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

Abstract

Background: Latex 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.

Results: The 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.

Conclusions: We 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.

Keywords: ATP citrate lyase; Acetoacetyl-CoA thiolase; Latex; Mevalonate pathway; Natural rubber; Sterols; Taraxacum; Triterpenes.

Fig. 1

Proposed biosynthesis of isoprenoid products via the MVA pathway with upstream reactions in T. brevicorniculatum latex. Enzymes or enzyme complexes are shown in blue and multiple/dashed arrows indicate multiple enzymatic steps. ACL, ATP citrate lyase; AACT, acetoacetyl-CoA thiolase; DMAPP, dimethylallyl pyrophosphate; FPP, farnesyl diphosphate; HMGR, 3-hydroxy-methyl-glutaryl-CoA reductase; IPP, isopentenyl pyrophosphate; MVA, mevalonate; SQE, squalene epoxidase

Fig. 2

Spatial and temporal ACLA/B and AACT mRNA expression profile in wild-type T. brevicorniculatum plants determined by qRT-PCR. The corresponding mRNA levels were normalized against the constitutive genes elongation factor 1 α (TbEF1α), glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH) and ribosomal protein L27 (TbRP) from T. brevicorniculatum. Bars represent standard errors of three independent wild-type plants per line. Normal distribution at p < 0.05 was assessed using the Kolmogorov-Smirnov test. Different letters indicate a significant difference (ANOVA with Tukey’s honest significant difference test (p < 0.01)). a TbACLA1 and TbACLA2 b TbACLB1 and TbACLB2 and c TbAACT1 and TbAACT2 mRNA levels in latex, roots, peduncles, leaves and flowers of 10-week-old wild-type T. brevicorniculatum plants. d TbACLA1 and TbACLA2, e TbACLB1 and TbACLB2 and f TbAACT1 and TbAACT2 mRNA levels in seedlings and root material of wild-type T. brevicorniculatum plants ranging in age from 4 to 32 weeks. Plants flowered in June

Fig. 3

Expression of A. thaliana ACL, AACT and HMGR in transgenic T. brevicorniculatum plants. a T-DNA constructs for the overexpression of AtACL(AB), AtAACT2 and Athmgrc1(S408A) in T. brevicorniculatum. NPTII, kanamycin resistance gene; BAR, bialaphos resistance gene; pMS, mannopine synthase promoter; pREF, rubber elongation factor promoter; LB, left border; RB, right border. b Protein extracts from the latex of 8-week-old transgenic and wild-type (Wt) plants analyzed by SDS-PAGE and western blot (upper part) with antibodies against the AtACLB subunit (α-ACL), AtAACT2 (α-AACT) and HMGR (α-HMGR), or Ponceau S staining after protein transfer (lower part). Numbers on the left refer to molecular weight markers. c Relative ACL, AACT and HMGR enzymatic activity in the latex of 8-week-old transgenic and wild-type plants. The values of the wild-type plants were set to 1. Bars represent standard errors of three independent transgenic plants per line. Normal distribution at p < 0.05 was assessed using the Kolmogorov-Smirnov test. Different letters indicate a significant difference (ANOVA with Tukey’s honest significant difference test (p < 0.01))

Fig. 4

Influence of AtACL(AB), AtAACT2 and Athmgrc1(S408A) overexpression in dandelion latex on isoprenoid end products in 12-week-old T. brevicorniculatum roots. a Quantitative analysis of triterpene precursors (squalene and 2–3-oxidosqualene), sterols (campesterol, stigmasterol and sitosterol) and pentacyclic triterpenes (taraxasterol, α-amyrin, β-amyrin and lupeol) by GC/MS in freeze-dried roots of transgenic and wild-type (Wt) T. brevicorniculatum plants. Bars represent standard errors of three independent transgenic plants per line. Normal distribution at p < 0.05 was assessed using the Kolmogorov-Smirnov test. Asterisks represent significant differences relative to wild-type (ANOVA with Tukey’s honest significant difference test (p < 0.01)). b Quantitative analysis of cis-1,4-isoprene by ¹H–NMR in freeze-dried roots of transgenic and wild-type T. brevicorniculatum plants. Bars represent standard errors of three independent transgenic plants per line. Normal distribution at p < 0.05 was assessed using the Kolmogorov-Smirnov test. Asterisks represent significant differences relative to wild-type (ANOVA with Tukey’s honest significant difference test (p < 0.01)). c Dolichol composition of freeze-dried root material determined by LC/MS. A mixture of dolichols (13–21 isoprene units) was used as a standard. One representative result for each transgenic line and a wild-type control is shown