Plant secondary metabolites have applications for the food, biofuel, and pharmaceutical industries. Recent advances in pathway elucidation and host expression systems now allow metabolic engineering of plant metabolic pathways to produce “new-to-nature” derivatives with novel biological activities, thereby amplifying the range of industrial uses for plant metabolites. Here we use a transient expression system in the model plant Nicotiana benthamiana to reconstitute the two-step plant-derived biosynthetic pathway for auxin (indole acetic acid) to achieve accumulation up to 500 ng/g fresh mass (FM). By expressing these plant-derived enzymes in combination with either bacterial halogenases and alternative substrates, we can produce both natural and new-to-nature halogenated auxin derivatives up to 990 ng/g FM. Proteins from the auxin synthesis pathway, tryptophan aminotransferases (TARs) and flavin-dependent monooxygenases (YUCs), could be transiently expressed in combination with four separate bacterial halogenases to generate halogenated auxin derivatives. Brominated auxin derivatives could also be observed after infiltration of the transfected N. benthamiana with potassium bromide and the halogenases. Finally, the production of additional auxin derivatives could also be achieved by co-infiltration of TAR and YUC genes with various tryptophan analogues. Given the emerging importance of transient expression in N. benthamiana for industrial scale protein and product expression, this work provides insight into the capacity of N. benthamiana to interface bacterial genes and synthetic substrates to produce novel halogenated metabolites.

P. sativum seeds (cultivar Cameor) were obtained from the Germplasm Resources Unit at the John Innes Centre UK and were grown in a glasshouse at 24/16 °C day/night temperature with a 14‐h photoperiod and at 70% relative humidity. The developing seeds were collected between 7 and 21 days after floral anthesis and the RNA was extracted using a RNeasy Plant mini kit (Qiagen). RNA sequencing was performed at the Earlham Institute (UK). Briefly, Illumina barcoded TruSeq RNA library were prepared and sequenced using Illumina HiSeq 2000/2500 in High-Output mode using 100 bp paired-end reads to generate at least 100 million pairs of reads per lane. FASTQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used on the reads to assess the quality. Trinity software⁴¹ was used to assembly a de novo transcriptome from the paired-end reads with the default parameters after removal of low-quality reads and trimming. PsYUC genes were identified by blasting the AtYUC6 sequence into the assembled transcriptome and retrieving the closest homologues.

Assembled transcriptomic dataset.