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Bacterial biosynthesis of abietane-type diterpene ferruginol from glucose

Microbial Cell Factories volume 24, Article number: 67 (2025) Cite this article

Abstract

Background

Microbial supply of plant extracts is a promising biomanufacturing strategy that requires engineering of metabolic pathways and enzymes. This study presents the engineering of Corynebacterium glutamicum for heterologous production of diterpenes miltiradiene and ferruginol.

Results

Through targeted metabolic pathway modifications, including inactivation of pyruvate carboxylase and phytoene synthase, the HL01 strain was optimized to enhance pyruvate and geranylgeranyl pyrophosphate (GGPP) pools. Overexpression of key MEP pathway enzymes (Dxs and Idi) and implementation of three GGPP synthase modules further boosted diterpene synthesis. Then, combining those modules with diterpene synthase (DiTPS) and intact P450 reductase modules (CYP76AH1 and CPR1) enabled production of miltiradiene (ferruginol equivalent) at 237.46 ± 34.8 mg/L and ferruginol at 107.34 ± 1.2 mg/L under constant glucose feeding, respectively.

Conclusions

Modular gene expression for heterologous metabolic pathway can be optimized for bacterial biosynthesis. This is the first demonstration of ferruginol production in bacteria. These findings pave the way for further optimization of diterpene biosynthesis through pathway engineering and module integration in bacterial systems.

Introduction

Ferruginol (C20H30O), an abietane-type tricyclic diterpene, is naturally obtained from the bark and roots of Chinese medicinal herb Salvia miltiorrhiza that has been widely used for the treatment of cerebrovascular and cardiovascular diseases for hundreds of years [1, 2]. Known for its valuable pharmacological properties, including anticancer activity [3], the sustainable production of diterpenes including ferruginol has garnered significant attention. However, traditional plant-based extraction methods are hindered by low yields, while climate change impacts plant cultivation, leading to supply instabilities [4]. Recent metabolic engineering and synthetic biology have demonstrated potential sustainable production of the diterpenes to overcome the limited supply [5, 6]. In addition, bacterial Cytochrome P450s (CYPs) using a kaurene-scaffold have facilitated high-throughput exploration of terpenoids and their chemical diversification [7]. This systematic approach could be beneficial to develop various bacterial cell factories for CYP-mediated products.

To biosynthesize ferruginol and its derivatives such as carnosic acid [1], forskolin [8], or tanshinones [9], microbial systems, including yeasts and bacteria, require the development of heterologous metabolic pathway engineering to optimize the mevalonate pathway or the methylerythritol 4-phosphate (MEP) pathway for efficient isopentenyl diphosphate (IPP) supply. Additionally, the expression of plant-derived enzymes is essential for constructing diterpene backbones and producing oxidized diterpenes [7]. Specifically, for production of ferruginol, geranylgeranyl pyrophosphate (GGPP) synthase combines one dimethylallyl diphosphate (DMAPP) and three IPP units to produce GGPP (Fig. 1) [10,11,12]. The cyclization of GGPP to copalyl diphosphate (CPP) is catalyzed by class II terpene synthase (CPS). This is followed by further cyclization of CPP, initiated by class I terpene synthase, specifically kaurene synthase-like enzymes, leading to the formation of tricyclic miltiradiene. Subsequently, ferruginol and its derivatives are produced through oxidation mediated by CYP enzymes.

Fig. 1
figure 1

Metabolic engineering for diterpene production in engineered C. glutamicum. The metabolic pathway for diterpene production in engineered C. glutamicum was divided into four distinct modules: OverMEP, GGPPS, DiTPS, and P450R. The parental strain, C. glutamicum HL01, was transformed with a series of plasmids, each encoding genes corresponding to these specific metabolic modules. The native pyc and crtB genes (shown in red) were inactivated in the strain HL01. See the Table 1 for the strains and plasmids used in this study. Abbreviations: Metabolite: G3P, glyceraldehyde 3-phosphate; Pyr, Pyruvate; DXP, 1-deoxy-D-xylulose 5-phosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; Enzyme: Dxs DXP synthase; Idi, isopentenyl diphosphate isomerase; GGPPS, Bts1, or IdsA, geranylgeranyl diphosphate synthase; ERG20(F96C), farnesyl diphosphate synthase; CPS, Class-II copalyl diphosphate synthase; KSL, Class-I kaurene synthase-like; CYP76AH1, cytochrome P450 oxidase; CPR, cytochrome P450 reductase

