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Article

Metabolic Engineering for the Biosynthesis of Pentalenene in the Rapidly Growing Bacterium Vibrio natriegens

by
Lujun Hu
1,†,
Rui Lin
1,†,
Hui Jiang
2,
Ge Yao
2,
Jiajia Liu
2,
Penggang Han
2,
Xiukun Wan
2,
Chang Chen
2,
Yunfei Zhang
2,
Shaoheng Bao
2,* and
Fuli Wang
2,*
1
College of Biological Engineering, Sichuan University of Science and Engineering, Yibin 644005, China
2
State Key Laboratory of NBC Protection for Civilian, Beijing 102205, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Fermentation 2025, 11(5), 249; https://doi.org/10.3390/fermentation11050249
Submission received: 21 March 2025 / Revised: 17 April 2025 / Accepted: 23 April 2025 / Published: 1 May 2025
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
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Abstract

:
Vibrio natriegens (V. natriegens) is an emerging synthetic biology chassis characterized by rapid growth, and its potential for the synthesis of sesquiterpenes (such as pentalenene) has not been developed. In this study, heterologous pentalenene biosynthesis was successfully established in V. natriegens via metabolic engineering. The optimization of gene dosage and culture conditions led to an increase in pentalenene yield from 0.75 mg/L to 39.4 mg/L, representing the highest titer reported in V. natriegens to date, though still markedly lower than yields achieved in conventional microbial hosts. Transcriptome analysis demonstrated that the exogenous mevalonate (MVA) pathway effectively activated terpenoid precursor synthesis, as evidenced by the up-regulation of key pathway genes. However, the endogenous methylerythritol 4-phosphate (MEP) pathway remained inactive, and genes involved in oxidative phosphorylation, the pentose phosphate pathway, and thiamine biosynthesis were down-regulated, leading to limited availability of ATP, NADPH, and acetyl-CoA. Competition for cofactors, particularly NADPH, further constrained precursor supply and pathway efficiency. This study confirmed the potential of V. natriegens as a pentalenene production platform and revealed its metabolic bottleneck, providing a theoretical basis for subsequent engineering optimization.

1. Introduction

V. natriegens is a non-pathogenic, facultative anaerobic bacterium derived from marine environments [1]. Among all known organisms, it exhibits the fastest reported growth rate [2]. The strain poses no health risk to humans and allows for facile genetic manipulation, making it a promising novel chassis for synthetic biology. Reports indicate that V. natriegens completes cell division within 10 min [3]. In a minimal glucose medium, its growth rate exceeds that of Escherichia coli (E. coli) by more than two-fold [4], primarily due to enhanced ribosomal biogenesis during exponential growth. On average, V. natriegens produces approximately 115,000 ribosomes per cell, significantly higher than the 70,000 ribosomes produced by E. coli [5]. Recent studies have demonstrated the organism’s strong potential for natural product biosynthesis. For example, Xing Liu employed V. natriegens to synthesize L-DOPA for the treatment of Parkinson’s disease. Notably, the strain V. natriegens Vmax-1 produced L-DOPA that exhibited a potency three-times greater than that generated by E. coli BL21-1 under identical conditions [6]. Smith 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 methylerythritol 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-erythritol-4-phosphate (MEP). Further conversion through a series of enzymes, including 2-C-methyl-D-erythritol-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.

2. Materials and Methods

2.1. Strains and Reagents

V. natriegens ATCC 14048 (Our laboratory) was used as the host strain for pentalenene overproduction, while E. coli S17-1 (Angyubio, Shanghai, China) was employed as the donor strain for conjugative plasmid transfer. Genomic and plasmid DNA extractions and PCR amplifications for Gibson assembly were performed using Q5 high-fidelity DNA polymerase (Vazyme, Nanjing, China). Taq polymerase for colony PCR was also obtained from Vazyme. Restriction endonucleases were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Gibson assembly was conducted using the ClonExpress® II One-Step Cloning Kit (Vazyme, Nanjing, China). Plasmid and gel extraction kits were acquired from TIANGEN (Beijing, China). The pentalenene standard was synthesized in house, and its structure was confirmed by nuclear magnetic resonance (NMR) spectroscopy [10].

