Erschienen:
Springer Science and Business Media LLC, 2010
Erschienen in:BMC Biochemistry
Sprache:
Englisch
DOI:
10.1186/1471-2091-11-11
ISSN:
1471-2091
Entstehung:
Anmerkungen:
Beschreibung:
<jats:title>Abstract</jats:title>
<jats:sec>
<jats:title>Background</jats:title>
<jats:p>Natural rubber is a biopolymer with exceptional qualities that cannot be completely replaced using synthetic alternatives. Although several key enzymes in the rubber biosynthetic pathway have been isolated, mainly from plants such as <jats:italic>Hevea brasiliensis</jats:italic>, <jats:italic>Ficus spec.</jats:italic> and the desert shrub <jats:italic>Parthenium argentatum</jats:italic>, there have been no <jats:italic>in planta</jats:italic> functional studies, e.g. by RNA interference, due to the absence of efficient and reproducible protocols for genetic engineering. In contrast, the Russian dandelion <jats:italic>Taraxacum koksaghyz</jats:italic>, which has long been considered as a potential alternative source of low-cost natural rubber, has a rapid life cycle and can be genetically transformed using a simple and reliable procedure. However, there is very little molecular data available for either the rubber polymer itself or its biosynthesis in <jats:italic>T. koksaghyz</jats:italic>.</jats:p>
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<jats:sec>
<jats:title>Results</jats:title>
<jats:p>We established a method for the purification of rubber particles - the active sites of rubber biosynthesis - from <jats:italic>T. koksaghyz</jats:italic> latex. Photon correlation spectroscopy and transmission electron microscopy revealed an average particle size of 320 nm, and <jats:sup>13</jats:sup>C nuclear magnetic resonance (NMR) spectroscopy confirmed that isolated rubber particles contain poly(<jats:italic>cis</jats:italic>-1,4-isoprene) with a purity >95%. Size exclusion chromatography indicated that the weight average molecular mass ("Equation missing"<!-- image only, no MathML or LaTex -->w) of <jats:italic>T. koksaghyz</jats:italic> natural rubber is 4,000-5,000 kDa. Rubber particles showed rubber transferase activity of 0.2 pmol min<jats:sup>-1</jats:sup> mg<jats:sup>-1</jats:sup>. <jats:italic>Ex vivo</jats:italic> rubber biosynthesis experiments resulted in a skewed unimodal distribution of [1-<jats:sup>14</jats:sup>C]isopentenyl pyrophosphate (IPP) incorporation at a "Equation missing"<!-- image only, no MathML or LaTex -->w of 2,500 kDa. Characterization of recently isolated <jats:italic>cis</jats:italic>-prenyltransferases (CPTs) from <jats:italic>T. koksaghyz</jats:italic> revealed that these enzymes are associated with rubber particles and are able to produce long-chain polyprenols in yeast.</jats:p>
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<jats:title>Conclusions</jats:title>
<jats:p>
<jats:italic>T. koksaghyz</jats:italic> rubber particles are similar to those described for <jats:italic>H. brasiliensis</jats:italic>. They contain very pure, high molecular mass poly(<jats:italic>cis</jats:italic>-1,4-isoprene) and the chain elongation process can be studied <jats:italic>ex vivo</jats:italic>. Because of their localization on rubber particles and their activity in yeast, we propose that the recently described <jats:italic>T. koksaghyz</jats:italic> CPTs are the major rubber chain elongating enzymes in this species. <jats:italic>T. koksaghyz</jats:italic> is amenable to genetic analysis and modification, and therefore could be used as a model species for the investigation and comparison of rubber biosynthesis.</jats:p>
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