## 3 Results and Analysis
### Optimization of Culture Conditions and Downstream Modules to Enhance Pentalenene Production  
The initial yield of pentalenene was limited to 0.75 mg/L when using plasmid pAC-6409-oriT-pents [16]. To enhance production, a series of optimization steps were performed, including adjustment of the medium composition, induction temperature, and IPTG concentration.  
Medium components such as carbon sources, nitrogen sources, and other nutrients play a critical role in regulating the production and expression of microbial secondary metabolites [23]. To improve pentalenene yield in _V. natriegens_, suitable fermentation media were screened for nutrient composition. As shown in Figure 2A, pentalenene production in the PPB medium reached 23.05 mg/L, representing a 30.7-fold increase compared to LBv2 and 2216E media, both yielding 0.75 mg/L. The enhanced performance of the PPB medium may be attributed to the inclusion of beef extract as a nitrogen source, which provides a higher nitrogen content than LBv2 and 2216E. This likely results in a carbon-to-nitrogen ratio in PPB that more closely approximates the optimal threshold for pentalenene synthesis [24]. In addition to nitrogen, beef extract supplies peptides, vitamins, and heme precursors that enhance the expression and enzymatic activity of the terpenoid biosynthetic pathway. Moreover, the buffering capacity provided by K\({}_{2}\)HPO\({}_{4}\) and citric acid in the PPB medium stabilizes the pH during fermentation. A stable pH environment favors the activity of secondary metabolic enzymes and supports sustained bacterial growth [25], further promoting the formation of pentalenene.  
Temperature strongly influences enzyme activity in microorganisms, thereby affecting biosynthetic capacity. As shown in Figure 2B, pentalenene production increased with rising induction temperature, reaching a peak at 30 \({}^{\circ}\)C (23.05 mg/L), and subsequently declined at higher temperatures. The observed trend may reflect the temperature sensitivity of key enzymes within the metabolic network [26; 27]. At lower temperatures, limited enzyme activity reduces the overall metabolic rate [28], constraining the supply of essential precursors such as acetyl-CoA and NADPH. As temperature increases, reaction rates accelerate, enhancing precursor availability and promoting pentalenene synthesis. However, excessive temperatures may lead to the partial inactivation of critical enzymes, resulting in metabolic bottlenecks restricting further production. A moderate temperature, such as 30 \({}^{\circ}\)C, enables more balanced metabolic activity and supports optimal terpenoid biosynthesis.  
Based on the identified optimal medium and temperature, the IPTG concentration was further optimized. Concentrations of 0.1, 0.3, 0.5, 0.8 and 1 mM were selected for induction. As shown in Figure 2C, pentalenene production reached a maximum of 25.3 mg/L at an IPTG concentration of 0.1 mM.  
Further improvement was achieved by increasing the copy number of the _PENTS_ gene in the downstream module. A dual-plasmid system was constructed, with the MVA pathway encoded on the upstream module. Under optimized fermentation conditions, strain _VN-1_ harboring the dual-plasmid system produced 30.8 mg/L of pentalenene. Upon amplification of the _PENTS_ gene copy number, the engineered strain _VN-2_ exhibited a marked increase in yield, reaching 39.4 mg/L (Figure 2D). The results indicate that enhancing the dosage of key downstream genes constitutes an effective strategy for boosting pentalenene biosynthesis in _V. natriegens_.