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Engineering Escherichia coli via introduction of the isopentenol utilization pathway to effectively produce geranyllinalool

Microbial Cell Factories volume 23, Article number: 292 (2024) Cite this article

Abstract

Background

Geranyllinalool, a natural diterpenoid found in plants, has a floral and woody aroma, making it valuable in flavors and fragrances. Currently, its synthesis primarily depends on chemical methods, which are environmentally harmful and economically unsustainable. Microbial synthesis through metabolic engineering has shown potential for producing geranyllinalool. However, achieving efficient synthesis remains challenging owing to the limited availability of terpenoid precursors in microorganisms. Thus, an artificial isopentenol utilization pathway (IUP) was constructed and introduced in Escherichia coli to enhance precursor availability and further improve terpenoid synthesis.

Results

We first constructed an artificial IUP in E. coli to enhance the supply of precursor geranylgeranyl diphosphate (GGPP) and then screened geranyllinalool synthases from plants to achieve efficient synthesis of geranyllinalool (274.78 ± 2.48 mg/L). To further improve geranyllinalool synthesis, we optimized various cultivation factors, including carbon source, IPTG concentration, and prenol addition and obtained 447.51 ± 6.92 mg/L of geranyllinalool after 72 h of shaken flask fermentation. Moreover, a scaled-up production in a 5-L fermenter was investigated to give 2.06 g/L of geranyllinalool through fed-batch fermentation. To the best of our knowledge, this is the highest reported titer so far.

Conclusions

Efficient synthesis of geranyllinalool in E. coli can be achieved through a two-step pathway and optimization of culture conditions. The findings of this study provide valuable insights into the production of other terpenoids in E. coli.

Background

Geranyllinalool is a naturally occurring terpene compound known for its distinctive floral and woody fragrances. It serves both as a fragrance itself and as a precursor in pharmaceutical synthesis for terpene-based drugs, notably including the broad-spectrum anti-ulcerative teprenone [1]. In plants, geranyllinalool plays a crucial role by serving as an intracellular intermediate metabolite in the biosynthesis of (E, E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, an essential insecticidal terpene derivative [2]. Moreover, using geranyllinalool as a precursor, 17-hydroxygeranyllinalool diterpene glycosides have been synthesized in Nicotiana species; these compounds have been shown to confer resistance against both specialist and generalist herbivore tobacco hornworms, i.e., Manduca sexta and Heliothis virescens, respectively [3, 4]. Geranyllinalool is predominantly obtained through chemical synthesis or plant extraction methods, which faces challenges such as the stringent conditions required for chemical synthesis [5] and limitations in achieving high extraction rates when using plants.

Terpenoids, composed of isoprene units, constitute a major class of natural secondary metabolites [6,7,8]. They have found extensive applications in the food, pharmaceutical, and cosmetics industries [9,10,11]. Isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP) serve as universal precursors for terpenoid synthesis, primarily synthesized through the methylerythritol phosphate (MEP) and mevalonate (MVA) pathways in nature [12]. However, these pathways have limitations in their synthesis capacities for terpenoids. In recent years, the development of the artificially constructed isopentenol utilization pathway (IUP) has facilitated efficient IPP synthesis, providing abundant precursors for downstream applications [13]. Compared with the MVA and MEP pathways, the IUP offers a simplified synthetic route with only two steps and uses a single cofactor (ATP). This highlights its potential for terpene compound synthesis in scientific research [14, 15].

In the IUP pathway, prenol (3-methyl-2-buten-1-ol) serves as a precursor and undergoes initial phosphorylation to form dimethylallyl phosphate (DMAP), which is then converted to DMAPP. Similarly, when isoprenol (3-methylbut-3-en-1-ol) is utilized, it undergoes phosphorylation to yield isopentenyl phosphate (IP), followed by conversion to IPP. Notably, the interconversion between IPP and DMAPP can be catalyzed via a biochemical reaction mediated by isopentenyl pyrophosphate isomerase (IDI) [16,17,18]. Researchers have successfully synthesized terpenoid compounds in E coli using the IUP, which operates independently of the natural MVA and MEP pathways and bypasses stringent cellular regulatory mechanisms, enabling efficient synthesis of IPP or DMAPP for downstream terpenoid production [19]. By optimizing precursor supply and refining the timing of isopentenyl alcohol addition, researchers have significantly improved terpenoid compound yields, including linalool, cis-abienol, and lycopene [20,21,22]. Therefore, E. coli was chosen as the host in this study to develop a biosynthetic pathway for geranyllinalool using the IUP. The fermentation conditions of the engineered strain were optimized to achieve a high yield of geranyllinalool synthesis, thereby providing new insights into the biological production of other terpenoid compounds.

Materials and methods

Plasmid construction and chromosomal manipulation

Recombinant DNA manipulation was performed as previously described [23]. The plasmid construction and cloning procedures were conducted using the E. coli TOP10 strain. Competent cells of E. coli were prepared using the Competent Cell Preparation Kit (Takara Bio Inc.) and subjected to the respective chemical transformation procedure according to the provided guidelines. The plasmids pEcCas and pEcgRNA, supplied by Prof. Sheng Yang from the Chinese Academy of Sciences, were employed for CRISPR/Cas9-mediated gene editing following the protocol outlined in the referenced literature [24]. The concentrations of ampicillin, chloramphenicol, and streptomycin sulfate used were 100, 30, and 50 µg/mL, respectively. Table 1 provides a comprehensive list of the specific plasmids and strains used in this study.

