Modular reconstruction and multilevel regulation of Saccharomyces cerevisiae to drive the efficient synthesis of asiatic acid
Chen Sang, Wenqian Wei, Xinran Yin, Qihang Chen, Xinglong Wang, Weizhu Zeng, Jingwen Zhou
TL;DR
Scientists engineered yeast to efficiently produce asiatic acid, a valuable compound used in food, medicine, and cosmetics.
Contribution
A high-yielding yeast platform was developed using modular reconstruction and multilevel metabolic engineering.
Findings
A Saccharomyces cerevisiae strain was engineered to produce 170.4 mg/L of asiatic acid in a 5 L bioreactor.
Metabolic engineering improved precursor supply and reduced cytotoxicity in the engineered yeast.
Mitochondrial and cofactor balancing enhanced metabolic flux and energy supply for efficient production.
Abstract
Asiatic acid, a pentacyclic triterpenoid compound, is highly valued in the fields of functional foods, pharmaceuticals, and cosmetics. However, its microbial synthesis has been constrained by an insufficient supply of key precursors. Additional challenges include cytotoxicity caused by intracellular terpenoid accumulation and imbalanced carbon metabolism. In this study, a high-yielding Saccharomyces cerevisiae platform strain was designed through modular reconstruction and multilevel metabolic engineering to enable efficient asiatic acid production. The supply of the critical precursor, α-amyrin, was enhanced by metabolic engineering, while cytotoxicity was alleviated via Raman spectroscopy-guided lipid droplet engineering. The catalytic efficiency of key P450 enzymes involved in ursolic acid synthesis was optimized, and mitochondrial engineering combined with cofactor balancing was…
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Taxonomy
TopicsMedicinal Plants and Neuroprotection · Plant biochemistry and biosynthesis · Microbial Metabolic Engineering and Bioproduction
Introduction
1
Asiatic acid is an ursane-type pentacyclic triterpenoid compound widely found in plants such as Centella asiatica and Prunella vulgaris, and exhibits diverse pharmacological properties, including anti-inflammatory, antioxidant, antidiabetic, and antitumor activities [[1], [2], [3], [4]]. It is regarded as the principal bioactive constituent of C. asiatica, holding considerable promise for applications in nutraceuticals, pharmaceuticals, and cosmetics [[5], [6], [7]]. Currently, the conventional approach for obtaining asiatic acid relies mainly on extraction from plant sources, which is often inefficient and time-consuming [8]. Thus, the development of microbial cell factories represents a crucial alternative for the sustainable production of high-value natural products [9,10]. Saccharomyces cerevisiae, a model eukaryotic microorganism, is well-known for its clear genetic background, ease of cultivation, extensive genetic toolbox, and efficient expression systems [11]. It has been successfully employed as a chassis organism for the synthesis of numerous natural metabolites [12].
The biosynthesis of asiatic acid begins with 2,3-oxidosqualene as the precursor, which undergoes cyclization catalyzed by α-amyrin synthase to form α-amyrin. Subsequent oxidative modifications of the carbon skeleton occur via the action of a C-28 oxidase, in conjunction with cytochrome P450 reductase, leading to the conversion to ursolic acid. Finally, ursolic acid is further oxidized at the C-2α and C-23 positions by specific oxidases to yield asiatic acid [[13], [14], [15]].Previous studies have identified a cytochrome P450 enzyme, CaCYP716A83, from C. asiatica, which catalyzes the oxidation at the C-28 position of the pentacyclic triterpenoid skeleton to produce ursolic acid [16,17]. Another enzyme derived from C. asiatica, CaCYP716C11, has been found to mediate specific hydroxylation at the C-2α position. In addition, CaCYP714E19, has been found to mediate specific hydroxylation at the C-23 position [18,19]. Zhao et al. catalyzed the synthesis of ursolic acid using CaCYP716A83 and CaCPR3, and achieved de novo biosynthesis of asiatic acid with a titer of 1.8 mg/L in S. cerevisiae CaCYP716A12 and CaCYP714E19 successively [20]. Yu et al. improved the efficiency of CaCYP714E19 and CaCYP716C11 catalytic cascades by adopting a PDZ-PDZlig-mediated protein self-assembly strategy, resulting in a titer of 30.09 mg/L in the shake flask [8]. Zhu et al. achieved a major breakthrough in titer by systematically enhancing the catalytic microenvironment of P450 enzymes through strategies such as cofactor-coupled cytochrome P450 engineering, reaching a production of 1068.9 mg/L in shake flasks [21]. These studies laid a key foundation for the establishment of microbial cell factories for the synthesis of asiatic acid.