2.2. Culture Medium

V. natriegens strains were cultured in an LB3 medium consisting of 5 g/L yeast extract, 10 g/L tryptone, and 30 g/L NaCl. LB6 solid medium (5 g yeast extract, 10 g tryptone, 60 g NaCl, 15 g agar per liter) was used for conjugation-based transformant selection. LBV2 medium (10 g tryptone, 5 g yeast extract, 22 g NaCl, 0.313 g KCl, 4.7 g MgCl2·6H2O per liter), 2216E medium (5.0 g tryptone, 1.0 g yeast extract, 0.1 g ferrous citrate, 19.45 g NaCl, 5.98 g MgCl2, 3.24 g Na2SO4, 1.8 g CaCl2, 0.55 g KCl, 0.16 g Na2CO3 per liter), and PPB medium (10 g tryptone, 5 g yeast extract per liter, 20 g glucose, 9.8 g K2HPO4, 5 g beef extract, 0.3 g ferric ammonium citrate, 0.06 g MgSO4, 1.9 g citric acid, 15 g NaCl, and 1 mL trace element solution [15]) were utilized for shake-flask fermentation.

2.3. Plasmid and Strain Construction

The plasmids, primers, strains, and gene sequences utilized in this research are detailed in Tables S1–S4.
The initial phase of the experiment aimed to transfer the pentalenene synthesis plasmid from E. coli into V. natriegens to enable production of the target compound. The plasmid pAC-6409-pents, previously constructed and reported by a research group [16], was selected for this purpose. Traditional chemical transformation and electroporation methods failed to introduce the high-molecular-weight plasmid into V. natriegens. In contrast, bacterial conjugation proved effective in mediating the transfer of large plasmids into the host strain. Successful conjugation required the presence of the origin of the transfer (oriT) sequence on the plasmid [17]. The oriT gene sequence was amplified from a laboratory source using Q5 High-Fidelity Polymerase with primer pair oriT_F/oriT_R. The plasmid pAC-6409-pents was linearized by Sac I digestion. Gibson assembly was performed using the ClonExpress II One-Step Cloning Kit to insert the oriT sequence, resulting in the recombinant plasmid pAC-6409-oriT-pents. A total of 10 μL of the ligation product was transformed into chemically competent E. coli DH5α cells.
The plasmid pAC-6409-oriT-pents served as a template for constructing pAC-6409-oriT-mva, which functions as the upstream module for the MVA biosynthetic pathway. The construction procedure followed the same protocol as described above.
To construct the downstream module required for pentalenene biosynthesis, the gene sequence FPPS-PENTS was introduced into the plasmid 3933-agGPPS-temp, a backbone vector previously developed in the laboratory. The FPPS-PENTS sequence was amplified from pAC-6409-oriT-pents using primer pair fpps-pents-F/fpps-pents-R. The vector backbone was amplified with primer pair 3933-temp-F/3933-temp-R. Gene amplification was conducted using Q5 High-Fidelity Polymerase. Gibson assembly following the aforementioned strategy yielded the plasmid 3933-fpps-pents.
Conjugative transformation mediated by E. coli S17-1 enabled the successful delivery of the constructed plasmids into V. natriegens ATCC 14048, resulting in the generation of engineered strains [18].

