Currently, heterologous production of natural products, especially terpenoids, by metabolic engineering of plants seems to reach its limit in productivity lagging far behind commercially viable level. Existing studies have shown that overexpression of hydroxymethylglutary CoA reductase (HMGR), isopentenyl diphosphate isomerase or farnesyl diphosphate synthase increases metabolic flux through mevalonate pathway and downstream thereafter thus improving terpenoids productivity. Here, we focused on the fact that acetyl-CoA is the common precursor for both fatty acid biosynthesis in plastid and terpene biosynthesis in cytosol, and the effect of increased plastidic acetyl-CoA production on the cytosolic terpene biosynthesis was examined. In tobacco leaf transient expression studies, the acetyl CoA carboxylase complex, which supplies malonyl CoA in plastid for fatty acid biosynthesis, was partially inhibited by overexpressing and incorporating the inactive form of biotin carboxyl carrier protein 1 (BCCP1). Produced terpenoids from biochemical reaction were quantitatively analyzed with selected ion monitoring mode on GC-MS (Agilent, model 5973N). Overexpression of BCCP1 showed 1.4–2.4-fold increase of sequiterpenes in cytosol; however, surprisingly overexpression of BCCP linked to truncated HMGR by a cleavable peptide 2A showed 20–40-fold increases of C15 sesquiterpenes (a-bisabolol, amorphadiene and valerenadiene) and a 6-fold increase of C30 b-amyrin in N. benthamiana transient expression system. Also, overexpression of BCCP1 accompanied 1.7-fold increase in ATP-citrate lyase transcripts, suggesting increased acetyl CoA pool in cytosol. The tHMGR-2A-BCCP1 co-expression strategy in this study could open up door towards commercial terpenoids production in plants.

Enhanced sesqui- and triterpene accumulation by co-expression of HMG-CoA reductase and biotin carboxyl carrier protein (BCCP1) in tobacco (Nicotiana benthamiana)

2019 Spring National Meeting

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