## 3 Gene Discovery and Gene Expression Analysis in the Pre-NGS Era
### Taxol  
Discovered in 1970 from bark of _Taxus brevifolia_, taxol (Figure 1), a diterpenoid also known as paclitaxel, is the best-selling natural anticancer drug [85]. However, the low yield at which taxol can be extracted and the unpredictable availability of natural sources represent a problem for the pharmaceutical industry. Hence, endophytic fungi from _Taxus_ spp. appeared to represent a more reliable source for industrial production of this drug, considering their fast growth, low costs, independence of environmental constraints, and possible improvements through genetic manipulation. The list of endophytic fungi recovered from different hosts and their taxol-producing capacities was recently updated by [PERSON] et al. [86]. However, the exploitation of endophytic fungi for industrial production of taxol has been impaired by reduction of yields depending on the downregulation in the expression of the encoding genes which occurs after subculturing and storage. Therefore, several next generation sequencing attempts were made to enable genome mining and transcriptome analysis to identify the taxol biosynthetic pathway and its rate-limiting steps in endophytic fungi. Following genome sequencing of _Penicillium aurantiogriseum_ NRRL 62431, gene candidates potentially involved in paclitaxel biosynthesis by this strain were identified through comparison with the 13 paclitaxel biosynthetic genes known in _Taxus_[64]. A protein search performed against the genome of NRRL 62431 revealed putative homologues to seven _Taxus_ genes encoding phenylalanine aminomatuse (PAM), geranylgeranyl diphosphate synthase (GGPPS), and five taxane hydroxyles (T2 OH, T5 OH, T7 OH, T10 OH, and T13 OH). Due to the low homology between the fungal paclitaxel biosynthetic gene candidates and those encoded by its host plant, it was postulated that gene candidates for taxol biosynthesis in NRRL 62431 evolved independently, and that in this case HGT is an unlikely explanation. In 2018, the transcriptome of strain MD2 of _Cladosporiulos_ undergoing drawdown in taxol production after subculturing was investigated for the first time with an RNA-sequencing technology to explore aspects related to gene expression [65]. KEGG analysis indicated that 40 unigenes in MD2 transcriptome were related to taxol biosynthesis and were homologous to the plant genes for taxadiene synthase (TS), taxadiene 5-alpha-hydroxylase (T5\(\alpha\)H), taxane 13-alpha-hydroxylase (T13\(\alpha\)H), and 2-alpha-hydroxytaxane 2-O-benzoyltransferase (TBT). However, no homologues to _TAT_ and _T10\(\beta\)H_ were detected in the transcriptome of this strain, suggesting that, unlike _Taxus_, it might not have this branch. Thus, the existence of a partial biosynthetic pathway from GGPP to 10-deacetylbaccatin III (10-DAB) in the MD2 transcriptome was speculated. Surprisingly, homologues of genes encoding enzymes functioning in the biosynthetic steps from 10-DAB to taxol, such as C-13 phenylpropanol side chain-CoA acyltransferase (BAPT), 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT), and 3\({}^{\prime}\)-N-debenzoyltaxol N-benzoyltransferase, were not detected. It is not known whether this was due to their low sequence homology with the host-counterparts or to the lack of transcription.