2.4. Bacterial Conjugation

High-molecular-weight plasmids were introduced into V. natriegens via bacterial conjugation, following a protocol adapted from previously reported methods [18]. Firstly, E. coli S17-1 was used as the donor bacterium, and the plasmid was transferred into E. coli S17-1 via ordinary chemical transformation. Transformed E. coli S17-1 and V. natriegens were then inoculated into an LB medium containing chloramphenicol and an LB3 medium, respectively. Cultures were incubated at 37 °C with shaking at 200 rpm until reaching the logarithmic growth phase. Cells were harvested by centrifugation, and the resulting pellets were each resuspended in 20 μL of LB2 medium. Equal volumes of the donor and recipient suspensions were mixed at a 1:1 ratio and spotted onto LB2 agar plates. Plates were incubated overnight at 37 °C. After incubation, a portion of the resulting colony mass was transferred into 100 μL of LB3 liquid medium. The suspension was then serially diluted and plated onto LB6 agar supplemented with 34 μg/mL chloramphenicol to select V. natriegens transformants harboring the target plasmid. In subsequent experiments, V. natriegens transformants carrying the chloramphenicol resistance gene were selected on 34 μg/mL chloramphenicol, while those harboring the ampicillin resistance gene were screened on 100 μg/mL ampicillin.

2.5. Shake Fermentation

Single colonies selected from antibiotic-containing plates were inoculated into an LB3 liquid medium and cultured at 37 °C for 10–12 h to prepare seed cultures. A 1% (v/v) aliquot of the seed culture was transferred into 10 mL of PPB medium and incubated at 37 °C with shaking at 220 rpm for 2–3 h. When the optical density at 600 nm (OD600) reached 0.8–1.0, expression of the target product was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM. An overlay of 2 mL n-decane was also added to the culture. Fermentation was then carried out at 30 °C with shaking for 48 h.

2.6. Identification and Quantitative Analysis of Products

After fermentation, the organic phase was collected from the shake flask and diluted 10-fold using ethyl acetate containing 5 mg/L caryophyllene oxide as an internal standard. The diluted sample was filtered through a 0.22 μm nylon membrane before analysis. Gas chromatography–mass spectrometry (GC-MS) was performed using a TRACE 1300 gas chromatograph coupled with a TSQ 9000 mass detector (Thermo Fisher Scientific, Waltham, MA, USA). The inlet temperature was set to 300 °C, with a carrier gas flow rate of 1 mL/min. The oven temperature program was as follows: initial temperature of 50 °C held for 1 min, ramped to 100 °C at 50 °C/min and held for 1 min, followed by a second ramp to 280 °C at 20 °C/min, with a final hold at 280 °C for 2 min.

2.7. Optimization of Expression Conditions for Pentalenene Production by V. natriegens

The recombinant strain was fermented under three types of medium conditions, PPB, LBv2 and 2216E, respectively. Based on the optimal medium, fermentation was further conducted at different temperatures (25 °C, 28 °C, 30 °C, 34 °C, and 37 °C) to identify the optimal cultivation temperature. Under the optimal temperature conditions, the recombinant strains were cultured and fermented at 0.1, 0.3, 0.5, 0.8 and 1 mmol/L IPTG concentration, respectively.

2.8. Transcriptomics Analysis Methods

Total RNA was extracted from V. natriegens single-cell pellets after the removal of genomic DNA. High-quality RNA was used for library preparation using the Illumina® Stranded mRNA kit [19]. Paired-end RNA sequencing was performed on the Illumina NovaSeq™ X Plus platform [20], and the resulting data were subjected to comprehensive bioinformatics analysis. Transcript abundance was quantified using the RSEM software package (Version 1.3.3), which supports both single-end and paired-end sequencing data [21]. Gene expression was quantified using FPKM (Fragments Per Kilobase of transcript per Million mapped reads) and TPM (Transcripts Per Million) methods. EdgeR (Version 4.0.2), DESeq2 (Version 1.42.0), and DESeq (Version 1.56.1) were employed for differential expression analysis [22]. Differentially expressed genes (DEGs) were identified based on a p-value (or FDR) below 0.05. Functional annotation of DEGs was carried out using KEGG PATHWAY enrichment via KOBAS, with statistical significance evaluated using Fisher’s exact test. A p-value ≤ 0.05 was considered indicative of significant pathway enrichment. All experiments were performed with three biological replicates. VN was used to denote the control strain without exogenous gene introduction, and VN-MVA referred to the recombinant strain carrying plasmid pAC-6409-oriT-pents. Final figures were assembled using Adobe Illustrator 2020.