Table 1 Plasmids and strains used in this study
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The primers used in this study are summarized in Additional file 1: Table S1. Primer sequences and heterologous genes, including MthIPK, TcGGPPS, and diverse geranyllinalool synthases, underwent codon optimization and synthesis, performed by Genscript Biotech Corporation. MthIPK gene fragments were polymerase chain reaction (PCR)-amplified from the pUC57-MthIPK plasmid using MthIPK-F/MthIPK-R primers. Additionally, the EcThiM gene fragment was amplified from E. coli BL21(DE3) genomic DNA using EcThiM-F/EcThiM-R primers. Subsequent purification of the PCR products resulted in successful acquisition of both MthIPK and EcThiM gene fragments. The EcThiM gene fragment was ligated into the pCDFDuet-1 plasmid using BamHI and HindII restriction enzymes to create the transformed plasmid designated pCDT. Similarly, ligation of the MthIPK gene fragment into the pCDT plasmid was achieved using NdeI and XhoI restriction enzymes, resulting in the transformed plasmid designated pCDTI. The TcGGPPS gene was PCR-amplified from the pUC57-TcGGPPS plasmid using the TcGGPPS-F/TcGGPPS-R primers. Similarly, the ScEGR20 and ScIDI gene fragments were amplified from the Saccharomyces cerevisiae S288C genome using ScEGR20-F/ScEGR20-R and ScIDI-F/ScIDI-R primers, respectively. Fusion of ScEGR20 and ScIDI was performed via overlap-PCR and then ligated into the pACYCDuet-1 plasmid using EcoRI and AflII enzymes to generate the recombinant plasmid pACEI. Ligation of the TcGGPPS gene fragment into pACEI was accomplished using NdeI and XhoI enzymes, followed by transformation to obtain the recombinant plasmid pACEIG.The NaGLS gene fragment was amplified through PCR using NaGLS-F/NaGLS-R primers and the pUC57-NaGLS plasmid as a template. Following purification, the fragment was ligated into the pETDueT-1 vector using BamHI and SacI restriction enzymes, and the ligated product was transformed into E. coli TOP10 cells to generate the recombinant plasmid designated pETDN. Similarly, we obtained other recombinant plasmids, including pETDNT, pETDNTO, pETDL, pETDS, and pETDC.

Fermentation

For shake flask fermentation, a single colony was carefully chosen and introduced into 50 mL of LB culture medium supplemented with appropriate antibiotics. The inoculated flask was then incubated overnight at 37 °C with shaking at 200 rpm to establish the initial seed culture. Subsequently, 1% of the seed culture was inoculated into 50 mL of TB medium (24 g/L yeast extract, 12 g/L peptone, 9.4 g/L K2HPO4, 2.2 g/L KH2PO4, and 10 g/L glycerol) supplemented with antibiotics. Cell optical density was measured at 600 nm using a spectrophotometer. The sample was diluted with water as an instrument blank to achieve absorbance within the range 0.1–1.0 units. Isopropyl β-D-thiogalactoside (IPTG; 0.5 mM) was added when the optical density at 600 nm (OD600) reached 2.5–3.0 at a controlled temperature of 25 °C held constant throughout the process. The incubation continued until the OD600 reached 8–9. Then, 2.5 g/L prenol was added, and samples were systematically collected every 24 h for subsequent analysis.

Fermentation in a 5 L fermenter followed the protocol established by [22], including the same medium and control strategy. IPTG was added to induce protein expression when the OD600 reached 30, and prenol supplementation followed at a concentration of 2.5 g/L when the OD600 reached 50. Prenol was replenished every 48 h, and samples were collected every 6 h from the initial substrate addition for subsequent analysis. As shown in Fig. 1, the synthesis of geranyllinalool requires three molecules of IPP and one molecule of DMAPP, totaling four moles of prenol to produce one mole of geranyllinalool. Consequently, the substrate conversion rate is expressed as a percentage representing the experimental yield of geranyllinalool relative to its theoretical yield based on the total substrate.

Optimization conditions for inducing expression

Following shake flask fermentation, the strain was cultured until it reached an OD600 of 2.5–3.0, after which IPTG was added. Induction was performed at temperatures of 16 °C, 20 °C, 25 °C, and 30 °C. Geranyllinalool production was then analyzed using gas chromatography (GC). Similarly, after culturing the strain to an OD600 of 2.5–3.0 and adding IPTG at final concentrations of 0–1.0 mM in increments of 0.2 mM, GC analysis was employed to further evaluate geranyllinalool production.

Soluble expression analysis of key recombinant enzymes in cells

Soluble expression levels of key enzymes in the engineered strain were validated using sodium dodecyl-sulfate polyacrylamide (SDS-PAGE), as described by [25]. A 15% separation gel and a 5% concentrated gel were prepared following the instructions provided with the SDS-PAGE Rapid Preparation Kit (Sangon Biotech) for protein solubility analysis.

Extraction and analysis of geranyllinalool

Cells were sonicated at 4 °C using a sonic cell disrupter at 600 W for 30 min in pulse mode (4 s on and 4 s off). The resulting cell lysate was subjected to solvent extraction with equal volumes of n-hexane and ethyl acetate, followed by vortexing for 30 min. Qualitative and quantitative detection of geranyllinalool was performed through GC-mass spectrometry (MS) and GC analysis of the extracted fractions. An HP-5MS column (0.25 μm × 60 m, 0.25 μm) was used for GC–MS analysis, with detection conditions adjusted based on a previous study [26]. The temperature program included an initial hold at 60℃ for 2 min, followed by a linear ramp at 8℃/min up to a final temperature of 280℃, and a final hold period of 10 min. GC analysis was performed using an HP-5 column (0.25 μm × 60 m, 0.25 μm) with helium as the carrier gas. The injection port and detector were maintained at a constant temperature of 250℃ throughout the analysis process. A split injection method with a split ratio of 10:1 was employed, and the injection volume was set at 1 µL with a column flow rate of 1 mL/min.

