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Team:TU Munich/Project/Limonene
From 2012.igem.org
Contents |
Limonene
Background and principles
Limonene
Limonene is a cyclic terpene and a major constituent of several citrus oils (orange, lemon, mandarin, lime and grapefruit). It is a chiral molecule; citrus fruits contain the (R)-enantiomer. The (R)-enantiomer smells like oranges, while the (S)-enantionmer has a piney, turpentine-like odor. D-Limonene is used as a component of flavorings and fragrances. It has been shown to inhibit rat mammary and other tumor development (Tsuda et al. 2004). Being an excellent solvent of cholesterol, d-limonene also has been used clinically to dissolve cholesterol-containing gallstones. Because of its gastric acid neutralizing effect and its support of normal peristalsis, it has also been used for relief of heartburn (Sun 2007).
Biosynthesis
Limonene is formed from geranyl pyrophosphat by limonene synthase. (R)-limonene synthase 1 consists of 606 aminoacids (sequence shown below, EC=4.2.3.20). (R)-limonene synthase needs magnesium or manganese as cofactor and catalyzes the following reaction: Geranyl pyrophosphate = (+)-(4R)-limonene + diphosphate.
Idea
Producing the flavoring substance limonene in our beer might result in a fresh, lemon-like taste on the one hand. On the other hand, we might have health beneficial effects like preventive activity against cancer, dissolution of gallstones and relief of heartburn.
Limonene can be produced by (R)-limonene synthase.
General remarks and issues
We should ask professor Schwab whether he has suitable plasmids for us or not.
Biobricks and sequences
File:SequenceLimoneneSynthase.jpg
Citrus limon (+)-limonene synthase 1 mRNA, complete cds
1 ggccaagtat aactgtaagc tagcttacac tacatctgta tatccaatgt cttcttgcat
61 taatccctca accttggtta cctctgtaaa tgctttcaaa tgtcttcctc ttgcaacaaa
121 taaagcagcc atcagaatca tggccaaata taagccagtc caatgcctta tcagcgccaa
181 atatgataat ttgacagttg ataggagatc agcaaactac caaccttcaa tttgggacca
241 cgattttttg cagtcattga atagcaacta tacggatgaa gcatacaaaa gacgagcaga
301 agagctgagg ggaaaagtga agatagcgat taaggatgta atcgagcctc tggatcagtt
361 ggagctgatt gataacttgc aaagacttgg attggctcat cgttttgaga ctgagattag
421 gaacatattg aataatatct acaacaataa taaagattat aattggagaa aagaaaatct
481 gtatgcaacc tcccttgaat tcagactact tagacaacat ggctatcctg tttctcaaga
541 ggttttcaat ggttttaaag acgaccaggg aggcttcatt tgtgatgatt tcaagggaat
601 actgagcttg catgaagctt cgtattacag cttagaagga gaaagcatca tggaggaggc
661 ctggcaattt actagtaaac atcttaaaga agtgatgatc agcaagaaca tggaagagga
721 tgtatttgta gcagaacaag cgaagcgtgc actggagctc cctctgcatt ggaaagtgcc
781 aatgttagag gcaaggtggt tcatacacat ttatgagaga agagaggaca agaaccacct
841 tttacttgag ctcgctaaga tggagtttaa cactttgcag gcaatttacc aggaagaact
901 aaaagaaatt tcagggtggt ggaaggatac aggtcttgga gagaaattga gctttgcgag
961 gaacaggttg gtagcgtcct tcttatggag catggggatc gcgtttgagc ctcaattcgc
1021 ctactgcagg agagtgctca caatctcgat agccctaatt acagtgattg atgacattta
1081 tgatgtctat ggaacattgg atgaacttga gatattcact gatgctgttg agaggtggga
1141 catcaattat gctttgaagc accttccggg ctatatgaaa atgtgttttc ttgcgcttta
1201 caactttgtt aatgaatttg cttattacgt tctcaaacaa caggattttg atttgcttct
1261 gagcataaaa aatgcatggc ttggcttaat acaagcctac ttggtggagg cgaaatggta
1321 ccatagcaag tacacaccga aactggaaga atacttggaa aatggattgg tatcaataac
1381 gggcccttta attataacga tttcatatct ttctggtaca aatccaatca ttaagaagga
1441 actggaattt ctagaaagta atccagatat agttcactgg tcatccaaga ttttccgtct
1501 gcaagatgat ttgggaactt catcggacga gatacagaga ggggatgttc cgaaatcaat
1561 ccagtgttac atgcatgaaa ctggtgcctc agaggaagtt gctcgtcaac acatcaagga