3. Results and Analysis

3.1. Production and Characterization of Pentalenene in V. natriegens

Pentalenene production was achieved using a metabolically engineered strain of V. natriegens. The plasmids 3933-fpps-pents and pAC-6409-oriT-mva (Figure 1A) were co-transformed into V. natriegens ATCC14048 via conjugative transformation.
Following shake-flask fermentation, the n-decane overlay was collected and analyzed by GC-MS. The chromatogram of the pentalenene standard exhibited a distinct peak at a retention time (RT) of 6.84 min (Figure 1C). A corresponding peak at 6.84 min was also observed in the fermentation sample (Figure 1B). Mass spectral analysis revealed that the molecular ion peak (204.2 m/z) and fragment ions in the sample (Figure 1B) matched those of the pentalenene standard (Figure 1C). Based on the retention time and mass spectral similarity, the compound was identified as pentalenene. These results confirmed that engineered V. natriegens is capable of producing pentalenene through heterologous expression of the biosynthetic pathway.

3.2. 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 K2HPO4 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 °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 °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.

3.3. Transcriptome-Based Analysis of the Key Regulatory Factors of Pentalenene Synthesis Efficiency

To investigate the potential causes of low pentalenene yield, genome-wide transcriptomic profiles of the recombinant strain VN-MVA (harboring the integrated MVA pathway and exhibiting pentalenene 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 pentalenene 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. natriegens 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 pentalenene biosynthesis. The lack of endogenous pathway participation may represent a key factor contributing to the relatively low pentalenene yield in the engineered V. natriegens [29,30].
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 exogenous MVA pathway for pentalenene 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). Several key genes involved in this pathway exhibited significant down-regulation (Table 1), including NDH (complex I: NADH dehydrogenase), petB, petC (complex III: cytochrome bc1 complex), cyoC, cyoB (complex IV: cytochrome oxidase), and atpA, atpC (ATP synthase) genes, indicating possible activation of the oxidative stress response [31]. Since oxidative phosphorylation is the primary route for ATP production, the down-regulation of ATP synthase genes may lead to ATP deficiency [32], limiting the activity of MVA pathway enzymes and restricting terpenoid precursor synthesis and transport. In the glutathione metabolism pathway, most DEGs were down-regulated, but the key gene GSR (glutathione reductase) in the pathway was up-regulated by 1.6-times. This response confirms the occurrence of oxidative stress, which was likely induced by the introduction of the heterologous pathway. The resulting oxidative stress triggered increased glutathione synthesis as an antioxidant defense mechanism [33], consequently leading to increased NADPH consumption [34]. In the pentose phosphate pathway, a large number of genes were down-regulated (up/down ratio = 3:11), including G6PDH (glucose-6-phosphate dehydrogenase), which showed a 2.3-fold reduction in expression. As G6PDH catalyzes the generation of NADPH, its repression leads to reduced NADPH availability [35]. NADPH is required for both the antioxidant defense functions of the glutathione metabolism pathway and the catalytic activity of HMGR in the MVA pathway. Limited NADPH availability may lead to competition between these two processes, resulting in cofactor imbalance and reduced efficiency in the synthesis of the terpenoid precursor IPP. In the thiamine synthesis pathway, key genes, such as DXS, thiM, tenA, thiI, thiG, and thiK, were significantly down-regulated (Table 1), indicating impaired thiamine production. As an essential coenzyme in cellular metabolism, thiamine is required for the activity of the pyruvate dehydrogenase complex. Thiamine deficiency, thus, limits the conversion of pyruvate to acetyl-CoA [36,37]. Given that acetyl-CoA serves as a critical precursor in the MVA pathway, its insufficient supply directly constrains pathway flux [38] and ultimately reduces pentalenene biosynthetic efficiency. In summary, the introduction of the heterologous MVA pathway disrupted the metabolic balance in V. natriegens, resulting in limited cofactor availability and precursor supply. These imbalances restricted MVA pathway flux and inhibited pentalenene synthesis.

