids have several beneficial effects on the human health. Microbial sources may offer economic and environmental-friendly processes to satisfy the increasing carotenoid demand of the food, feed, pharmaceutical and cosmetic industries. Mucor circinelloides is a -carotene accumulating filamentous fungus. The availability of efficient transformation systems and the capacity to express exogenous genes make this fungus an attractive object of biotechnological developments. Our aim is to investigate the biological requirements of the application of M. circinelloides as a carotenoid producer and construct strains and methods serving as a basis for further applied research. Carotenoids are isoprenoid compounds. In fungi, their biosynthesis branches from the general acetate-mevalonate pathway, of which HMG-CoA reductase is a key enzyme catalysing the formation of mevalonate. In this study, expression of the three HMG-CoA reductase genes (hmgR1, hmgR2 and hmgR3) and their effects on the carotenoid content were examined. Transcription levels during the life cycle of the fungus and under various cultivation conditions were analysed by quantitative real-time PCR. Results suggest that the three genes have different functions. HmgR1 showed a constitutively low transcription in these studies, while hmgR2 and hmgR3 seems to be involved in the general metabolism and the mycelial development, respectively. The genes were also used in transformation experiments to over-express them by elevating their copy numbers in the recipient strains. Gene dose effect led to increased carotene content in