Quantification of major byproducts in the carbon metabolic pathways

The concentrations of glycerol, glucose, acetic acid, lactic acid, and ethanol in the fermentation broth were quantified using high-performance liquid chromatography with refractive index detection, following the method described by [27]. A SilGreen GH0830078H column, measuring 300 mm × 7.8 mm × 8 μm, was employed for separation, using a refractive index detector. The mobile phase comprised 9 mM sulfuric acid, and the injection volume was 10 µL. The flow rate was set at 0.5 mL/min, and the column temperature was maintained at 50℃.

Results

Design and construction of the geranyllinalool biosynthetic pathway

This study outlines a biosynthetic pathway for geranyllinalool derived from the IUP, using prenol as the substrate and undergoing six enzymatic steps to produce geranyllinalool (Fig. 1). Key enzymes in this pathway include EcThiM (GenBank ID: BAA15972.1) from E. coli BL21(DE3) [28], MthIPK (GenBank ID: AAB84554.1) from Methanothermobacter thermautotrophicus [29], ScIDI (GenBank ID: 855986), and ScEGR20(F96C) (GenBank ID: 853272) from S. cerevisiae [30, 31], and TcGGPPS (GenBank ID: AAD16018.1) from Taxus canadensis. Among them, EcThiM and MthIPK play pivotal roles in catalyzing DMAPP/IPP generation from prenol in the IUP. ScIDI, ScEGR20(F96C), and TcGGPPS contribute to the production of the diterpene precursor GGPP from DMAPP/IPP. The genetically modified strain E. coli LSC05, engineered by our research group through specific deletion of aphA (acid phosphatase), yqaB (fructose-1-P and 6-phosphogluconate phosphatase) [32], and phoA (alkaline phosphatase) [33], effectively reduces DAMP, IP, IPP, and geranyl diphosphate (GPP) degradation while enhancing terpene product synthesis. Introduction of plasmids pCDTI and pACEIG into E. coli LSC05 resulted in enhanced GGPP production, yielding the strain designated E. coli GP.

Fig. 1
figure 1

Development of a biosynthetic pathway for geranyllinalool production

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Metabolite abbreviations: G3P, glycerol-3-phosphate; DHAP, dihydroxyacetone phosphate; GAP glyceraldehyde 3-phosphate; DHA, dihydroxyacetone; 1,3-2PG, 1,3-diphosphoglycerate; PGA, 3-phosphoglycerate; PEP, phosphoenolpyruvate; PYR, pyruvate; AcCoA, acetyl-CoA; OAA, oxaloacetate; DXP, 1-deoxy-D-xylulose-5-phosphate; MEP, 2 C-methyl-D-erythritol-4-phosphate; CDP-ME, 4-diphosphocytidyl-2 C-methyl-D-erythritol; CDP-MEP, 4-diphosphocytidyl-2 C-methyl-D-erythritol-2-phosphate; MEcPP, 2 C-methyl-D-erythritol 2,4-cyclodiphosphate; HMBPP, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate; IP, isopentenyl monophosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate. Genes involved in the pathway: dxs, DXP synthase; ispC, DXP reductoisomerase; ispD, CDPME synthase; ispE, CDPME kinase; ispF, MEcPP synthase; ispG, HMBPP synthase; ispH, HMBPP reductase. EcThiM, hydroxyethylthiazole kinase; MthIPK, hydroxyethylthiazole kinase; ScIDI, isopentenyl diphosphate isomerase; ScEGR20(F96C), farnesyl pyrophosphate synthase mutant; TcGGPPS, geranylgeranyl diphosphate synthase; NaGLS, geranyllinalool synthase; aphA, acid phosphatase gene; yqaB, fructose-1-phosphate phosphatase gene; phoA, alkaline phosphatase gene.

To identify a highly active geranyllinalool synthase, we evaluated six enzymes: NaGLS [2], NtGLS, NtoGLS, SdGLS, LfGLS, and CaGLS. These enzymes possess characteristic structural domains typical of terpene synthases, including the DDXXD motif and the DTE/NSE motif [LLND(L/I)X(S)XXXE] (Additional file 1: Fig S2). Incorporating these six geranyllinalool synthases into the E. coli GP strain generated six engineered strains, designated E. coli GL01–06, for screening high-activity geranyllinalool synthases through shake flask fermentation. Figure 2 illustrates the variations in geranyllinalool production among these strains after 72 h of fermentation. Among the strains, E. coli GL01 exhibited the highest geranyllinalool production at 274.78 ± 2.48 mg/L after 72 h, prompting further experimentation with this strain.

Fig. 2
figure 2

Geranyllinalool synthases screened at an induction temperature of 25℃ with 0.5 mM IPTG for geranyllinalool biosynthesis enhancement. Asterisk denotes statistically significant differences: *p < 0.05; **p < 0.01; ***p < 0.001

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Verification of geranyllinalool products

Fermentation of E. coli GL01 was conducted in shake flasks, and the resulting broth was subjected to GC and GC–MS analysis. Using geranyllinalool as a reference standard, the fermentation broth was examined via GC and GC–MS analysis. Results conclusively demonstrated that the elution time of the fermentation broth sample precisely matched that of the geranyllinalool standard (Additional file 1: Fig S3A). In addition, detailed comparison between the sample and geranyllinalool standard using GC–MS revealed chromatographic consistency (Additional file 1: Fig S3B). These findings provide evidence supporting the E. coli GL01 strain’s capability to synthesize geranyllinalool effectively while validating the rational selection of key enzymes in the artificially designed biosynthetic pathway as well as the pathway’s accurate assembly.

