Metabolic engineering of dhurrin in transgenic Arabidopsis plants with marginal inadvertent effects on the metabolome and transcriptome

Abstract

Focused and nontargeted approaches were used to assess the impact associated with introduction of new high-flux pathways in Arabidopsis thaliana by genetic engineering. Transgenic A. thaliana plants expressing the entire biosynthetic pathway for the tyrosine-derived cyanogenic glucoside dhurrin as accomplished by insertion of CYP79A1, CYP71E1, and UGT85B1 from Sorghum bicolor were shown to accumulate 4% dry-weight dhurrin with marginal inadvertent effects on plant morphology, free amino acid pools, transcriptome, and metabolome. In a similar manner, plants expressing only CYP79A1 accumulated 3% dry weight of the tyrosine-derived glucosinolate, p-hydroxybenzylglucosinolate with no morphological pleitropic effects. In contrast, insertion of CYP79A1 plus CYP71E1 resulted in stunted plants, transcriptome alterations, accumulation of numerous glucosides derived from detoxification of intermediates in the dhurrin pathway, and in loss of the brassicaceae-specific UV protectants sinapoyl glucose and sinapoyl malate and kaempferol glucosides. The accumulation of glucosides in the plants expressing CYP79A1 and CYP71E1 was not accompanied by induction of glycosyltransferases, demonstrating that plants are constantly prepared to detoxify xenobiotics. The pleiotrophic effects observed in plants expressing sorghum CYP79A1 and CYP71E1 were complemented by retransformation with S. bicolor UGT85B. These results demonstrate that insertion of high-flux pathways directing synthesis and intracellular storage of high amounts of a cyanogenic glucoside or a glucosinolate is achievable in transgenic A. thaliana plants with marginal inadvertent effects on the transcriptome and metabolome.DNA microarrays ͉ metabolic engineering ͉ metabolite profiling ͉ channeling ͉ substantial equivalence M etabolic engineering offers the possibility to design plants that produce desired compounds (1-3). Knowledge of the impact of insertion of metabolic pathways on preexisting metabolic pathways is typically scarce, and is assessed by targeted approaches, but not by more global and unbiased approaches. We have previously reported transfer of the entire pathway for synthesis of the tyrosine-derived cyanogenic glucoside dhurrin from Sorghum bicolor to Arabidopsis thaliana by coexpression of three S. bicolor cDNAs: two multifunctional cytochromes P450, CYP79A1 and CYP71E1, and a family 1 glucosyltransferase, UGT85B1 (Fig. 1). The transgenic A. thaliana plants accumulated up 4% of plant dry matter of dhurrin (4). Transgenic A. thaliana plants in which only CYP79A1 was introduced accumulated up to 3% dry matter of the tyrosine derived p-hydroxybenzylglucosinolate, a glucosinolate that does not occur naturally in A. thaliana (5). The accumulation of tyrosine derived p-hydroxybenzylglucosinolate in A. thaliana is the result of metabolic crosstalk, where the p-hydroxyphenylacetaldoxime produced by CYP79A1 is efficiently used by the post-oximemetabolizing enzymes CYP83B1 or CYP83A1 in the endogenous glucosinolate biosynthetic pathway (5, 6) (Fig. 1).In this study,...

Xenobiotic response in Drosophila melanogaster: Sex dependence of P450 and GST gene induction

“…Thus, "detox chips" probably should become a benchmark in the routine diagnosis of transcriptional changes in the detoxifi cation pathways of any organism with a fully sequenced genome. Such thematic arrays have already proven their utility in vertebrate toxicology (Gerhold et al, 2001) and plant physiology Kristensen et al, 2005).…”

Nutritionally Enhanced Staple Food Crops

“…However, use of transgene still has some practical limitations, for example, mechanism and pattern of gene integration, dosage effect due to variable copy insertion, interaction between transgenes, rearrangements and silencing, promoter choice as a function of gene number (i.e., one promoter vs. more promoters when integrating multiple genes) as repetitive promoters may in some cases have negative impact on transgene stability and expression, or proper coordination of all enzymes involved in the metabolic pathway. Transfer of an incomplete pathway induces significant changes in plant morphology, variable expression of transgene effects in different generation and some lines with more expression than others in later generations etc (Kristensen et al 2005;Naqvi et al 2009a, b;Dietz-Pfeilstetter 2010;Peremarti et al 2010).…”

Exogenous glucosinolate produced by Arabidopsis thaliana has an impact on microbes in the rhizosphere and plant roots

“…We focused on root compartment, as studies on glucosinolate content of CYP79A1 and wild-type plants concerned leaves (Bak et al, 1999;Kristensen et al, 2005). Glucosinolates content of six replicates of seedling root tissues at post-germination stage reveal a different profile for CYP79A1 plants.…”

“…We chose a transgenic A. thaliana plant in which CYP79A1 gene from sorghum (Sorghum bicolor) was introduced, which led to the accumulation of up to 3% dry matter of p-hydroxybenzylglucosinolate, an aliphatic glucosinolate derived from tyrosine (Bak et al, 1999;Kristensen et al, 2005). We report that the aliphatic hydroxybenzylglucosinolate is only produced in transgenic A. thaliana plant roots and that the modifications of glucosinolate profiles by introduction of CYP79A1 gene leads to specific changes in the active microbial community on the roots but also in the rhizosphere of A. thaliana growing in natural soil.…”