Although heterologous biosynthesis of asiatic acid in S. cerevisiae has been demonstrated in several studies, achieving industrial-level production remains a significant challenge [15]. The introduction of exogenous pathways often leads to substantial cellular stress, retarded growth, and insufficient supply of key precursors, thereby limiting carbon flux toward the desired product [22]. Furthermore, two critical factors impede efficient synthesis. One is the accumulation of cytotoxic terpenoid intermediates that could compromise the cellular capacity for terpene biosynthesis [23]. Another is the inadequate supply of essential cofactors required for biosynthesis, which further constrains efficient production [24]. Therefore, enhancing precursor availability, alleviating cytotoxicity, and balancing metabolic flux with cofactor supply are essential strategies for improving asiatic acid titers.
In this study, a modularly engineered S. cerevisiae chassis was constructed for efficient asiatic acid production through systematic optimization of the key precursors, α-amyrin and ursolic acid (Fig. 1). First, multilevel metabolic engineering was implemented to enhance the α-amyrin synthesis pathway, accompanied by a Raman spectroscopy-based and lipid droplet regulation-assisted strategy to alleviate cytotoxicity and improve cellular fitness. Then, the catalytic efficiency of the pivotal P450 enzyme responsible for ursolic acid production was improved through the maintenance of heme homeostasis and the application of a protein fusion strategy. Furthermore, mitochondrial engineering and cofactor supply coordination were employed to balance metabolic flux and energy provision. Finally, successful biosynthesis of asiatic acid was achieved using this optimized platform strain, reaching a titer of 46.2 mg/L in shake-flask cultures and 170.4 mg/L in fed-batch fermentation with a 5 L bioreactor. The engineered platform developed in this study provides valuable insights and technical support for future industrial-scale production.Fig. 1. Construction of asiatic acid-producing S. cerevisiae.Biosynthetic pathways of asiatic acid in S. cerevisiae. The blue arrow represents the path to overexpression. The pink arrow represents the path to weaken. ADH2, alcohol dehydrogenase; ALD6, acetaldehyde dehydrogenase; tHMG1, truncated hydroxyme thylglutaryl-CoA reductase; IDI1, isopentenyl diphosphatedelta-isomerase; ERG1^V249H/L343A^, squalene epoxidase mutant; IaAS1, α-AS from Ilex asprella; CYP716A83, oxidase at position C-28; CaCYP714E19, oxidase at position C-23; CaCYP716C11, oxidase at position C-2α; ERG7, lanosterol synthase; POS5, NADH kinase; IDP2, peroxisome NADP-dependent isocitrate dehydrogenase; MAE1, malate dehydrogenase; DGA1 acyl-CoA, diacylglycerol acyltransferase; ARE1, sterol acyltransferase; ARE2, sterol acyltransferase; YEH1: sterol ester hydrolase; YEH2, sterol ester hydrolase.Fig. 1
Materials and methods
2
Strains, genes, and standards
2.1
In this study, the squalene-producing S. cerevisiae strain SQ1 was used as the chassis strain [25], and the metabolically engineered strains are listed in Table S3. Escherichia coli JM109 is used for gene construction and plasmid propagation. The genes used in this study were synthesized by Sangon Biotech Co., Ltd (Shanghai, China), with the nucleotide sequences provided in Table S2. All synthesized genes were optimized for S. cerevisiae codons [26]. All standards were purchased from Shanghai Yuanye Biological Technology Co., Ltd (Shanghai, China).
Plasmid and strain construction
2.2
The PY26 plasmid and PCT series plasmid constructed in this study are listed in Table S4 and amplified in Escherichia coli JM109. Gene fragments were inserted into the JM109 vector using a Gibson assembly kit (Nanjing Vazyme Biotech Co., Ltd, Nanjing, China). The primers used in this study are listed in Table S5. All primers and plasmids passed Sanger sequencing by Sangon Biotechnology Co., Ltd.
The single gene editing system of S. cerevisiae utilizes the CRISPR-Cas9 system, and the prediction and design of sgRNAs is performed using the Benchling (https://benchling.com) website. The length of the homology arms for the homologous DNA fragments used for genomic integration is 500 bp [27]. Verification methods for gene knockout or gene knock-in include S. cerevisiae colony PCR and genome sequencing. The elimination of plasmids is carried out using passaging and plate photocopying methods. The multicopy integration in S. cerevisiae integrates histidine, leucine, uracil, and tryptophan with the gene of interest, with the homology arm length of the homologous DNA fragment also being 500 bp. Yeast nitrogen base (YNB) medium lacking relevant amino acids was selected for screening high-copy S. cerevisiae strains of the target gene.