Optimal fermentation conditions in a shake flask

To confirm the intracellular localization of geranyllinalool, we subjected the fermentation broth, supernatant, and cell precipitate to GC analysis (Fig. 3A). Results revealed that a minor fraction of geranyllinalool (22.87 ± 4.14 mg/L) was excreted into the extracellular environment, whereas the majority (245.43 ± 5.59 mg/L) remained intracellular. Thus, geranyllinalool can be considered a predominantly intracellular product. For subsequent characterization, we employed cell lysis and extraction applied to the whole fermentation broth.

Given that both prenol and isoprenol can be metabolized in the IUP pathway, 2.5 g/L of prenol, 2.5 g/L of isoprenol, and a 1:1 mixture of these two substrates were separately added to the fermentation broth. The addition of 2.5 g/L prenol resulted in the highest yield of geranyllinalool at 271.62 ± 6.12 mg/L. These results show that prenol as the sole substrate achieved optimal geranyllinalool production (Fig. 3B), consistent with the findings of a previous study on higher toxicity of isoprenol [22]. Considering the potential cytotoxicity of high prenol concentrations [22], we assessed the effects of varying prenol concentrations on geranyllinalool production. Results revealed an increase in geranyllinalool production as prenol concentration increased from 1.0 g/L to 2.5 g/L. However, a significant decrease in geranyllinalool production was observed when prenol concentration was further increased from 2.5 g/L to 5.0 g/L (Fig. 3C). Thus, the optimal concentration of prenol was determined to be 2.5 g/L.

The application of two-phase bioprocess fermentation has been extensively studied for enhancing the production of various diterpenoids, including its impact on host strain productively [22, 30, 34]. In this study, the effect of incorporating 10% (v/v) isopropyl myristate, dodecane, and oleyl alcohol on geranyllinalool production by the engineered strain E. coli GL01 was investigated [35], alongside a control culture lacking solvents. However, results indicated that introducing organic solvents into two-phase culture did not enhance geranyllinalool biosynthesis. Compared with the single-phase, all two-phase bioprocess fermentations significantly reduced geranyllinalool yield. Furthermore, there was no significant reduction observed in cell growth in biphasic cultures compared to single-phase conditions. (Additional file 1: Fig S5). Considering the potential cytotoxic effects of geranyllinalool at high concentrations, we assessed its cytotoxicity at varying concentrations [36]. Results revealed that geranyllinalool had no discernible impact on cell growth (Additional file 1: Fig S4), indicating the absence of cytotoxic effects on E. coli. These findings confirm that two-phase bioprocess fermentation is unsuitable for geranyllinalool biosynthesis, prompting the adoption of single-phase fermentation for subsequent experiments.

Fig. 3
figure 3

Optimization of fermentation conditions for geranyllinalool production by E. coli GL01. (A) Geranyllinalool distribution within the cellular milieu assessed following induction at 25℃ with 0.5 mM IPTG. (B) Effects of various substrates on geranyllinalool production at an induction temperature of 25℃ with 0.5 mM IPTG. (C) Influence of different concentrations of prenol on geranyllinalool production was examined under induction conditions at 25℃ with 0.5 mM IPTG. *p < 0.05; **p < 0.01; ***p < 0.001

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Effect of induction conditions on geranyllinalool production

For E. coli fermentation, glycerol and glucose are commonly used as carbon sources. Therefore, in a carbon source-free TB medium, glycerol and glucose were separately supplemented at concentrations of 10 g/L to investigate their respective impacts on the biosynthesis of geranyllinalool (Fig. 4A). Results showed significantly higher yield when glycerol was used as the primary carbon source, reaching 282.98 ± 5.00 mg/L after 72 h of fermentation, approximately 1.9-fold higher than that achieved with glucose. These findings revealed that glycerol as a carbon source enhanced geranyllinalool production. This phenomenon may be attributed to the improved generation of reducing equivalents required for the biosynthetic pathway of geranyllinalool, reduced aggregation of heterologous enzymes, or changes in membrane phospholipid composition to facilitate expanded membrane space for geranyllinalool accumulation [37]. Temperature markedly influences various factors in microbial processes, including strain growth, plasmid stability, and protein solubility [38]. In this study, we investigated the impact of various induction temperatures on geranyllinalool production. Results showed that at a 20℃ induction temperature, geranyllinalool production peaked at 334.78 ± 8.11 mg/L after 72 h of fermentation, with higher temperatures leading to a gradual decrease in geranyllinalool production (Fig. 4B). Lower temperatures enhance the solubility and expression capacity of enzymes from yeast to plants, whereas excessively low temperatures (< 16℃) adversely impact both cell growth and enzymatic activity, hindering product biosynthesis efficiency. Therefore, 20℃ was identified as the optimal induction temperature for subsequent experiments.

The concentration of IPTG used for induction significantly affects soluble recombinant protein expression and product synthesis in E. coli [39]. Thus, we investigated the IPTG concentration at the recommended induction temperature. As shown in Fig. 4C, geranyllinalool yield reached 447.46 ± 6.94 mg/L after 72 h of fermentation with an IPTG induction concentration of 0.4 mM. Further increases in IPTG concentration resulted in decreased yield. This decline may be attributed to cytotoxic effects at higher IPTG levels, inhibiting cell growth, or excessive protein synthesis rates, leading to misfolding or inclusion body formation, thereby reducing catalytic efficiency. Based on our findings, 0.4 mM IPTG was chosen as the optimal concentration for subsequent experiments.