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Table 1 Bacteria strains and plasmids used in this study
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For microbial supply of diterpenes and its oxidized products, Saccharomyces cerevisiae has been engineered to overproduce miltiradiene [10, 11, 13], ferruginol [8, 14], carnosic acid [1, 12, 15], forskolin [8], and tanshinones [9]. Similarly, engineered Escherichia coli have been demonstrated heterologous production of taxadiene and its oxidized derivative, taxadien-5α-ol [16, 17]. E. coli has been utilized to biosynthesize kaurene and its oxidized product, steviol [18]. Regardless of species and strains, the production of diterpenes and their oxidized derivatives primarily depends on the optimization of key factors. These include enhancing the metabolic pathway for IPP supply, achieving efficient terpene synthase expression through optimized truncation of the protein sequence or metabolite channeling, and identifying specific pairs of CYP enzymes and their cognate CYP reductases (CPR). However, no studies to date have reported the production of miltiradiene or its oxidized derivatives in E. coli or other bacterial systems.

Corynebacterium glutamicum, a non-pathogenic industrial host widely used for amino acid production, has been extensively studied as one of microbial cell factories for the production of both natural products (natural-inherent; natural-noninherent) and non-natural products (non-natural-noninherent; non-natural-created) [19, 20]. Amino acids, diamines, alcohols, organic acids, biopolymers, and terpenoids have been produced in metabolically engineered C. glutamicum. In addition, strain-specific genetic tools have been developed to facilitate host engineering [21, 22], alongside synthetic expression vectors and modular DNA parts [23]. Furthermore, C. glutamicum exhibits low susceptibility to phages, thanks to the development of the prophage-free strain C. glutamicum MB001, which lacks three prophages (CGP1, CGP2, and CGP3) [24]. Thus, engineered C. glutamicum serves as a promising cell factory for the phage-free scale-up production of natural compounds, including terpenoids.

Therefore, in the present study, we metabolically engineered C. glutamicum to produce miltiradiene and ferruginol from glucose using a metabolic engineering approach to overcome the native metabolic pathway. In addition, we investigated whether combinatorial modular engineering to increase GGPP pools and to produce miltiradiene and ferruginol in C. glutamicum.

Methods

Bacterial strains and growth condition

E. coli DH5α [25] and C. glutamicum ATCC 13,032 (wild-type) were used in this study (Table 1). E. coli strains were grown in Lysogeny Broth (LB) medium (10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl) at 37 ºС on a rotary shaker at 200 rpm. When appropriate, the medium was supplemented with 30 µg/mL chloramphenicol or 50 µg/mL kanamycin. For strain development, C. glutamicum ATCC 13,032 and its derivatives were cultivated in brain heart infusion sorbitol-supplemented (BHIS) medium at 30 ºС on a rotary shaker at 120 rpm [21, 23]. When appropriate, the medium was supplemented with 7.5 µg/mL chloramphenicol, or 25 µg/mL kanamycin, and/or 50 µg/mL spectinomycin.

To construct gene inactivation mutants using the high-fidelity cytosine base editor for C. glutamicum (HF-CBE-STOP [21]), pCoryne-BE3-R132E was co-transformed to C. glutamicum WT with either pCoryne2-sgRNA-pyc(Q279Ter) or pCoryne2-sgRNA-crtB(Q4Ter), obtained from the laboratory sgRNA collection for C. glutamicum. After performing base editing to inactivate the pyc and crtB genes, we obtained the C. glutamicum HL01 strain and confirmed the genomic sequence at the targeted editing regions (Table 1). The various plasmids for gene expression for diterpene biosynthesis were introduced into C. glutamicum by electroporation, and strain validation was performed using colony PCR and DNA sequencing [26].

For diterpene production, C. glutamicum ATCC 13,032 derivative strains were pre-cultivated overnight in Brain Heart Infusion-supplemented with 91 g/L sorbitol (BHIS) medium at 30 °C and 250 rpm in 15 mL glass tubes. Following pre-cultivation, the strains were transferred to a baffled 96-deep well plate (DWP; Tomas Scientific, NJ, USA) and incubated aerobically at 30 °C and 1200 rpm in 0.6 mL of CgXII defined medium per well, containing 2% (w/v) glucose as the initial carbon source [27]. The DWP was covered with a gas-permeable sealing film (AeraSeal, Excel Scientific, Inc., CA, USA) to prevent contamination and to allow uniform gas exchange. When necessary, the medium was supplemented with 7.5 µg/mL of chloramphenicol (Cm), or 50 µg/mL of kanamycin (Km). Cell cultivation was carried out with the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce gene expression for diterpene production, introduced 4 h after inoculation.

