A plant-specific biogenic amine, serotonin, was produced by heterologous expression of two key biosynthetic genes, tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H), in Escherichia coli. The native T5H, a cytochrome P450 enzyme, was unable to be functionally expressed in E. coli. Through a series of N-terminal deletions or additions of tagging proteins, we generated a functional T5H enzyme construct (GST∆37T5H) in which glutathione S transferase (GST) was translationally fused with the N-terminal 37 amino acid deleted T5H. Dual expression of GST∆37T5H and TDC using a pCOLADuet-1 E. coli vector produced serotonin at concentrations of approximately 24 mg l⁻¹ in the culture medium and 4 mg l⁻¹ in the cells. An optimum temperature of approximately 20 °C was required to achieve peak serotonin production in E. coli because the low induction temperature gave rise to the highest soluble expression of GST∆37T5H.

No abstract

PAL5, a tomato (Lycopersicon esculentum Mill.) plant defense gene that encodes phenylalanine ammonia-lyase, is known to respond to a variety of environmental stresses including pathogen infection and wounding. A shiva-1 gene recombinant that encodes a small synthetic antibacterial peptide under the PAL5 gene promoter was transformed into potato (Solanum tuberosum L.) and its ability to induce resistance to Erwinia carotovora was compared with a construct under the control of the constitutive and widely used cauliflower mosaic virus (CaMV) 35S promoter. The shiva-1 peptide, an analog of natural cecropin B, was shown previously to have high bactericidal activity in vitro, but when expressed in vivo under the control of the CaMV 35S promoter, the effects were very inconsistent. As observed previously, in the present studies a few transformants with the CaMV 35S promoter were highly resistant when assayed for susceptibility to soft rot disease. In marked contrast the majority of transformants with the PAL5 gene promoter were highly resistant. More-detailed analyses of the incorporated DNA indicated that most of the transformants with the CaMV 35S promoter contained multiple copies of the transforming DNA while all of the PAL5 recombinants contained single copies. The highly resistant CaMV 35S recombinant also was present as a single copy. The results indicate that, at least in this instance, a constitutive promoter may not be ideal for the effective expression of a foreign gene and suggest that multiple insertions may have negative consequences.

No abstract

The upstream regulatory region of an efficiently expressed phenylalanine ammonia‐lyase (PAL5) gene from tomato (Lycopersicon esculentum) was characterized by DNA sequencing and S1‐nuclease‐protection studies. Two transcription initiation sites giving rise to a long and a short transcript were identified, each corresponding to a different putative TATA sequence element with multiple conserved upstream regulatory motifs. When tomato plants were challenged with three different environmental stimuli, wounding, light and Verticillium infection, this gene was observed to utilize the alternate initiation sites differentially, in a tissue‐dependent and stimulus‐dependent manner. In general, basal levels of the shorter transcript were very low but preferentially and strongly stimulated by the environmental factors. In contrast, the longer transcript was less affected, essentially representing a constitutive expression of this gene. The results suggest that the multiple regulatory sites in the PAL5 gene promoter provide for both an essential constitutive expression of phenylalanine ammonia‐lyase as well as an appropriate response to different environmental stresses.