## 3 Gene Discovery and Gene Expression Analysis in the Pre-NGS Era
### Resveratrol  
Resveratrol (Figure 1) is a phenolic compound known for multiple beneficial functions, including neuroprotection, without major adverse effects [87]. High quantities of this compound are required for applications in functional food [88]. At present resveratrol is mainly extracted from plant by-products, with limited availability [67; 89].  
De novo transcriptome sequencing of _Alternaria_ sp. MG1, an endophytic strain isolated from grape which is able to synthesize resveratrol from glucose, identified 20 genes for glycolysis, 10 for phenylalanine biosynthesis, and 4 genes for phenylpropanol and still-benoid biosynthesis [67]. Phenylalanine ammonia-lyase (PAL), cytochrome P-450 CYP3A, 4-coumarate coenzyme A ligase (4 CL), and chalcone synthase (CHS) were annotated for converting L-phenylalanine into resveratrol. Resveratrol synthase (also known as stilbene synthase, STS), the key enzyme catalyzing the last step of biosynthesis of this compound in plants, was not annotated; thus CHS, an isoenzyme of resveratrol synthase, was identified for its involvement in biosynthesis by this strain. The identified enzymes indicated that the reasonable biosynthesis pathway from glucose to resveratrol occurred via glycolysis, phenylalanine, and phenylpropanol biosynthesis, and stilbenoid pathways.  
Apart from producing resveratrol, _Alternaria_ sp. MG1 has been found to produce its analogues pterostilbene and piceatannol. Pterostilbene is a dimethyl ether that exhibits cancer chemopre, antioxidant, antifungal, and hypolipidemic properties [90]. Piceatannol has displayed antitumor and immunosuppressive properties, a stronger antioxidant activity, and it is considered a potential antiarrhythmic agent [91]. Whole genome sequencing of strain MG1 has outlined its high metabolic diversity and led to the discovery of 21 proteins in the phenylpropanol pathways, each encoded by more than one gene, some of which are involved in the biosynthesis of stilbenes, flavonoids, and lignins, and are indicative of the resveratrol biosynthesis pathway [68]. Particularly, the key genes encoding 4-coumarate coenzyme A ligase (4 CL) and cinnamate 4-hydroxylase (C4H) were identified in the upstream metabolic pathway. As in the previous study, STS was not annotated, while CHS could be identified. The enzymes catalyzing conversion of resveratrol into pterostilbene and piceatannol were also successfully annotated in MG1, respectively as trans-resveratrol di-O-methyltransferaseanferase (ROMT) and piceatannol synthase (which is a cytochrome P450 enzyme, CYP1B1).  
Additional enzymes could be annotated, indicating that strain MG1 may provide diverse gene resources for the production of functional compounds, especially those normally produced by plants at low levels. Those are the key enzymes involved in the flavonoid biosynthesis pathway from p-coumaric CoA to naringenin chalcone, and shiki-mate O-hydroxycinnamoyltransferase (HCT), the enzyme responsible for driving metabolic flux from p-coumaric CoA to the lignin biosynthesis pathway; conversely, ferulate-5-hydroxylase, which is involved in the lignin biosynthetic pathway in _Vitis vinifera_, was not found [68].