Plasmid construction

All of the plasmids used for in this study were listed in Table 1. For the supplying GGPP pools (GGPPS module), idsA gene was PCR-amplified from the genomic DNA of C. glutamicum. Also, the truncated GGPPS gene [28] from Taxus candensis, in which hydrophobic N-terminal 294 amino-acid residues for soluble expression and Bts1-[GGT GGT GGT TCC]-ERG20(F96C) genes [29, 30] from S. cerevisiae were codon-optimized for C. glutamicum (Genscript) and were cloned into a CoryneBrick vector [23]. To overexpress key enzymes of the MEP pathway (OverMEP module, DI), the dxs gene, the idi gene from C. glutamicum, were cloned into the plasmid (pTctGGPPS or pIdsA or pBE) using the standard BglBrick cloning method [23], yielding GGPPS-OverMEP module plasmids (Table 1). For the biosynthesis of miltiradiene (DiTPS module), the KSL1 and CPS1 genes [11] from S. miltiorrhiza were codon-optimized for C. glutamicum (Genscript, USA) and were cloned into a high-copy origin CoryneBrick vector (pBbEC1k-RFP), resulting in plasmids pKC and pCK. A KSL and CPS were fused together with a GGGS linker [11]. Additionally, truncated versions of the KSL and CPS genes was also cloned into a pBbEC1k-RFP, yielding ptKtC. Truncations of KSL and CPS were performed, removing amino acids M1 to C47 in SmKSL and M1 to S35 in SmCPS, respectively. A start codon (ATG) was introduced to ensure proper translation of the truncated versions. DNA sequences used in this study available in Supplementary Table S1.

For biosynthesis of ferruginol (P450R module), the CYP76AH1 (GenBank accession no. JX422213) and CPR1 (no. CBX24555) genes [14] from S. miltiorrhiza were codon-optimized for C. glutamicum (Genscript, USA) and they were cloned into a plasmid ptKtC, yielding ptKtC-CP450CR. Additionally, truncated CYP76AH1 (M1-F22) and SmCPR (M1-W60) gene were modified, constructing ptKtC-tCP450tCR. Transmembrane helices of CYP76AH1 and SmCPR were predicted by the TMHMM 2.0 [31] (Supplementary Fig. S1). A start codon (ATG) was introduced to ensure proper translation of the truncated versions.

To investigate the localization of CYPs in C. glutamicum, the CYP76AH1 gene and a truncated version of CYP76AH1 (M1-F22) were cloned into the vector pBbEC1k-RFP. The egfp gene [23] was fused to both the CYP76AH1 and truncated CYP76AH1 genes by removing the stop codon of CYP76AH1 and the start codon of egfp, generating pCP450-eGFP and ptCP450-eGFP, respectively.

Quantification of miltiradiene, abietatriene, and ferruginol

Cultured cells (1 mL) were harvested by centrifugation at 3600 × g for 10 min. After discarding the supernatant, the cell pellets were washed with distilled water. For extraction, the cell pellets were resuspended in 1 mL of distilled water. Subsequently, 1 mL of hexane (spiked with 1-eicosene as an internal standard at a final concentration of 10 mg/L) was added to the sample. The mixture was then transferred to 2 mL screw-top tubes containing 250 mg of glass beads (150–212 μm; Sigma-Aldrich). After disrupting the cells using a bead beater (Taco™ Prep, GeneReach Biotechnology Corp., Taichung City, Taiwan), the extraction mixture was centrifuged at 3600 × g for 10 min, and the supernatant from hexane layer was collected for further analysis. The sample was identified and quantified by both gas chromatography-mass spectrometry (GC-MS) and GC-Flame Ionization Detector (FID). In particular, GC-FID was employed for routine analyses after GC-MS analysis. GC-MS (Agilent 5977B GC/MSD) is equipped with an Agilent HP-5MS column (30 m \(\:\times\:\) 0.25 mm, thickness 0.25 μm): Carrier gas, He (1 mL/min). The initial oven temperature was set at 50 oC followed by a 40 oC/min gradient to 170 oC, and 20 oC/min in gradient to 240 oC, 40 oC /min gradient to 300 oC, and a final 1 min hold. The mass spectrometer was operated at 70 eV with selected ion monitoring (SIM), and the mass range was m/z 29 − 300. The ion source temperature was maintained at 230 °C. GC-FID (Agilent 7890B, Waldbronn, Germany) is equipped with an Agilent 122–5032 DB-5 column (30 m \(\:\times\:\) 0.2 mm, thickness 0.11 μm): Carrier gas, N2 (1mL/min). The initial oven temperature was set at 50 oC for 2 min followed by a 40 oC/min gradient to 170 oC, and 20 oC/min in gradient to 240 oC, 40 oC /min gradient to 300 oC, and a final 1 min hold.

