Metabolic Engineering for the Biosynthesis of Pentalene in the Rapidly Growing Bacterium _Vibrio natriegens_  
[PERSON]  
1 College of Biological Engineering, Sichuan University of Science and Engineering, Yibin 644005, China; [EMAIL_ADDRESS] (L.H.); [EMAIL_ADDRESS] (R.L.)  
[PERSON]  
1 College of Biological Engineering, Sichuan University of Science and Engineering, Yibin 644005, China; [EMAIL_ADDRESS] (L.H.); [EMAIL_ADDRESS] (R.L.)  
[PERSON]  
2 State Key Laboratory of NIBC Protection for Civilian, Beijing 102205, China; [EMAIL_ADDRESS] (H.J.); [EMAIL_ADDRESS] (G.Y.); jiaj  
by _E. coli_ BL21-1 under identical conditions [6]. [PERSON] demonstrated the application of _V. natriegens_ in melanin production, achieving a volumetric rate of 473 mg/(L-h), one of the highest efficiencies reported to date [7].  
Pentalenene is a tricyclic sesquiterpene composed of three isoprene units. Initially isolated from various species of _Streptomyces_[8], the compound serves as a key intermediate in the biosynthesis of antibiotics such as pentalenolactone [9]. In addition to its pharmaceutical relevance, pentalenene exhibits superior fuel-related properties, including high energy density and a low freezing point [10]. The energy released from the combustion of its hydrogenation product is estimated to be comparable to that of commercial jet fuel A-1 [11], indicating its potential value as an aviation fuel feedstock.  
Isoprene biosynthesis proceeds predominantly through two distinct metabolic routes: the mevalonate (MVA) pathway, mainly present in eukaryotic organisms, and the methylerythntol phosphate (MEP) pathway, commonly found in prokaryotes [12]. In the MVA pathway, acetyl-CoA serves as the starting substrate, where two molecules condense to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), subsequently reduced to mevalonic acid by HMG-CoA reductase (HMGR). Mevalonate then undergoes sequential phosphorylation and decarboxylation to generate isopentenyl pyrophosphate (IPP), which is isomerized by IPP isomerase (IDI) to form dimethylallyl pyrophosphate (DMAPP). In contrast, the MEP pathway utilizes pyruvate and glyceraldehyde-3-phosphate (G3P) as initial substrates. Under the action of 1-deoxy-D-xylulose-5-phosphate synthase (DXS), these intermediates are converted into 1-deoxy-D-xylulose-5-phosphate (DXP), which is subsequently reduced and rearranged by 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) to form 2-C-methyl-D-erythntol-4-phosphate (MEP). Further conversion through a series of enzymes, including 2-C-methyl-D-erythntol-4-phosphate cytidylyltransferase (CMK) and 4-hydroxy-3-methylbut-2-enyl-diphosphate synthase (GCPE), leads to the production of both IPP and DMAPP. These two isoprene units constitute the universal building blocks of all terpenoids. Subsequent condensation yields farnesyl pyrophosphate (FPP), the direct precursor of sesquiterpenes, which are synthesized via specific sesquiterpene synthases.  
Compared with the MEP pathway, the MVA pathway usually has higher terpenoid synthesis efficiency [13]. In the present study, an engineered _V. natriegens_ strain was developed to produce pentalenene via a heterologous MVA pathway, achieving a final yield of 39.4 mg/L, which is the highest yield of pentalenene detected in _V. natriegens_ to date. However, significant limitations remain. Most notably, the yield remains substantially lower than the levels achieved in conventional microbial platforms such as _E. coli_ and _Saccharomyces cerevisiae_[14]. This disparity highlights the need for further optimization of metabolic flux distribution, precursor availability, and host-specific regulatory networks in _V. natriegens_. In addition, this study has not yet explored the amplification challenges and economic feasibility of the production system compared with the traditional platform. Overall, although _V. natriegens_ offers considerable potential as an alternative microbial chassis, substantial improvements in pentalenene production efficiency are required to satisfy the demands of industrial application.