Fig. 4
figure 4

Optimization of induction conditions for geranyllinalool production by E coli GL01. (A) Effects of various carbon sources on geranyllinalool production with an induction temperature of 25℃ and 0.5 mM IPTG. (B) Effects of IPTG temperature on geranyllinalool production with 10 g/L initial glycerol and 0.5 mM IPTG. (C) Effects of IPTG concentration on geranyllinalool production with 10 g/L initial glycerol and induction at 20℃. Asterisk denotes statistically significant differences: *p < 0.05; **p < 0.01; ***p < 0.001

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Protein solubility analysis of recombinant enzymes

To investigate the solubility of significantly overexpressed enzymes, protein properties were analyzed. Recombinant plasmids pCDTI, pACEIG, and pETDN (Additional file 1: Fig S1) were introduced into E. coli LSC05 strain to generate the engineered strains E. coli TI, E. coli EIG, and E. coli DN, respectively. Subsequently, these strains were separately cultured, and cells were harvested after optimal induction for 24 h. Bacterial cells were reconstituted in phosphate-buffered saline (PBS) buffer solution to eliminate residual culture medium before being suspended and sonicated in fresh PBS buffer solution. SDS-PAGE was conducted on the cell lysate, cell precipitate, and cell lysate–precipitate mixture to validate soluble expression of key enzymes in the geranyllinalool synthesis pathway (Additional file 1: Fig S6).

NaGLS recombinant protein (100.9 kDa) was observed in lanes 1–3, corresponding to the respective components of the cell lysate, supernatant, and precipitate. Similarly, MthIPK (29.1 kDa) and EcThiM recombinant proteins (27.2 kDa), detected in lanes 4–6, represent distinct fractions of the cell lysate, supernatant, and precipitate, respectively. Appearing in lanes 7–9, TcGGPPS protein (32.4 kDa) was observed within different fractions of the cell lysate, supernatant, and precipitate. These findings validate robust expression of genetically engineered proteins achieved through optimized induction conditions facilitating effective geranyllinalool synthesis.

Fed-batch production of geranyllinalool in a 5-L bioreactor

To further validate the engineered strain’s ability to biosynthesize geranyllinalool, fed-batch fermentation in a 5-L bioreactor was conducted using glycerol as the carbon source. Concentrations of glycerol, geranyllinalool, and major carbon metabolic byproducts, including acetic acid, lactic acid, and ethanol, were continuously monitored throughout fermentation. The dissolved oxygen-linked supplementation method was employed for carbon source replenishment, maintaining dissolved oxygen levels at 10–40%, with glycerol concentrations kept below 0.5 g/L. The substrate, totaling 7.5 g/L, was added in three separate batches. Figure 5 provides a summary of the fermentation data.

Fermentation proceeded for 150 h, during which the maximum cell density (OD600) reached 85.4 at 132 h. At 144 h, a high geranyllinalool yield of 2.06 g/L was achieved. Furthermore, the prenol substrate’s conversion rate was calculated at approximately 32.6%. A previous study showed that acetic acid concentrations of 5–10 g/L significantly inhibit specific growth rate, cell concentration, and protein yield during aerobic culture [40]. Furthermore, with glycerol used as a carbon source alongside an oxygen-dependent feeding strategy, the synthesis of carbon metabolic byproducts, such as acetic acid (0.95 g/L at 138 h), lactic acid (0.24 g/L at 150 h), and ethanol (0.63 g/L at 120 h), was effectively mitigated, maintaining these byproducts at relatively low levels throughout fermentation.

Fig. 5
figure 5

Fed-batch fermentation of recombinant E. coli GL01 in a 5-L bioreactor for geranyllinalool production (A) and by-product formation (B) with glycerol as the carbon source

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Discussion

The IUP, distinct from the naturally occurring MVA and MEP pathways, involves only two phosphorylation steps. Each IPP or DMAPP molecule requires only two ATP molecules, making the pathway more efficient compared with the MVA pathway, requiring two NADPH and three ATP molecules, and the MEP pathway, requiring three NADPH and two ATP molecules. Furthermore, implementing this IUP pathway not only streamlines the biosynthesis of terpenoid compounds but also overcomes challenges in enhancing their production through metabolic and enzyme engineering, as it minimally disrupts bacterial carbon metabolism. In the present study, through enhanced precursor supply, reduced metabolic pathway competition, and optimized fermentation, geranyllinalool yield reached an unprecedented level of 2.06 g/L in a 5-L batch fermenter. These results indicate that introducing the IUP not only enhances geranyllinalool biosynthesis relative to traditional pathways but also provides new insights for the biosynthesis of other terpenoids.

The strain employed in this study achieved a prenol conversion rate of only 32.6%. Future efforts to enhance geranyllinalool production could focus on the following strategies. (1) In the present study, a limited number of phosphatases were selectively knocked out in the cell. The endogenous phosphatases AphA and YqaB are capable of hydrolyzing DMAP and IP, whereas PhoA can hydrolyze GPP [33, 41, 42]. Furthermore, certain intermediate metabolites within the geranyllinalool pathway are subject to hydrolysis. For instance, the endogenous glucose-1-phosphatase (Agp) has the capacity to hydrolyze IP, whereas dihydroneopterin triphosphate diphosphatase (NudB) can catalyze the conversion of IPP into prenol [32]. In addition, the enzymes undecaprenyl-diphosphatase (YbjG) and phosphatidylglycerol phosphatase B (PgpB) exhibit hydrolytic activity, converting FPP to farnesenol [43, 44]. Future investigations should focus on enhancing geranyllinalool yield by deactivating or inhibiting other pyrophosphatases, such as endogenous dITP/XTP pyrophosphatase (RdgB), 8-oxo-dGTP diphosphatase (MutT), NAD-capped RNA hydrolase (NudC), ADP-ribose pyrophosphatase (NudF), and phosphatase NudJ, which have previously been reported to efficiently hydrolyze isoprenoid substrates [41]. (2) Experimental analysis revealed that geranyllinalool predominantly localizes within cells, and its production yield was not improved by biphasic culture. Membrane engineering, a pivotal technology, aims to optimize the production and tolerance of cells toward lipophilic compounds by precisely modulating microbial membrane structure and functionality [45]. An innovative engineering approach involved using E coli to reconstruct the endogenous outer membrane vesicle (OMV) system, effectively facilitating the extracellular secretion of liposoluble carotenoids, such as lycopene and other carotenoids [46,47,48]. Consequently, mutations in Tol–Pal complex proteins (TolA, TolQ, and TolR) may compromise outer membrane integrity and trigger OMV generation, thereby augmenting the release of lipophilic compounds. Future studies should aim to improve geranyllinalool yield by deleting genes encoding transmembrane proteins such as TolA, TolQ, and TolR to enhance both intracellular storage of hydrophobic metabolites and production of external membrane vesicles [49,50,51].