After GC analysis, the concentrations of miltiradiene and abietatriene were determined by converting their peak areas to ferruginol equivalent normalized by internal standard, using a standard curve prepared with ferruginol (Merck, USA) (CAS:3452-07-01, Cat.: 184411). For quantification of ferruginol, an authentic standard of ferruginol (CAS: 514-62-5, Cat.: TBZ2395), purchased from ChemFaces (Wuhan, China), was utilized to generate a corresponding standard curve.

Quantification of glucose

Glucose in the supernatant was quantified using high-performance liquid chromatography (HPLC) as described previously [32]. Briefly, the culture supernatant was passed through a syringe filter (pore size = 0.45 \(\:\mu\:\)m). The concentation of glucose was detected by a HPLC (Agilent 1260, Waldbronn, Germany) equipped with refractive index detector (RID) and a Hi-Plex H, 7.7 \(\:\times\:\:\)300 mm column (Agilent Technologies) under the following conditions: sample volume of 20 µL, mobile phase of 5 mM H2SO4, flow rate of 0.6 mL/min, and column temperature of 65 oC.

Confocal microscopy

Cells were cultivated in the CgXII medium with 2% (w/v) glucose and harvested 48 h after inoculation. After harvesting, the cells were washed three times with 6.5 mM phosphate-buffered saline (PBS, pH 7.4) and resuspended in the same buffer. To immobilize the cells and prevent movement, the suspension was mixed with 0.25% agarose. The prepared samples were then imaged using a confocal microscope (Zeiss LSM 980 laser scanning microscope with Airyscan 2 module, Zeiss, Germany) with an excitation filter at 490 nm and an emission filter at 520 nm. Images were acquired and processed using ZEN software.

Results and discussion

Synthetic metabolic pathway modules in C. glutamicum for diterpene productions

Previously, we demonstrated that inactivating both the pyruvate carboxylase (PC), which converts pyruvate to oxaloacetate, and the major geranylgeranyl pyrophosphate synthase (IdsA), which competes with squalene synthase, significantly increased triterpene squalene production in C. glutamicum [32, 33]. Similarly, for diterpene production, the native metabolic pathway of C. glutamicum was engineered to enhance the pools of pyruvate and geranylgeranyl pyrophosphate (GGPP) instead of farnesyl pyrophosphate (FPP). This was achieved by inactivating the pyc gene (pyc::Q279Ter) encoding PC and the crtB gene (crtB::Q4Ter) encoding phytoene synthase (an enzyme that converts GGPP to phytoene) using a cytosine base editor [21]. The base editor introduced premature translational termination codons in these target genes. This engineering resulted in the construction of strain HL01 (Table 1).

Next, to further enhance the pools of isopentenyl pyrophosphate (IPP) biosynthesized from glyceraldehyde 3-phospahte and pyruvate, a C5 isoprenoid building block for diterpene synthesis, we overexpressed key enzymes of the methylerythritol 4-phosphate (MEP) pathway [32, 34, 35]. In the OverMEP module, Dxs (1-deoxy-xylulose-5-phosphate synthase) and Idi (isopentenyl diphosphate isomerase) were selected for overexpression in C. glutamicum HL01 (Fig. 1). To synthesize geranylgeranyl pyrophosphate (GGPP), the substrate for diterpene synthase derived from IPP, we developed three distinct GGPP synthase (GGPPS) modules. The first GGPPS module involved the overexpression of the native idsA gene, which is known as the primary GGPPS in C. glutamicum and exhibits high catalytic efficiency with DMAPP and IPP [36]. The second GGPPS module featured the co-expression of GGPPS from Canadian yew Taxus canadensis (used for production of taxanes), which has been utilized for diterpene levopimaradiene production in E. coli [37]. The third GGPPS module utilized a fusion protein of Bts1 and the ERG20(F96C) variant [29], which has been previously expressed in S. cerevisiae for miltiradiene production and demonstrated significant improvements in production efficiency [11]. To optimize this system for C. glutamicum, we constructed a codon-optimized Bts1-ERG20(F96C) fusion GGPPS. In combination with the OverMEP and GGPPS modules, we constructed three distinct plasmids: pTctGGPPS-DI, pIdsA-DI, and pBE-DI.