4. Discussion

V. natriegens, 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. natriegens 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. natriegens-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 G6PDH 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 metabolic homeostasis and created conflicts in energy and cofactor allocation. Despite successful and significant up-regulation of key MVA pathway genes (such as HMGR, FPPS), cofactor limitation and the suppression of energy metabolism emerged as major bottlenecks. As a result, the efficiency of pentalenene biosynthesis remained constrained.
Compared with E. coli, the high metabolic rate of V. natriegens may aggravate the metabolic pressure after the introduction of heterologous pathways. Although transcriptome analysis provided a preliminary explanation for the observed low yield, further investigation is required to elucidate the strain’s dynamic response to oxidative stress, strategies for cofactor rebalancing, and the potential benefits of thiamine supplementation. Future research may enhance the production performance of the V. natriegens chassis by dynamically regulating key gene expression, optimizing cofactor availability (e.g., through overexpression of NADPH regeneration pathways), or introducing modules to enhance thiamine biosynthesis, thereby improving metabolic flux distribution and overall pathway efficiency.

5. Conclusions

In conclusion, pentalenene overproduction was achieved by integrating the complete FPP biosynthetic pathway and the pents gene into an engineered V. natriegens. A novel microbial chassis based on V. natriegens was successfully constructed and applied to sesquiterpene biosynthesis. Under optimized culture conditions, shake-flask fermentation yielded 39.4 mg/L of pentalenene. Transcriptomic analysis indicated that strategies such as dynamic regulation and cofactor supply optimization hold promise for further improving production efficiency. These findings provide a foundation for the development of V. natriegens as an alternative chassis for sesquiterpene biosynthesis.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation11050249/s1, Table S1. Plasmids used in this study. Table S2. Strains used in this study. Table S3. Primers used in this study. Table S4. Genes used in this study. Reference [16] is cited in the supplementary materials.

Author Contributions

Data curation, R.L., G.Y. and P.H.; formal analysis, F.W.; investigation, G.Y., J.L., X.W., C.C. and Y.Z.; methodology, H.J., J.L., P.H., X.W., C.C. and Y.Z.; supervision, L.H., H.J. and F.W.; validation, L.H.; writing—original draft, R.L.; writing—review and editing, S.B. All authors have read and agreed to the published version of the manuscript.

Funding

This study did not receive external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no known competing financial interests.

