# Unveiling Monoterpene Biosynthesis in _Taiwania cryptorneioides_ via Functional Characterization
## 2 Results and Discussion
### In Vitro Enzymatic Assay  
To understand the biosynthetic product of newly discovered _Tc_TPSs, heterologous protein expression, purification, and in vitro reaction were used. Because of the volatile properties of monotereponoids, we used solid phase microextraction (SPME) to absorb the headspace of each reaction.  
From the GC-MS analysis results, _Tc_TPS13 and _Tc_TPS14 shared the same product profile (Figure 3, RT 12.46/12.48 min), and geraniol was detected according to the comparison of spectral libraries and authentic standards. The product of _Tc_TPS15 had different retention times (Figure 4A, RT 8.44 min), and after comparison with standards, it had identical retention times and mass spectra to linalool. _Tc_TPS16, which was phylogenetically close to _C. formosensis_\(\alpha\)-pinene synthase, shared the same metabolite profile as \(\beta\)-pinene (Figure 4B, 5.61 min). However, _Tc_TPS17 lacks a monotereponoid product, although its expression can be observed by Western blot, and it may result from abnormal protein folding, which forms a functionless protein. As described above, _Tc_TPSs 13 and 14 are geraniol synthases, _Tc_TPS15 is linalool synthase, and _Tc_TPS16 is \(\beta\)-pinene synthase.  
Figure 2: Phylogenetic analysis of selected terpene synthases. Each branch was colored according to its function and species orientation. Green: diterpene synthases; blue: tri-domain sesquiterpene from gymnosperms, deep blue: mono-/sesquietpene synthases from angiosperms; pink: sesquiterpene from gymnosperms; orange: monoterepne synthases from gymnosperms. The Cupressaceae-associated subgroups are marked with a colored background.  
The plasticity of the monoterpene synthase catalytic pocket was shaped by neighboring residues, and their location across helix D, F, G, H, J, and J/L loops (Figure 5), which can be grouped into four major plasticity regions identified via previous mutation assays [17]. For the biosynthesis of hydroxylated acrylic monoterpneoids, the intervention caused by water molecules in the reaction is the key. In limonene synthase from _Mentha spicata_, the lone side chain of M458 and H579 at region 3 and the J/K loop stabilize the cyclic intermediate, and the reaction was intervened by water once it was mutated to a less bulky amino acid, which formed hydroxylated acrylic monoterpene [15]. In _Tc_TPS13 and 15, the Val located at the M458-related location (Figure 5) indicates that a smaller amino acid could be essential to geraniol and linalool formation. However, the same Val was observed at M458 on _Tc_TPS 16 (\(\alpha\)-pinene synthase); therefore, the key residues that determine the water interruption still need to be discovered.  
Figure 4: GC-MS analysis of the enzymatic products of _Tc_TPS15 and 16. **(A)** The metabolic profile of the _Tc_TPS15 product and linalool standard; **(B)** the metabolic profile of _Tc_TPS16 and \(\beta\)-pinene standard.  
Figure 3: GC-MS analysis of the enzymatic products of _Tc_TPS13 and _Tc_TPS14 and comparison with the geraniol standard.  
On the other hand, from the comparison results of acyclic monoterpene synthase and bicyclic monoterpene synthase, we were able to identify Phe in _Tc_TPS13 and _Tc_TPS15 at the associated site of Y327 on _Tc_PinS (Figure 4), which could terminate the second cyclization of cations [21]. The conserved Tyr residues among conifer pinene synthases indicate that it is crucial to pinyl cation formation. On the other hand, some residues were characterized in determining the orientation of the pinyl cation from _Abies grandis_ pinene synthase, and C372S and F579W preferred \(\alpha\)-pinene formation instead of \(\beta\)-pinene [28]. However, the _T. cryptomerioides_ pinene synthases exhibited seemingly opposite seanario: Cys on \(\alpha\)-pinene synthase and Ser on \(\beta\)-pinene synthase, but the detailed mechanism needs to be verified in future experiments.