The DiTPS and P450R modules were designed to enable the biosynthesis of ferruginol from GGPP. The DiTPS module facilitates the conversion of GGPP to miltiradiene using diterpene synthase, while the P450R module catalyzes the hydroxylation of miltiradiene via CYP monooxygenase in conjunction with a CYP reductase. In the DiTPS module, we selected the CPS gene, encoding copalyl diphosphate synthase, and the KSL gene, encoding a kaurene synthase-like enzyme, both derived from the Chinese medicinal herb S. miltiorrhiza [11]. Subsequently, we evaluated different expression orders of KSL and CPS to optimize their functionality because different translational order in an operon could cause slightly different protein abundance in a host. The first gene has more time for translation then the second gene [38]. We constructed two different versions of the plasmid: pKC and pCK. In the P450R module, the CYP76AH1 and SmCPR genes from S. miltiorrhiza, which have been demonstrated to facilitate ferruginol production in yeast systems [14], were integrated into the DiTPS module to produce ferruginol from GGPP.

Heterologous production of miltiradiene and abietatriene in engineered C. glutamicum

To produce the miltiradiene in C. glutamicum, we constructed the strain HL01 harboring two distinct vectors, representing six different combinations of the GGPPS, OverMEP, and DiTPS modules (Fig. 2). First, we cultivated the six strains using 2% (w/v) glucose as the sole carbon source for 48 h. Analysis of the extracted samples revealed the presence of a miltiradiene peak, along with a peak corresponding to abietatriene, a by-product formed through the spontaneous oxidation of miltiradiene (Fig. 2A). Subsequently, due to the unavailability of commercially purchased miltiradiene and abietatriene, we used ferruginol as a structurally relevant equivalent standard to quantify the relative levels of miltiradiene and abietatriene in the engineered strains. This approach enabled us to measure the relative levels of miltiradiene and abietatriene across the six module combinations (Fig. 2B). As a result, the combination of pBE-DI and pCK modules yielded 4.5 ± 1.0 mg miltiradiene (ferruginol equivalent) per gDCW and 0.63 ± 0.1 mg abietatriene (ferruginol equivalent) per gDCW in C. glutamicum HL01 at 48 h. Regardless of the module constructs, the abietatriene level consistently accounted for 19% of the miltiradiene level. Interestingly, the ratio of abietatriene to miltiradiene was higher in engineered C. glutamicum compared to engineered yeast [1, 11, 39]. This difference is likely attributed to the small-scale cultivation used in our study, which involved microplates agitated at 1200 rpm, providing higher oxygen levels that facilitated spontaneous oxidation. The GGPPS module incorporating a fusion protein of Bts1 and the ERG20(F96C) variant demonstrated better miltiradiene production compared to other GGPPS modules (Student t-test, p-value < 0.05; Fig. S2). In the DiTPS module, the gene expression order of KSL and CPS did not significantly affect diterpene production (Fig. S2).

Fig. 2
figure 2

Quantification of miltiradiene and abietatriene in engineered C. glutamicum. (A) GC-MS measurement of miltiradiene (C20H32) and abietatriene (C20H30) produced in engineered C. glutamicum. 1-eicosene (C20H40) was injected into sample as internal standard and ferruginol was used for quantification of miltiradiene and abietatriene (ferruginol equivalent mg/gDCW). (B) Heterologous diterpenes (miltiradiene and abietatriene) were produced in engineered C. glutamicum strains harboring various plasmids. A two-plasmid system was used for gene expressions: a pBL1-origin vector harboring GGPPS and OverMEP modules and a pCG1-origin vector harboring DiTPS module. Miltiradiene (grey) and abietatriene (white) were quantified as ferruginol equivalent mg/gDCW using GC-FID. Data represent mean values of at least triplicated cultivations, and error bars represent standard deviations. LK stands for a linker

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To investigate the impact of carbon source availability on production, 1% (w/v) glucose was added after the depletion of the initial 2% (w/v) glucose. This supplementation with an additional carbon source resulted in increased diterpene production. The combination of pBE-DI and pKC modules resulted in 7.61 ± 1.0 mg miltiradiene (ferruginol equivalent) per gDCW and 1.02 ± 0.05 mg abietatriene (ferruginol equivalent) per gDCW in C. glutamicum HL01. So, we selected C. glutamicum HL01 harboring pBE-DI and pKC as the diterpene-producing strain for the next improvement.