With the rapid advancement of synthetic biology, particularly in modifying microbial host cells, designing and implementing novel pathways to synthesize target metabolites, as well as applying microbial fermentation technology in terpene compound biosynthesis, offer numerous advantages. These include shorter production cycles, operational simplicity, environmental friendliness, high efficiency, and scalability. Such benefits align with the growing emphasis on sustainable manufacturing practices and the necessary direction for advancing biological synthesis.

Data availability

No datasets were generated or analysed during the current study.

References

  1. Formighieri C, Melis A. Heterologous synthesis of geranyllinalool, a diterpenol plant product, in the cyanobacterium Synechocystis. Appl Microbiol Biotechnol. 2017;101:2791–800.

    Article  CAS  PubMed  Google Scholar 

  2. Heiling S, Llorca LC, Li J, Gase K, Schmidt A, Schafer M, et al. Specific decorations of 17-hydroxygeranyllinalool diterpene glycosides solve the autotoxicity problem of chemical defense in Nicotiana attenuata. Plant Cell. 2021;33:1748–70.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Li J, Halitschke R, Li D, Paetz C, Su H, Heiling S, et al. Controlled hydroxylations of diterpenoids allow for plant chemical defense without autotoxicity. Science. 2021;371:255–60.

    Article  CAS  PubMed  Google Scholar 

  4. Heiling S, Llorca LC, Li J, Gase K, Schmidt A, Schäfer M, et al. Specific decorations of 17-hydroxygeranyllinalool diterpene glycosides solve the autotoxicity problem of chemical defense in Nicotiana attenuata. Plant Cell. 2021;33:1748–70.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Svatoš A, Urbanová K, Valterová I. The first synthesis of Geranyllinalool Enantiomers. Collect Czech Chem Commun. 2002;67:83–90.

    Article  Google Scholar 

  6. Nagel R, Schmidt A, Peters RJ. Isoprenyl diphosphate synthases: the chain length determining step in terpene biosynthesis. Planta. 2019;249:9–20.

    Article  CAS  PubMed  Google Scholar 

  7. Gershenzon J, Dudareva N. The function of terpene natural products in the natural world. Nat Chem Biol. 2007;3:408–14.

    Article  CAS  PubMed  Google Scholar 

  8. Kanwal A, Bilal M, Rasool N, Zubair M, Shah SAA, Zakaria ZA. Total synthesis of terpenes and their biological significance: a critical review. Pharmaceuticals (Basel). 2022;15:1392.

    Article  CAS  PubMed  Google Scholar 

  9. Yao S, Tan X, Huang D, Li L, Chen J, Ming R, et al. Integrated transcriptomics and metabolomics analysis provides insights into aromatic volatiles formation in Cinnamomum cassia bark at different harvesting times. BMC Plant Biol. 2024;24:84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Fajdek-Bieda A, Pawlinska J, Wroblewska A, Lus A. Evaluation of the antimicrobial activity of Geraniol and selected Geraniol Transformation products against Gram-positive Bacteria. Molecules. 2024;29:950.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Trepa M, Sulkowska-Ziaja K, Kala K, Muszynska B. Therapeutic potential of fungal terpenes and terpenoids: application in skin diseases. Molecules. 2024; 29:1183.

  12. Schempp FM, Drummond L, Buchhaupt M, Schrader J. Microbial cell factories for the production of Terpenoid Flavor and Fragrance compounds. J Agric Food Chem. 2018;66:2247–58.

    Article  CAS  PubMed  Google Scholar 

  13. Wang YZ, Jing HY, Li X, Zhang F, Sun XM. Rapid construction of Escherichia coli chassis with genome multi-position integration of isopentenol utilization pathway for efficient and stable terpenoid accumulation. Biotechnol J. 2023;18:e2300283.

    Article  PubMed  Google Scholar 

  14. Chatzivasileiou AO, Ward V, Edgar SM, Stephanopoulos G. Two-step pathway for isoprenoid synthesis. Proc Natl Acad Sci U S A. 2019;116:506–11.

    Article  CAS  PubMed  Google Scholar 

  15. Hayakawa H, Motoyama K, Sobue F, Ito T, Kawaide H, Yoshimura T, et al. Modified mevalonate pathway of the archaeon Aeropyrum pernix proceeds via trans-anhydromevalonate 5-phosphate. Proc Natl Acad Sci U S A. 2018;115:10034–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lund S, Hall R, Williams GJ. An Artificial Pathway for Isoprenoid Biosynthesis decoupled from native Hemiterpene metabolism. ACS Synth Biol. 2019;8:232–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Clomburg JM, Qian S, Tan Z, Cheong S, Gonzalez R. The isoprenoid alcohol pathway, a synthetic route for isoprenoid biosynthesis. Proc Natl Acad Sci U S A. 2019;116:12810–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Rico J, Duquesne K, Petit JL, Mariage A, Darii E, Peruch F, et al. Exploring natural biodiversity to expand access to microbial terpene synthesis. Microb Cell Fact. 2019;18:23.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Ma T, Shi B, Ye Z, Li X, Liu M, Chen Y, et al. Lipid engineering combined with systematic metabolic engineering of Saccharomyces cerevisiae for high-yield production of lycopene. Metab Eng. 2019;52:134–42.

