## 3 Results and Analysis
### Transcriptome-Based Analysis of the Key Regulatory Factors of Pentaleneene Synthesis Efficiency  
To investigate the potential causes of low pentalene yield, genome-wide transcriptomic profiles of the recombinant strain _VN-MVA_ (harboring the integrated MVA pathway and exhibiting pentalene production capacity) were compared with those of the wild-type control strain _VN_. Transcriptome analysis identified 1622 DEGs, including 752 up-regulated and 870 down-regulated genes (Figure 3B), following plasmid introduction. Functional expression of the exogenous MVA pathway in the engineered strain was first evaluated. DEGs were annotated and mapped to the terpenoid backbone biosynthesis pathway using KEGG enrichment analysis. As illustrated in Figure 4A, both the endogenous MEP pathway and the exogenous MVA pathway for pentalene biosynthesis were examined in _VN-MVA_. Genes associated with the endogenous MEP pathway did not exhibit overall transcriptional activation. Notably, _DXS_ and _GCPE_ were significantly down-regulated, with log2(FC) of \(-0.96\) and \(-0.51\), respectively (\(p<0.05\)). In contrast, the nine genes (_IDI_, _HMGS_, _HMGR_, _FPPS_, _MK_, _PMK_, _PMD_, _ATOB_ and _PENTS_) introduced via the exogenous MVA pathway were significantly up-regulated (Figure 4B), indicating successful transcriptional activation of the heterologous pathway and compatibility with the _V. natrigens_ expression system. The results indicated that the endogenous MEP pathway in the engineered strain did not contribute to the synthesis of terpenoid precursors. Instead, terpenoid precursor supply was established exclusively via the exogenous MVA pathway, enabling pentalene biosynthesis. The lack of endogenous pathway participation may represent a key factor contributing to the relatively low pentalene yield in the engineered _V. natrigens_[29; 30]. Figure 2: The optimization of pentalene production by _V. natrigens_ was conducted. (**A**) Comparison of the yield of pentalene synthesized through different culture media. (**B**) Comparison of the production yields of pentalene synthesized under different induction temperatures. (**C**) Comparison of the yield of pentalene synthesized under different concentrations of inducer. (**D**) Comparison of pentalene yields before and after the increase in the copy number of the _pents_ gene. Note: Significance (\(p\)-value) was assessed using a two-tailed \(t\)-test (** \(p<0.01\); *** \(p<0.001\), *** \(p<0.0001\)), n = 3. ns, statistically non-significant. DEGs were further classified and enriched into the top 20 KEGG pathways (Figure 5). Up-regulated genes were primarily enriched in the terpenoid backbone biosynthesis and ribosome pathways, while down-regulated genes were predominantly associated with oxidative phosphorylation, pentose phosphate pathway, and thiamine metabolism related to cofactor and energy metabolism. These findings suggest that introduction of the exoge  
Figure 4: (**A**) Synthesis of pentalene within _V. natriegens_ via the natural MEP pathway with the heterologous MVA pathway, followed by two-step transformation with FPPS and PENTS. Red genes indicate significantly up-regulated expression. (**B**) Statistical graph of gene expression of heterologous MVA pathway genes versus endogenous MEP pathway genes. Note: Significance (_p_-value) was assessed using a two-tailed _t_-test (*_p_\(<\) 0.05; **_p_\(<\) 0.01; ***_p_\(<\) 0.001), n.s., statistically non-significant. Figure 3: (**A**) Overall differential PCA plot. (**B**) MA plot of differentially expressed genes after plasmid-induced expression, with red dots indicating up-regulated genes, dark green dots indicating down-regulated genes, and blue dots indicating non-differentially expressed genes. nous MVA pathway for pentalene biosynthesis may disrupt cellular metabolism, growth, and energy balance. Within the oxidative phosphorylation pathway, a greater proportion of genes were down-regulated (up/down ratio = 6:17).