Improvement of diterpene production using constant glucose feeding

Since we observed increased diterpene production with additional glucose feeding, we implemented a fed-batch fermentation strategy to enhance diterpene yields [40, 41]. First, we measured cell growth and diterpene production (miltiradiene and abietatriene) in the C. glutamicum strain HL01 harboring pBE-DI and pKC plasmids during cultivation with a single glucose feeding (Fig. 3A). Interestingly, the titers and contents of miltiradiene and its derivative, abietatriene, decreased after glucose depletion during cultivation. C. glutamicum has cytochrome P450 enzymes involved in various oxidation reactions. For instance, certain aromatic oxygenases, such as cytochrome P450 hydroxylases, can oxidize compounds like indole at specific positions, leading to further oxidation products [42]. However, C. glutamicum lacks potential C20-oxidases (CYPs) with promiscuous activity that could convert miltiradiene into oxygenated derivatives. Then, we speculated that miltiradiene undergoes non-enzymatic degradation during deep well plate cultivation at 1200 rpm, possibly driven by an unidentified mechanism. To direct carbon flux toward miltiradiene production and prevent glucose depletion in the cells, we implemented continuous glucose feeding into the medium. This approach resulted in an increase in the concentration of miltiradiene (ferruginol equivalent) at 126.7 ± 0.9 mg/L at 96 h, although the relative content of miltiradiene was slightly lower at that time point.

Fig. 3
figure 3

Miltiradiene and abietatriene productions in engineered C. glutamicum (A) A fed-batch cultivation of engineered strains (C. glutamicum HL01 harboring pBE-DI pKC) was conducted, starting with 2% (w/v) initial glucose, followed by a single glucose supplementation (1%, w/v) during the cultivation (left panel). Optical densities at 600 nm (OD600, black square) and glucose concentrations (g/L, blue circle) in the medium were measured (upper panel). Relative diterpene contents (ferruginol equivalent mg/gDCW) and concentrations (ferruginol equivalent mg/L) profiles were measured for miltiradiene (black bar, square symbol) and abietatriene (red bar, circle symbol), respectively (lower panel). A fed-batch cultivation of the engineered strain was conducted, starting with 2% (w/v) initial glucose, followed by multiple glucose supplementations (1%, w/v) during the cultivation (right panel). Data represent mean values of at least triplicated cultivations, and error bars represent standard deviations. (B) A fed-batch cultivation of engineered strains (C. glutamicum HL01 harboring pBE-DI ptKtC) was conducted with truncation versions of KSL and CPS. LK stands for a linker. See the details above

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To further enhance the production titer, we investigated the channeling of GGPP to miltiradiene via CPP by expressing fused KSL and CPS enzymes. Additionally, we employed a truncated KSL (tKSL) lacking the chloroplast transit peptide (M1-C47) and a truncated CPS (tCPS) lacking the chloroplast transit peptide N-terminal region (M1-S35) to improve catalytic efficiency and promote soluble protein expression [10, 15]. This strategy has been demonstrated to benefit heterologous diterpene production by increasing the local concentration of intermediates through enhanced metabolic channeling. As a result, we observed a 1.26-fold increase in miltiradiene production at 48 h in C. glutamicum HL01 harboring pBE-DI and ptKtC (10.3 ± 0.8 ferruginol equivalent mg/gDCW), compared to C. glutamicum HL01 harboring pBE-DI and pKC (8.2 ± 2.4 ferruginol equivalent mg/gDCW) (Fig. 3B). With continuous glucose feeding, C. glutamicum HL01 harboring pBE-DI and ptKtC maintained consistent production throughout the cultivation period, achieving a final miltiradiene (ferruginol equivalent) titer of 154.5 ± 11.8 mg/L at 96 h. A 10% non-enzymatic degradation of miltiradiene and abietatriene occurred between 48 h and 96 h in C. glutamicum HL01 strain harboring pBE-DI ptKtC.

Heterologous production of ferruginol in C. glutamicum

Next, we sought to co-express the P450R module in C. glutamicum HL01 harboring pBE-DI and ptKtC for miltiradiene production. Among six CYP genes identified in the rhizome of S. miltiorrhiza, CYP76AH1 demonstrated catalytic activity in converting miltiradiene to ferruginol when co-expressed with CPR in S. cerevisiae [14]. Based on this, we selected the CYP76AH1 gene and its cognate CPR1 as the P450R module for ferruginol production in C. glutamicum. Additionally, we evaluated a truncated version of CYP76AH1 and SmCPR to enhance soluble expression and improve ferruginol production by testing either C. glutamicum HL01 pBE-DI ptKtC-CP450CR or C. glutamicum HL01 pBE-DI ptKtC-tCP450tCR.