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Fermentation 11 00249 g001
Figure 1. (A) Illustration of the metabolic engineering vector assembly. (B) Represents the total ion chromatograms (TIC) and mass spectrum, respectively, of pentalenene detected in the samples obtained after inducing fermentation following the introduction of the pentalenene biosynthetic pathway into V. natriegens. (C) Represents the total ion chromatogram (TIC) and mass spectrum of pentalenene standard, respectively (from left to right).
Figure 1. (A) Illustration of the metabolic engineering vector assembly. (B) Represents the total ion chromatograms (TIC) and mass spectrum, respectively, of pentalenene detected in the samples obtained after inducing fermentation following the introduction of the pentalenene biosynthetic pathway into V. natriegens. (C) Represents the total ion chromatogram (TIC) and mass spectrum of pentalenene standard, respectively (from left to right).
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Figure 2. The optimization of pentalenene production by V. natriegens was conducted. (A) Comparison of the yield of pentalenene synthesized through different culture media. (B) Comparison of the production yields of pentalenene synthesized under different induction temperatures. (C) Comparison of the yield of pentalenene synthesized under different concentrations of inducer. (D) Comparison of pentalenene 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.
Figure 2. The optimization of pentalenene production by V. natriegens was conducted. (A) Comparison of the yield of pentalenene synthesized through different culture media. (B) Comparison of the production yields of pentalenene synthesized under different induction temperatures. (C) Comparison of the yield of pentalenene synthesized under different concentrations of inducer. (D) Comparison of pentalenene 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.
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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.
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.
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Figure 4. (A) Synthesis of pentalenene 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 4. (A) Synthesis of pentalenene 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.
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Figure 5. The top 20 KEGG pathways enriched after plasmid-induced expression with the number of differential genes.
Figure 5. The top 20 KEGG pathways enriched after plasmid-induced expression with the number of differential genes.
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Table 1. Annotation of DEGs in oxidative phosphorylation and glutathione metabolism pathways.
Table 1. Annotation of DEGs in oxidative phosphorylation and glutathione metabolism pathways.
PathwayGene NameAnnotationlog2 (FC)Up or Down
Oxidative phosphorylationNDHNADH dehydrogenase−1.13 *Down
petBcytochrome B−0.74 *Down
petCcytochrome C−0.41 *Down
cyoBcytochrome o ubiquinol oxidase subunit I−1.69 *Down
cyoCcytochrome o ubiquinol oxidase subunit III−2.27 *Down
cyoDCytochrome o ubiquinol oxidase−2.14 *Down
atpAATP F0F1 synthase subunit alpha−0.52 *Down
atpCATP synthase F0F1 subunit epsilon−0.93 *Down
atpDATP synthase F0F1 subunit beta−0.75 *Down
atpGATP F0F1 synthase subunit gamma−0.88 *Down
Glutathione metabolismGSRglutathione reductase0.64 *Up
Pentose phosphate pathwayG6PDHglucose-6-phosphate dehydrogenase−0.87 *Down
Thiamine metabolismDXS1-deoxy-D-xylulose-5-phosphate synthase−0.96 *Down
thiHthiamine biosynthesis protein ThiH−1.63 *Down
thiMhydroxyethylthiazole kinase−1.61 *Down
tenAhypothetical protein−1.68 *Down
thiItRNA s(4)U8 sulfurtransferase−0.64 *Down
thiGthiazole synthase−0.93 *Down
thiKthiamine kinase−0.62 *Down
Note: In the table, differentially expressed genes (DEGs) were identified using a significance threshold of p < 0.05, with asterisks (*) denoting statistically significant differences.
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MDPI and ACS Style

Hu, L.; Lin, R.; Jiang, H.; Yao, G.; Liu, J.; Han, P.; Wan, X.; Chen, C.; Zhang, Y.; Bao, S.; et al. Metabolic Engineering for the Biosynthesis of Pentalenene in the Rapidly Growing Bacterium Vibrio natriegens. Fermentation 2025, 11, 249. https://doi.org/10.3390/fermentation11050249

AMA Style

Hu L, Lin R, Jiang H, Yao G, Liu J, Han P, Wan X, Chen C, Zhang Y, Bao S, et al. Metabolic Engineering for the Biosynthesis of Pentalenene in the Rapidly Growing Bacterium Vibrio natriegens. Fermentation. 2025; 11(5):249. https://doi.org/10.3390/fermentation11050249

Chicago/Turabian Style

Hu, Lujun, Rui Lin, Hui Jiang, Ge Yao, Jiajia Liu, Penggang Han, Xiukun Wan, Chang Chen, Yunfei Zhang, Shaoheng Bao, and et al. 2025. "Metabolic Engineering for the Biosynthesis of Pentalenene in the Rapidly Growing Bacterium Vibrio natriegens" Fermentation 11, no. 5: 249. https://doi.org/10.3390/fermentation11050249

APA Style

Hu, L., Lin, R., Jiang, H., Yao, G., Liu, J., Han, P., Wan, X., Chen, C., Zhang, Y., Bao, S., & Wang, F. (2025). Metabolic Engineering for the Biosynthesis of Pentalenene in the Rapidly Growing Bacterium Vibrio natriegens. Fermentation, 11(5), 249. https://doi.org/10.3390/fermentation11050249

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