    Article  CAS  PubMed  Google Scholar 

  20. Luo Z, Liu N, Lazar Z, Chatzivasileiou A, Ward V, Chen J, et al. Enhancing isoprenoid synthesis in Yarrowia Lipolytica by expressing the isopentenol utilization pathway and modulating intracellular hydrophobicity. Metab Eng. 2020;61:344–51.

    Article  CAS  PubMed  Google Scholar 

  21. Zhang Y, Cao X, Wang J, Tang F. Enhancement of linalool production in Saccharomyces cerevisiae by utilizing isopentenol utilization pathway. Microb Cell Fact. 2022;21:212.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Zhang XY, Zhu KX, Shi H, Wang X, Zhang Y, Wang F, et al. Engineering for effective and economic production of cis-abienol by optimizing isopentenol utilization pathway. J Clean Prod. 2022;351:131310.

    Article  CAS  Google Scholar 

  23. Wang G, Wang M, Yang J, Li Q, Zhu N, Liu L et al. De novo Synthesis of 2-phenylethanol from glucose by metabolically Engineered Escherichia coli. J Ind Microbiol Biotechnol. 2022;49:kuac026.

  24. Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang S. A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin (Shanghai). 2021;53:620–7.

    Article  CAS  PubMed  Google Scholar 

  25. Hernandez-Benitez LJ, Ramirez-Rodriguez MA, Hernandez-Santoyo A, Rodriguez-Romero A. A trimeric glycosylated GH45 cellulase from the red abalone (Haliotis rufescens) exhibits endo and exoactivity. PLoS ONE. 2024;19:e0301604.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Tong Y, Hu T, Tu L, Chen K, Liu T, Su P, et al. Functional characterization and substrate promiscuity of sesquiterpene synthases from Tripterygium Wilfordii. Int J Biol Macromol. 2021;185:949–58.

    Article  CAS  PubMed  Google Scholar 

  27. Barroso RGMR, Damaso MCT, Machado F, Gonçalves SB. Lactic acid production by is improved by cell recycling and pH control. Fermentation-Basel. 2024;10:149.

    Article  CAS  Google Scholar 

  28. Du C, Wang Y, Gong S. Regulation of the ThiM riboswitch is facilitated by the trapped structure formed during transcription of the wild-type sequence. FEBS Lett. 2021;595:2816–28.

    Article  CAS  PubMed  Google Scholar 

  29. Qiu C, Liu Y, Wu Y, Zhao L, Pei J. Functional characterization and screening of promiscuous kinases and Isopentenyl Phosphate Kinases for the synthesis of DMAPP via a one-Pot Enzymatic Cascade. Int J Mol Sci. 2022;23:12904.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Li L, Wang X, Li X, Shi H, Wang F, Zhang Y, et al. Combinatorial Engineering of Mevalonate Pathway and Diterpenoid synthases in Escherichia coli for cis-abienol production. J Agric Food Chem. 2019;67:6523–31.

    Article  CAS  PubMed  Google Scholar 

  31. Dong H, Chen S, Zhu J, Gao K, Zha W, Lin P, et al. Enhance production of diterpenoids in yeast by overexpression of the fused enzyme of ERG20 and its mutant mERG20. J Biotechnol. 2020;307:29–34.

    Article  CAS  PubMed  Google Scholar 

  32. Kang A, George KW, Wang G, Baidoo E, Keasling JD, Lee TS. Isopentenyl diphosphate (IPP)-bypass mevalonate pathways for isopentenol production. Metab Eng. 2016;34:25–35.

    Article  CAS  PubMed  Google Scholar 

  33. Liu W, Zhang R, Tian N, Xu X, Cao Y, Xian M, et al. Utilization of alkaline phosphatase PhoA in the bioproduction of geraniol by metabolically engineered Escherichia coli. Bioengineered. 2015;6:288–93.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Zhang LH, Fan C, Yang HQ, Xia YY, Shen W, Chen XZ. Biosynthetic pathway redesign in non-conventional yeast for enhanced production of cembratriene-ol. Bioresour Technol. 2024;399:130596.

  35. Wang X, Xu X, Liu J, Liu Y, Li J, Du G et al. Metabolic Engineering of Saccharomyces cerevisiae for efficient Retinol synthesis. J Fungi (Basel). 2023;9:512.

  36. Shah AA, Wang C, Yoon SH, Kim JY, Choi ES, Kim SW. RecA-mediated SOS response provides a geraniol tolerance in Escherichia coli. J Biotechnol. 2013;167:357–64.

    Article  CAS  PubMed  Google Scholar 

  37. Yang D, Park SY, Lee SY. Production of Rainbow colorants by metabolically Engineered Escherichia coli. Adv Sci (Weinh). 2021;8:e2100743.

    Article  PubMed  Google Scholar 

  38. Wepukhulu M, Wachira P, Huria N, Sifuna P, Essuman S, Asamba M. Optimization of Growth Conditions for Chlorpyrifos-Degrading Bacteria in Farm Soils in Nakuru County, Kenya. Biomed Res Int 2024, 2024:1611871.

