Engineering 4-hydroxyisoleucine biosynthesis by ectopic expression of bacterial IDO gene in fenugreek and tobacco

Abstract There is enough evidence that 4-hydroxyisoleucine (4-HIL), the main ingredient of fenugreek plant for the treatment of type 2 diabetes, has significant influences on the secretion of and resistance to insulin in human cells. However, its content in the leaves and seeds of fenugreek seems insufficient for industrial and pharmaceutical exploitation. In order to boost 4-HIL content in fenugreek and promote its heterologous expression in the model tobacco plant, the encoding gene of isoleucine dioxygenase (IDO), a key enzyme in the 4-HIL biosynthesis pathway, was cloned from Bacillus thuringiensis and ectopically expressed under the control of CaMV-35S promoter in fenugreek (Trigonella foenum-graecum L.) using a viral vector (pER2L5R5) and in tobacco (Nicotiana tabacum L.) via Agrobacterium tumefaciens-mediated transformation method. Transgene expression was confirmed at the mRNA level through RT-PCR and qRT-PCR, and 4-HIL content was measured by High Performance Liquid Chromatography (HPLC). The obtained results showed that the amount of 4-hydroxyisoleucine in transfected fenugreek plants was higher than that in the control plants. Also, heterologous production of 4-HIL in transgenic tobacco plants was achieved and foliar application of isoleucine on these plants could increase the amount of this valuable amino acid derivative.