First, we tested whether the CYP76AH1 protein or its truncated version were expressed in the cytoplasmic membrane or the cytosol, respectively. A single transmembrane helix of CYP76AH1 was predicted (P5-F22; Fig. S1). To investigate its soluble expression, a truncated version of CYP76AH1, lacking the M1-F22 region and incorporating the start codon ATG, was expressed in the C. glutamicum wild-type. Confocal microscopy analysis confirmed that the intact CYP76AH1 was expressed in a membrane-associated manner, while the truncated CYP76AH1 protein was correctly localized to its intended cytosol. (Fig. 4). Subsequently, based on the transmembrane analysis using the TMHMM 2.0 [31], SmCPR was truncated by removing the M1-W60 region. The truncated version of SmCPR, incorporating the start codon ATG, was used for further production studies without confocal analysis, as its topology was simpler compared to CYP76AH1 (Fig. S1).

Fig. 4
figure 4

Confocal microscopy analysis of Cytochrome CYP76AH1 expression in engineered C. glutamicum. The C. glutamicum HL01 strains harboring a plasmid of either pCP450-eGFP (A) or ptCP450-eGFP (B) were prepared. Fluorescence imaging was performed using a Zeiss LSM 980 laser scanning microscope equipped with an Airyscan 2 module (Zeiss, Germany). Images were acquired and processed with ZEN software, utilizing an excitation filter at 490 nm and an emission filter at 520 nm. Scale bar is 2 μm

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Next, we cultivated engineered strains (C. glutamicum HL01 pBE-DI ptKtC-CP450CR, a intact P450R module; C. glutamicum HL01 pBE-DI ptKtC-tCP450tCR, a truncated P450R module) with glucose as sole carbon source and measured the diterpene and ferruginol productions throughout the cultivation period. As a result, ferruginol was successfully produced in engineered bacteria for the first time (Fig. 5). Notably, only the C. glutamicum HL01 strain harboring pBE-DI ptKtC-CP450CR module produced ferruginol. In addition, the strain produced miltiradiene and abietatriene as well (Fig. 6). Then, we analyzed the relative production levels of diterpene and the absolute production levels of ferruginol in the heterologous metabolic pathway. Under both single and multiple glucose feeding conditions, the relative levels of miltiradiene produced in the ferruginol-producing strain were significantly decreased at 48 h (Fig. 6A), compared to the parental miltiradiene-producing strain (Fig. 3B). When we calculated the molar conversion of ferruginol from the abietatriene and miltiradiene contents quantified as ferruginol equivalent, we found that 13.0% of the diterpene contents was converted to ferruginol between 24 h and 96 h of cultivation (Fig. 6A). This conversion in C. glutamicum HL01 strain harboring pBE-DI ptKtC-CP450CR was higher than the unidentified non-enzymatic degradation of abietatriene, which resulted in a 10% loss from 48 h to 96 h in C. glutamicum HL01 strain harboring pBE-DI ptKtC (Fig. 3B). The increase in ferruginol production was attributed to the catalytic activity of the P450R module, demonstrating a spontaneous oxidation-coupled enzymatic conversion of miltiradiene to ferruginol since the molar conversion of sole abietatriene to ferruginol exceeded 100% between 24 h and 96 h of cultivation.

Fig. 5
figure 5

Identification of ferruginol produced in engineered C. glutamicum The chromatographic profile and mass fragments of the sample were analyzed using GC-MS. The sample was extracted from engineered cells, and the mass fragment ions (m/z) obtained at retention time 38.3 min were compared with the NIST mass spectrum library (entry #161789, ferruginol)

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Fig. 6
figure 6

Ferruginol production in engineered C. glutamicum. (A) A fed-batch cultivation of engineered strains (C. glutamicum HL01 harboring pBE-DI ptKtC-CP450CR) was conducted, starting with 2% (w/v) initial glucose, followed by a single glucose supplementation (1%, w/v) during the cultivation (left panel). Optical densities at 600 nm (OD600, black square) and glucose concentrations (g/L, blue circle) in the medium were measured. Relative diterpene contents (ferruginol equivalent mg/gDCW) profiles were measured for miltiradiene (black bar, square symbol) and abietatriene (red bar, circle symbol), respectively (left Y-axis). Ferruginol (C20H30O) content profiles were measured (mg/gDCW, blue bar, triangle symbol) (left Y-axis). Concentration of diterpene (ferruginol equivalent mg/L) and ferruginol (mg/L) profiles were measured, respectively (right Y-axis). A fed-batch cultivation of the engineered strain was conducted, starting with 2% (w/v) initial glucose, followed by multiple glucose supplementations (1%, w/v) during the cultivation (right panel). Data represent mean values of at least triplicated cultivations, and error bars represent standard deviations. (B) A fed-batch cultivation of engineered strains (C. glutamicum HL01 harboring pBE-DI ptKtC-tCP450tCR) was conducted with truncation versions of CYP76AH1 and SmCPR. LK stands for a linker. See the details above