  39. Bhatwa A, Wang W, Hassan YI, Abraham N, Li XZ, Zhou T. Challenges Associated with the formation of recombinant protein inclusion bodies in Escherichia coli and Strategies to address them for Industrial Applications. Front Bioeng Biotechnol. 2021;9:630551.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Gecse G, Labunskaite R, Pedersen M, Kilstrup M, Johanson T. Minimizing acetate formation from overflow metabolism in Escherichia coli: comparison of genetic engineering strategies to improve robustness toward sugar gradients in large-scale fermentation processes. Front Bioeng Biotechnol. 2024;12:1339054.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Lei D, Qiu Z, Wu J, Qiao B, Qiao J, Zhao GR. Combining metabolic and Monoterpene Synthase Engineering for De Novo Production of Monoterpene Alcohols in Escherichia coli. ACS Synth Biol. 2021;10:1531–44.

    Article  CAS  PubMed  Google Scholar 

  42. Wang X, Wu J, Chen J, Xiao L, Zhang Y, Wang F, et al. Efficient biosynthesis of R-(-)-Linalool through adjusting the expression strategy and increasing GPP Supply in Escherichia coli. J Agric Food Chem. 2020;68:8381–90.

    Article  CAS  PubMed  Google Scholar 

  43. Wang C, Park JE, Choi ES, Kim SW. Farnesol production in Escherichia coli through the construction of a farnesol biosynthesis pathway - application of PgpB and YbjG phosphatases. Biotechnol J. 2016;11:1291–7.

    Article  CAS  PubMed  Google Scholar 

  44. Wang J, Zhu L, Li Y, Xu S, Jiang W, Liang C, et al. Enhancing Geranylgeraniol Production by Metabolic Engineering and Utilization of Isoprenol as a substrate in Saccharomyces cerevisiae. J Agric Food Chem. 2021;69:4480–9.

    Article  CAS  PubMed  Google Scholar 

  45. Wu T, Jiang J, Zhang H, Liu J, Ruan H. Transcending membrane barriers: advances in membrane engineering to enhance the production capacity of microbial cell factories. Microb Cell Fact. 2024;23:154.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Wu T, Li SW, Ye LJ, Zhao DD, Fan FY, Li QY, et al. Engineering an Artificial membrane vesicle trafficking system (AMVTS) for the excretion of β-Carotene in Escherichia coil. ACS Synth Biol. 2019;8:1037–46.

    Article  CAS  PubMed  Google Scholar 

  47. Fordjour E, Bai ZH, Li SH, Li SJ, Sackey I, Yang YK, et al. Improved membrane permeability via Hypervesiculation for in situ recovery of Lycopene in Escherichia coli. ACS Synth Biol. 2023;12:2725–39.

    Article  CAS  PubMed  Google Scholar 

  48. Song FQ, Qin ZJ, Qiu K, Huang ZS, Wang L, Zhang H, et al. Development of a vitamin B5 hyperproducer in Escherichia coli by multiple metabolic engineering. Metab Eng. 2024;84:158–68.

    Article  CAS  PubMed  Google Scholar 

  49. Wu T, Ye L, Zhao D, Li S, Li Q, Zhang B, et al. Engineering membrane morphology and manipulating synthesis for increased lycopene accumulation in Escherichia coli cell factories. 3 Biotech. 2018;8:269.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Gao Q, Chen H, Wang G, Yang W, Zhong X, Liu J, et al. Highly efficient production of Menaquinone-7 from glucose by metabolically Engineered Escherichia coli. ACS Synth Biol. 2021;10:756–65.

    Article  CAS  PubMed  Google Scholar 

  51. Wu T, Li S, Ye L, Zhao D, Fan F, Li Q, et al. Engineering an Artificial membrane vesicle trafficking system (AMVTS) for the excretion of beta-carotene in Escherichia coli. ACS Synth Biol. 2019;8:1037–46.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

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Funding

This work was supported by the Key Research Projects of the Science and Technology Department of Henan Province (232102311136, 232102320129 and 242102320122) and Key Research and Development Projects of Tobacco Corporation, China (110202102020).

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Authors and Affiliations

  1. Laboratory of Biotransformation and Biocatalysis, School of Tobacco Science and Engineering, Zhengzhou University of Light Industry, No.136 Ke Xue Avenue, Zhengzhou, Henan, 450002, People’s Republic of China

    Jin Chang, Xinduo Wei, Deyu Liu, Qian Li, Chong Li, Jianguo Zhao & Guanglu Wang

  2. Laboratory of Synthetic Biology, Shandong Binzhou Animal Science and Veterinary Medicine Academy, Research Institution of Veterinarian, No.777 Chang Jiang 5th Road, Binzhou, Shandong Province, 256600, China

    Likun Cheng

Authors
  1. Jin Chang
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  2. Xinduo Wei
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  3. Deyu Liu
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  4. Qian Li
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  5. Chong Li
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  6. Jianguo Zhao
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  7. Likun Cheng
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  8. Guanglu Wang
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Contributions

Jin Chang: Writing original draft, investigation, laboratory and fermentation experiments. Xinduo Wei: Investigation, laboratory and fermentation experiments. Deyu Liu: Fermentation experiments and data analysis. Qian Li: Investigation; analysis. Chong Li: Genetic designs, reviewing and editing original draft. Jianguo Zhao: Conceptualization, reviewing and editing the original draft. Likun Cheng: Conceptualization, methodology and manuscript preparation. Guanglu Wang: Project management and experimental design, reviewing, editing original draft and funding acquisition. All authors read and approved the final manuscript.

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Correspondence to Likun Cheng or Guanglu Wang.

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Chang, J., Wei, X., Liu, D. et al. Engineering Escherichia coli via introduction of the isopentenol utilization pathway to effectively produce geranyllinalool. Microb Cell Fact 23, 292 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12934-024-02563-2

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Keywords

Microbial Cell Factories

ISSN: 1475-2859

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