## 3 Results
### Heterologous Expression of STSs Encoding Genes in Yeast Produced Diverse Sesquiterpenes  
To elucidate the sesquiterpenes (STs) potentially produced by the strain CPCC 401429, we first attempted to reconstitute STs production using the _S. cerevisiae_ Bj5464 strain as a heterologous host. Two predicted STSs (DtSTS9, DtSTS10) were codon optimized, cloned into the YET vector and verified by sequencing. The expression plasmids were transformed into the BJ5464 strain [28]. For this, 100 mL of cultures were grown to mid log phase and then solid phase micro-extraction (SPME) coupled with Gas Chromatography Mass Spectrometry (GC-MS) was used to extract and analyze the volatiles in the headspace. Subsequent analyses of terpenoids produced by the resulting recombinant transformants carrying corresponding STSs expression cassette were performed. The two DtSTSs gave rise to diverse sesquiterpenes in liquid cultures of the corresponding yeast transformant (Table 3), while no sesquiterpenes was detected from the control strain BJ-CK. Terpene compounds were identified by comparing retention indices and mass spectra to National Institute of Standards and Technology (NIST) Standard Reference Database (v20, 2023-01, [[https://webbook.nist.gov/](https://webbook.nist.gov/)]([https://webbook.nist.gov/](https://webbook.nist.gov/)), accessed on 25 November 2018). Comparison of the GC-MS profiles of the yeast cultures expressing DtSTS9 and DtSTS10 against the empty vector yeast control revealed the putative sesquiterpene (_m/z_ = 204, C\({}_{15}\)H\({}_{24}\)), or sesquiterpene alcohols (_m/z_ = 240, C\({}_{15}\)H\({}_{26}\)O) as products (Table 3). Wide varieties of sesquiterpenes were detected in the yeast culture expressing DtSTS9 and DtSTS10. Besides the noticeable product cis-thujopsene (11.25% of the peak area), \(\beta\)-himachalene (4.82%) as well as small amounts of \(\alpha\)-cubebene (4.4%), acoradiene, \(\alpha\)-bulnesene, 4,10-dimethyl-7-isopropyl [4,4,0]-bicyclo-1,4-decadiene, \(\beta\)-chamigrene, thuiopsene-(I2), and widrold were also detected in the headspace of DtSTS9 cultures. In addition, _S. cerevisiae_ strain expressing DtSTS10 gave rise to \(\alpha\)-himachalene as the main product (83.59% of the peak area), together with small amounts of \(\alpha\)-bisabolol (4.62%), \(\beta\)-sesquiphellandrene (2.9%), (+)-cuparene (2.0%), \(\beta\)-himachalene, cis-thujopsene, \(\alpha\)-cedrene, acoradiene, \(\gamma\)-muvurolene and \(\delta\)-elemene. Interestingly, minor wildrol (2.14%) and bisabolol (4.62%) were detected in DtSTS10 culture based on GC-MS analysis, respectively. [PERSON] et al., characterized the structures of (+)-torreylo, \(\alpha\)-cardinol, and 1-napthalenol in genetically engineered _S. cerevisiae_ cultures derived from _L. rhinocretis_ STSs [11]. The results suggested that the sesquiterpene alcohols might be generated from sesquiterpene precursors with the aid of endogenous cytochrome P450 monooxygenase of yeast [11].  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Rt (min)** & **Peak Area (\%)** & **Name/Structure** & **Rt (min)** & **Peak Area (\%)** & **Name/Structure** \\ \hline \multirow{4}{*}{33.563} & BJ-At\_STS9 & & & BJ-At\_STS10 \\  & & & & \\  & & & 35.938 & 1.86 \\  & & \(\alpha\)-bulnesene & & cis-thujopsene \\  & & & 38.618 & 0.54 \\  & & (+)-aromadendrene & & & \\ \hline \hline \end{tabular}
\end{table}
Table 3: Heterologous production of sesquiterpenes in yeast expressing STSs.