Characterization of rubber particles and rubber chain elongation in Taraxacum koksaghyz

<p>Abstract</p> <p>Background</p> <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 <it>Hevea brasiliensis</it>, <it>Ficus spec. </it>and the desert shrub <it>Parthenium argentatum</it>, there have been no <it>in planta </it>functional studies, e.g. by RNA interference, due to the absence of efficient and reproducible protocols for genetic engineering. In contrast, the Russian dandelion <it>Taraxacum koksaghyz</it>, 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 <it>T. koksaghyz</it>.</p> <p>Results</p> <p>We established a method for the purification of rubber particles - the active sites of rubber biosynthesis - from <it>T. koksaghyz </it>latex. Photon correlation spectroscopy and transmission electron microscopy revealed an average particle size of 320 nm, and ¹³C nuclear magnetic resonance (NMR) spectroscopy confirmed that isolated rubber particles contain poly(<it>cis</it>-1,4-isoprene) with a purity >95%. Size exclusion chromatography indicated that the weight average molecular mass (<inline-formula><graphic file="1471-2091-11-11-i1.gif"/></inline-formula>w) of <it>T. koksaghyz </it>natural rubber is 4,000-5,000 kDa. Rubber particles showed rubber transferase activity of 0.2 pmol min^-1mg^-1. <it>Ex vivo </it>rubber biosynthesis experiments resulted in a skewed unimodal distribution of [1-¹⁴C]isopentenyl pyrophosphate (IPP) incorporation at a <inline-formula><graphic file="1471-2091-11-11-i1.gif"/></inline-formula>w of 2,500 kDa. Characterization of recently isolated <it>cis</it>-prenyltransferases (CPTs) from <it>T. koksaghyz </it>revealed that these enzymes are associated with rubber particles and are able to produce long-chain polyprenols in yeast.</p> <p>Conclusions</p> <p><it>T. koksaghyz </it>rubber particles are similar to those described for <it>H. brasiliensis</it>. They contain very pure, high molecular mass poly(<it>cis</it>-1,4-isoprene) and the chain elongation process can be studied <it>ex vivo</it>. Because of their localization on rubber particles and their activity in yeast, we propose that the recently described <it>T. koksaghyz </it>CPTs are the major rubber chain elongating enzymes in this species. <it>T. koksaghyz </it>is amenable to genetic analysis and modification, and therefore could be used as a model species for the investigation and comparison of rubber biosynthesis.</p

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