Full size image

Interestingly, the level of abietatriene in the ferruginol-producing strain was higher than that in the miltiradiene-producing strain, regardless of glucose feeding conditions. Co-expression of the intact P450R could promote spontaneous oxidation of miltiradiene to form abietatriene and catalyzes a single oxidation at C12 position of abietatriene to form ferruginol [14, 15, 43]. On the contrary, the strain carrying a truncated version of the P450R module (C. glutamicum HL01 pBE-DI ptKtC-tCP450tCR) did not produce ferruginol although lower miltiradiene levels and higher abietatriene levels (Fig. 6B) compared to the miltiradiene-producing strain (C. glutamicum HL01 harboring pBE-DI ptKtC) (Fig. 3B). Thus, ferruginol production requires a functional expression of the P450R module with its membrane association.

For ferruginol production, miltiradiene and abietatriene levels declined following glucose depletion after 48 h, whereas the level of ferruginol remained stable. Under constant glucose feeding, the level of ferruginol increased, reaching a content of 6.23 ± 0.2 mg/gDCW and a concentration of 107.34 ± 1.2 mg/L at 96 h (Fig. 6A). Further optimization on fermentation conditions and scale-up processes will be required to achieve feasible and efficient production of ferruginol in engineered C. glutamicum. Similarly, in an engineered yeast, the GGPPS, DiTPS, and P450R modules has been utilized with an optimized mevalonate pathway and led to a heterologous production of ferruginol at 10.5 mg/L in a flask scale [14]. Further pathway engineering of yeasts with various P450 expressions have allowed the carnosic acid productions [1, 15]. Systematic expression of bacterial CYPs and CPRs for diversifying terpene production [7], incorporation of functional compartmentalization to achieve targeted protein localization [44], and the application of metabolic channeling through protein fusions to enhance P450 reaction efficiency [45] have all been successfully demonstrated in bacteria. Leveraging these strategies in engineering C. glutamicum holds the potential to significantly enhance diterpene production and expand applications to the biosynthesis of other terpenoids.

Conclusions

In this study, we successfully developed a bacterial strain capable of heterologous miltiradiene production by incorporating the GGPPS, DiTPS, and OverMEP modules for the first time. Furthermore, the integration of heterologous membrane-associated plant P450 and reductase enzymes enabled the production of ferruginol in bacteria. Subsequent pathway engineering efforts, including the exploration of various P450 and reductase proteins, will focus on optimizing the sustainable production of natural diterpenes for biotechnological applications.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

The authors thank to Jigyeong Son, Jun Sung Park, You Lim Lee for the technical support.

Funding

This study is supported by the National Research Foundation of Korea funded by the Korean Government (Ministry of Science and ICT) (RS-2022-NR070265 to HMW, 2022M3J5A1062990 to HMW, 2022R1A4A5032720 to HMW, RS-2022-NR067345 to HMW, 2021R1I1A1A01057622 to HJL).

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  1. BioFoundry Research Center, Institute of Biotechnology and Bioengineering, Sungkyunkwan University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon, 16419, Republic of Korea

    Hyun Jeong Lee, Chaeyeon Kim, Yu Been Heo, Seong-Eun Kim & Han Min Woo

  2. Department of Food Science and Biotechnology, Sungkyunkwan University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon, 16419, Republic of Korea

    Chaeyeon Kim, Yu Been Heo, Seong-Eun Kim & Han Min Woo

  3. Department of MetaBioHealth, Sungkyunkwan University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon, 16419, Republic of Korea

    Han Min Woo

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Contributions

H.J.L. and H.M.W. designed the experiments, analyzed the data, and wrote the manuscript. H.J.L. and C. K. performed the experiments. H.J.L., C.K., Y.B.H., S.-E.K. and H.M.W. guided the scope of the project and provided critical input for the manuscript. All authors read and approved the final manuscript.

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Correspondence to Han Min Woo.

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Lee, H.J., Kim, C., Heo, Y.B. et al. Bacterial biosynthesis of abietane-type diterpene ferruginol from glucose. Microb Cell Fact 24, 67 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12934-025-02691-3

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Microbial Cell Factories

ISSN: 1475-2859

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