Culture conditions
2.3
S. cerevisiae strains were activated on defective YNB plates, then inoculated in YNB (YPD) medium for primary seed solution, incubated in primary seed solution for 24 h, and inoculated in 20 mL of YNB (YPD) medium at 2 % inoculation rate for fermentation culture. The samples were incubated in a shaker at 30 °C and 220 r/min for 120 h, and the samples were treated for product determination after fermentation.
In batch fermentation with a 5 L bioreactor, several fresh S. cerevisiae monocolonies were picked, inoculated into 25 mL YPD medium, and incubated at 30 °C, 220 r/min for 24 h as a primary seed solution. Next, 5 % (v/v) of the primary seed solution was transferred to 200 mL of YPD medium and incubated for an additional 24 h to prepare the secondary seed solution. The secondary seed solution was then inoculated into a 5 L fermenter containing 2.5 L of YPD medium. The temperature was maintained at 30 °C and the dissolved oxygen (DO) level was controlled to 15 % by cascading agitation (200−900 rpm) with an airflow rate of 2 vvm (airflow/working volume/min). The pH was adjusted to 5.3–5.6 with 6 mol/L NaOH. The feed medium consisted of 800 g/L glucose, 18 g/L KH_2_PO_4_, 10.24 g/L MgSO_4_·7H_2_O, 0.4 g/L FeSO_4_·7H_2_O, and 7 g/L K_2_SO_4_.
Raman spectroscopy sample detection preparation
2.4
A small amount of activated bacteria or standard powder was scraped onto a glass slide, after which a normal drop of saline was dopped on the slide. The sample was then allowed to dry completely, after which another glass slide pressed onto the sample to generate a double clip slide. Finally the slide was put into focus under a brightfield microscope and suitable field was determined for analysis.
Extraction method for the product
2.5
Extraction method for α-amyrin: 1 mL of fermentation broth was added to a 2 mL crushing tube, after which the broth was centrifuged at 12000 r/min for 10 min. The supernatant was then discarded, the pellet collected, and an equal volume of grinding beads (0.5 mm) added. Next 1 mL of n-hexane was added add to lyse the cells using the S. cerevisiae FastPrep program for six cycles, after which the supernatant collected, stored in a liquid phase bottle for future membrane treatment.
Extraction method for ursolic acid: 1 mL of fermentation broth was added to a 2 mL crushing tube, after which the broth was centrifuged at 12000 r/min for 10 min. The supernatant was then discarded, the pellet collected, and an equal volume of grinding beads (0.5 mm) added. Next, 1 mL of ethyl acetate was added to lyse the cells using the S. cerevisiae FastPrep program for six cycles, and after crushing, the supernatant was centrifuged and placed in a vacuum concentrator to steam dry. After, 200 μL of a 1:1 mixture of pyridine and N,O-bis(methylsilyl)trifluoroacetamide was added to the dried supernatant, after which it was placed in alkylate in a water bath at 80 °C for 30 min. Finally, the supernatant was collected in a liquid phase flask for detection after film treatment.
Extraction method for asiatic acid: Take 1 mL of fermentation broth was added to a 2 mL crushing tube, after which the broth was centrifuged at 12000 r/min for 10 min. The supernatant was then discarded, the pellet collected, and an equal volume of grinding beads (0.5 mm) added. Next 1 mL of methanol was added to lyse the cells using the S. cerevisiae FastPrep program for six cycles, and after crushing, the supernatant was centrifuged and collected in a liquid phase bottle for detection after membrane treatment.
Detection method for the product
2.6
α-Amyrin gas chromatography mass spectrometry (GC–MS) detection conditions: chromatographic column DB-5ms, helium flow rate 1 mL min^−1^, injection temperature 300 °C, split ratio 1:10, injection volume 1 μL. Specific procedures: 80 °C for 1 min, 40 °C·min^−1^ to 280 °C for 5 min, 20 °C·min^−1^ to 300 °C for 10 min. The ion source temperature was 250 °C and the ion scanning range 50–600 m/z [28].
Ursolic acid GC–MS detection conditions: column DB-5ms, helium flow rate 1 mL min^−1^, injection temperature 300 °C, split ratio 1:30, injection volume 1 μL; Specific procedure: The starting temperature for heating was 80 °C, after which the temperature was raised to 320 °C at a rate of 20 °C/min and maintained for 28 min. The ion scanning range was 45–750 m/z [29].
