## 4 Discussion  
_V. natrigens_, the fastest-growing known bacterium with a doubling time of less than 10 min, has attracted growing interest as a potential alternative to _E. coli_ as a microbial chassis [39]. As a marine bacterium, _V. natrigens_ offers several advantageous features, including high metabolic activity, rapid proliferation, broad carbon source utilization, and ease of genetic manipulation. In the present study, a _V. natrigens_-based chassis system was successfully developed for the biosynthesis of pentalenene, a sesquiterpene compound, thereby demonstrating its potential as a novel microbial host. Although the integration of heterologous MVA pathway and fermentation optimization significantly increased pentalenene production, the final production level remained lower than that achieved in the _E. coli_ system. Transcriptomic analysis identified an imbalance in the engineered strain's metabolic network as the primary factor limiting pentalenene biosynthesis. First, key genes in the endogenous MEP pathway (such as _DXS_, GCPE) were either not activated or significantly down-regulated, indicating a complete shift in terpenoid precursor supply from the endogenous route to the exogenous MVA pathway. The extensive down-regulation of oxidative phosphorylation pathway genes (such as _NDH_, _atpA_) leads to the limitation of ATP production, which directly affects the enzyme activity of the MVA pathway and the efficiency of precursor transport. Concurrently, significant down-regulation of _G6 PDH_ in the pentose phosphate pathway reduced NADPH availability. As a critical cofactor, NADPH is required for both the HMGR-catalyzed step in the MVA pathway and glutathione reductase (GSR)-mediated oxidative stress defense, leading to competitive cofactor consumption and metabolic burden. In addition, the down-regulation of genes in the thiamine synthesis pathway (such as _DXS_, _thiM_) resulted in reduced acetyl-CoA availability, further limiting the flux through the MVA pathway. The observed cascade of metabolic imbalances indicates that, although the introduction of a heterologous pathway effectively redirected metabolic flux toward target compound synthesis, it disrupted native \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Pathway** & **Gene Name** & **Annotation** & **log2 (FC)** & **Up or Down** \\ \hline Oxidative phosphorylation & _NDH_ & NADH dehydrogenase & \(-1.13\)* & Down \\  & _petB_ & cytochrome B & \(-0.74\)* & Down \\  & _petC_ & cytochrome C & \(-0.41\)* & Down \\  & _cyoB_ & cytochrome o ubiquinol oxidase subunit I & \(-1.69\)* & Down \\ Oxidative & _cyoC_ & cytochrome o ubiquinol oxidase subunit III & \(-2.27\)* & Down \\  & _cyoD_ & Cytochrome o ubiquinol oxidase & \(-2.14\)* & Down \\  & _atpA_ & ATP FOF1 synthase subunit alpha & \(-0.52\)* & Down \\  & _atpC_ & ATP synthase FOF1 subunit epsilon & \(-0.93\)* & Down \\  & _atpD_ & ATP synthase FOF1 subunit beta & \(-0.75\)* & Down \\  & _atpA_ & ATP FOF1 synthase subunit gamma & \(-0.88\)* & Down \\ \hline Glutathione metabolism & \(GSR\) & glutathione reductase & \(0.64\)* & Up \\ \hline Pentose phosphate pathway & _G6 PDH_ & glucose-6-phosphate dehydrogenase & \(-0.87\)* & Down \\ \hline Thiamine metabolism & _DXS_ & 1-deoxy-D-xylulose-5-phosphate synthase & \(-0.96\)* & Down \\  & _thiH_ & thiamine biosynthesis protein ThiH & \(-1.63\)* & Down \\  & _thiM_ & hydroxyethylthiazol kinase & \(-1.61\)* & Down \\  & _tentaA_ & hypothetical protein & \(-1.68\)* & Down \\  & _thiI_ & tRNA s(4)U8 suffurtransferase & \(-0.64\)* & Down \\  & _thiG_ & thiazole synthase & \(-0.93\)* & Down \\  & _thiK_ & thiamine kinase & \(-0.62\)* & Down \\ \hline \hline \end{tabular} Note: In the table, differentially expressed genes (DEGs) were identified using a significance threshold of \(p<0.05\), with asterisks (*) denoting statistically significant differences.  
\end{table} 
Table 1: Annotation of DEGs in oxidative phosphorylation and glutathione metabolism pathways. metabolic homeostasis and created conflicts in energy and cofactor allocation.