Isoprenoid biosynthesis in dandelion latex is enhanced by the overexpression of three key enzymes involved in the mevalonate pathway

Abstract

BackgroundLatex from the dandelion species Taraxacum brevicorniculatum contains many high-value isoprenoid end products, e.g. triterpenes and polyisoprenes such as natural rubber. The isopentenyl pyrophosphate units required as precursors for these isoprenoids are provided by the mevalonate (MVA) pathway. The key enzyme in this pathway is 3-hydroxy-methyl-glutaryl-CoA reductase (HMGR) and its activity has been thoroughly characterized in many plant species including dandelion. However, two enzymes acting upstream of HMGR have not been characterized in dandelion latex: ATP citrate lyase (ACL), which provides the acetyl-CoA utilized in the MVA pathway, and acetoacetyl-CoA thiolase (AACT), which catalyzes the first step in the pathway to produce acetoacetyl-CoA. Here we isolated ACL and AACT genes from T. brevicorniculatum latex and characterized their expression profiles. We also overexpressed the well-characterized HMGR, ACL and AACT genes from Arabidopsis thaliana in T. brevicorniculatum to determine their impact on isoprenoid end products in the latex.ResultsThe spatial and temporal expression profiles of T. brevicorniculatum ACL and AACT revealed their pivotal role in the synthesis of precursors necessary for isoprenoid biosynthesis in latex. The overexpression of A. thaliana ACL and AACT and HMGR in T. brevicorniculatum latex resulted in the accumulation of all three enzymes, increased the corresponding enzymatic activities and ultimately increased sterol levels by ~5-fold and pentacyclic triterpene and cis-1,4-isoprene levels by ~2-fold. Remarkably high levels of the triterpene precursor squalene were also detected in the triple-transgenic lines (up to 32 mg/g root dry weight) leading to the formation of numerous lipid droplets which were observed in root cross-sections.ConclusionsWe could show the effective expression of up to three transgenes in T. brevicorniculatum latex which led to increased enzymatic activity and resulted in high level squalene accumulation in the dandelion roots up to an industrially relevant amount. Our data provide insight into the regulation of the MVA pathway in dandelion latex and can be used as a basis for metabolic engineering to enhance the production of isoprenoid end products in this specialized tissue.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-017-1036-0) contains supplementary material, which is available to authorized users.

Engineering linear, branched‐chain triterpene metabolism in monocots

“…sterols or terpenes from heterologously‐expressed terpene synthases) (Chappell et al ., ; Harker et al ., ; Wu et al ., ). Similar results have been observed in other species such as Taraxacum brevicorniculatum (Pütter et al ., ) and N. benthamiana (Reed et al ., ).…”

Evidence that the <i>Arabidopsis thaliana</i> 3-hydroxy-3-methylglutaryl-CoA reductase 1 is phosphorylated at Ser577 <i>in planta</i>

“…by the mevalonate pathway such as phytosterols by removing the potential phosphorylation site to avoid negative regulation of the enzyme activity has also been an emerging strategy (Hey et al 2006;Pütter et al 2017). Although the negative regulation of HMGR through phosphorylation is likely to occur in planta, it has not been experimentally verified.…”

Identification of oxidosqualene cyclases from the medicinal legume tree Bauhinia forficata: a step toward discovering preponderant α‐amyrin‐producing activity

“…We co‐expressed BfOSC3 with AtHMGR1cd‐S577A, which lacks the inactivating phosphorylation site and has greater activity in vitro than AtHMGR1cd (Robertlee et al ., , ). The HMGR1 catalytic domain with the S577A mutation enhances the production of isoprenoids (latex) in dandelion (Pütter et al ., ). Moreover, co‐expression of the HMGR catalytic domain with triterpenoid biosynthetic genes increases triterpenoid production in an N. benthamiana transient expression system (Reed et al ., ).…”