1621 tatgatgaga cagatgtgga agaaggtgaa tgcatacaca gccgataaag actctccctt
1681 gactggaaca actactgagt tcctcttgaa tcttgtgaga atgtcccatt ttatgtatct
1741 acatggagat gggcatggtg ttcaaaacca agagactatc gatgtcggtt ttacattgct
1801 ttttcagccc attcccttgg aggacaaaca catggctttc acagcatctc ctggcaccaa
1861 aggctgatga tgaattataa tgcatgatgc gttgcgaatt cccagagagt gcagtttcag
1921 ttgatgttgg cctccacttt tctttcttct gagggatctc ttttcgataa taaaataaat
1981 tccctcattc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
2041 aaa
| Name | Length | RFC10 | RFC25 | Codon Usage | NCBI |
| (+)-Limonene Synthase 1 | 2043bp | 2x EcoRI (497-503/1896-1902) 1x SpeI (671-677) 1x PstI(1024-1030) 1x XbaI (1450-1456) | ok after RFC10 | 1 AS < 10% | AF514287.1 |
Lavandula limonene synthase, plasmid provided by Prof. Schwab
GAAACCCGACGCTCCGGAAACTACAACCCTACCGCTTGGGATTT CAACTACATCCAATCCCTCGACAATCAGTATAAGAAAGAGAGGTACTCGA CAAGACACGCTGAGCTGACTGTGCAAGTGAAGAAGCTGCTGGAGGAAGAA ATGGAAGCGGTTCAAAAGTTGGAATTGATTGAGGATTTGAAGAACCTGGG AATATCTTACCCATTTAAGGACAATATCCAACAGATTTTAAATCAAATAT ATAATGAGCACAAATGTTGCCACAACAGTGAAGTGGAAGAAAAGGATTTG TATTTCACGGCTCTTCGATTCCGACTCCTTAGACAACAGGGTTTTGAAGT CTCTCAAGAAGTATTTGATCATTTCAAGAACGAGAAGGGTACAGATTTCA AGCCAAACCTTGCTGACGATACTAAAGGACTATTGCAACTTTACGAAGCA TCTTTCCTATTAAGAGAAGCTGAAGATACACTTGAGTTAGCTCGACAATT TTCAACCAAATTACTGCAGAAAAAAGTCGATGAGAATGGTGATGATAAAA TAGAGGATAATCTATTATTATGGATTCGCCGTTCTTTGGAGCTCCCGCTT CATTGGAGGGTGCAAAGGCTAGAAGCAAGAGGGTTCTTGGATGCTTACGT TAGAAGGCCCGACATGAATCCAATTGTTTTTGAGCTCGCCAAACTCGACT TCAATATTACCCAAGCAACACAACAAGAAGAACTGAAAGATCTCTCGAGG TGGTGGAATAGTACAGGCCTTGCCGAAAAACTCCCATTCGCGAGGGATCG GGTTGTTGAGTCCTACTTCTGGGCAATGGGAACCTTTGAGCCTCATCAAT ATGGTTATCAGAGAGAACTTGTCGCCAAGATTATTGCTCTAGCAACAGTT GTAGATGATGTTTACGATGTATATGGTACGTTAGAGGAACTGGAACTATT TACAGATGCCATTCGGAGATGGGATCGTGAATCAATCGACCAACTTCCTT ACTACATGCAGCTATGCTTTTTGACTGTCAACAACTTTGTTTTCGAGCTT GCTCATGATGTTCTTAAGGATAAGAGTTTCAACTGCTTACCACATTTACA GAGATCGTGGCTAGACTTGGCTGAAGCATATCTTGTCGAGGCTAAGTGGT ACCACAGTAGATATACACCGAGCCTCGAGGAATATCTCAATATTGCAAGA GTTTCAGTTACGTGTCCCACTATAGTTTCACAAATGTACTTTGCATTACC AATTCCGATAGAGAAACCGGTCATCGAGATCATGTACAAATACCACGACA TACTTTACCTCTCAGGAATGCTTCTAAGGCTTCCTGATGATCTAGGAACA GCATCGTTTGAGTTGAAGAGAGGTGATGTGCAAAAAGCAGTCCAGTGTTA TATGAAGGAAAGAAATGTTCCTGAAAATGAGGCACGAGAACATGTGAAGT TTCTGATTCGGGAGGCGTCGAAGCAGATAAACACCGCGATGGCGACCGAT TGTCCATTTACTGAAGATTTTGCTGTGGCTGCAGCGAATCTTGGAAGAGT GGCGAATTTTGTGTACGTCGACGGAGATGGTTTTGGCGTGCAACACTCAA AAATATATGAACAGATTGGAACCCTGATGTTCGAGCCATATCCCTAA
| Name | Length | RFC10 | RFC25 | Codon Usage | NCBI |
| lavendula angustifolia limonene synthase | 1641 bp | 2x PstI | ok after RFC10 | 4 AS < 10% | [1] |
(pET29a and pGEX4T1 plasmids contain synthase gene)
Note: The posted sequence above still contains an N- terminal signal sequence for transport in plastids (of plants), which we do not need. The plasmids from Prof. Schwab do not have these signal sequences and therfore they do not have any start codons, either. We will have to generate the ATG start- codon by adequate primer- design. (Roman)
References
Herrero O, Ramón D, Orejas M: Engineering the Saccharomyces cerevisiae isoprenoid pathway for de novo production of aromatic monoterpenes in wine. Metab Eng 2008 10:78-86.
Lücker J, Tamer M, Schwab W, Verstappen F, Van der Plas L, Bouwmeester H and Verhoeven H: Monoterpene biosynthesis in lemon (Citrus limon) cDNA isolation and functional analysis of four monoterpene synthases. Eur J Biochem 2002 269:3160-3171.
Sun J: D-Limonene: Safety and Clinical Applications. Altern Med Rev 2007 12(3):259-264.
Tsuda H, Ohshim Y, Nomoto H, Fujita K, Matsuda E, Iigo M, Takasuka N, Moore M: Cancer Prevention by Natural Compounds. Drug Metab Pharmacokin 2004 19(4):245-263.