Asiatic acid liquid chromatography mass spectrometry (LC–MS) detection conditions: A Thermo Fisher (Hypers11 ODS 250 mm × 4.6 mm, 5 μm) C18 column was used under a mobile phase using acetonitrile (A) and 0.2 % phosphate water (B). The ratio was 50:50, the elution time was 20 min, the flow rate was 1 mL min^−1^, the column temperature was 40 °C, and the detection wavelength was 210 nm [30].
Results
3
Construction and optimization of the α-amyrin biosynthesis pathway
3.1
S. cerevisiae SQ1 was constructed as a platform strain, which had been engineered for multi-copy integration at the Ty1 sites to overexpress tHMG1 and IDI1 for squalene overproduction [25]. qPCR results confirmed that five copies of the tHMG1 and IDI1 genes had been integrated into the Ty1 sites of the SQ1 strain (Fig. S1).To achieve the synthesis of α-amyrin, IaAS1 derived from Ilex cornuta was introduced, and the native ERG1 gene was replaced with the mutant ERG1^V249H/L343A^ to enhance the conversion of the precursor squalene [31]. The results showed that the titer of α-amyrin in the SQ1-2 strain was 10.52 mg/L (Fig. S2). To further improve the expression efficiency of the key enzyme IaAS1 and increase α-amyrin accumulation, six promoters of varying strengths (P_GAL1, PGAL7, PGAL10, PTDH1, PTDH3, PPGK1) with different intensities were selected to express IaAS1. These constructs were integrated into the BTS1 locus of S. cerevisiae to generate stable genomically integrated strains A01–A06. The constitutive strong promoter PPGK1_ was the best promoter for the expression of IaAS1, and the accumulation of α-amyrin for the A06 strain was 51.25 mg/L after fermentation for 120 h (Fig. 2A). It is worth noting that no α-amyrin was detected when IaAS1 was expressed with P_TDH1_ as the promoter, likely because the expression strength of this promoter is insufficient for the heterologous α-amyrin synthase. This enzyme requires high-level expression to compete effectively with endogenous pathways. To further increase the titer of target compounds, the ERG1^V249H/L343A^ and IaAS1 genes were integrated into the multicopy TY3 genomic sites. The results showed that the titer of α-amyrin in the A10 strain reached 79.85 mg/L, which was 55.8 % higher than that of the A06 strain (Fig. 2B). qPCR results confirmed that three copies of the ERG1^V249H/L343A^ and IaAS1 genes had been integrated into the Ty3 sites of the A10 strain (Fig. S1).Fig. 2. Optimization of the α-amyrin biosynthetic metabolic network. A: Screening for the best promoter of IaAS1 to synthesize α-amyrin. B: High-copy integrated expression enhances α-amyrin titer. C: Downregulated metabolic map of the competitive pathway and ethanol assimilation pathway. D: Screening for the optimal N-degron to downregulate the endogenous sterol biosynthesis pathway. E: Enhanced expression of ethanol assimilation pathway genes synthesized α-amyrin titer. Results from three independent experiments are presented as averages and standard deviations.Fig. 2
The endogenous sterol pathway competes with the α-amyrin synthesis pathway for the common precursor 2,3-oxidosqualene. It has been reported that downregulating ERG7 through an N-degron-dependent protein degradation strategy can channel more flux toward the triterpenoid biosynthesis pathway [32]. The ubiquitin gene Ubi4a, together with four N-degron tags (K3K15, K15, KN113, and KN119), was ligated to the N-terminus of ERG7 to construct strains A13, A14, A15, and A16 (Fig. 2C). The results showed that the accumulation of α-amyrin in the A14 strain carrying the K15 N-degron tag increased significantly to 137.9 mg/L, which was 72.7 % higher than that of the A10 strain (Fig. 2D). To alleviate the metabolic inhibition caused by high ethanol concentrations and promote the accumulation of acetyl-CoA, the expression of the alcohol dehydrogenase gene ADH2 was enhanced using the P_TEF_ promoter, and the expression of the aldehyde dehydrogenase gene ALD6 was enhanced using the P_TDH1_ promoter [25] (Fig. 2C). The results showed that the accumulation of α-amyrin of the A17 strain was 159.65 mg/L (Fig. 2E).
A regulatory switch for lipid droplet dynamics enhances cellular fitness
3.2
Because the accumulation of triterpenoids has a toxic effect on S. cerevisiae cells, which will inhibit cell growth and greatly limit the synthesis of products. Raman spectroscopy can differentiate compounds based on their characteristic peaks, thereby revealing their distribution within cells [33] (Fig. S3). Analysis indicates that α-amyrin was primarily stored in lipid droplets (Fig. 3A). Based on these findings, dynamic and fine-tuned regulation of lipid droplets was implemented. Dynamic regulation of lipid droplets was achieved by enhancing the expression of genes involved in lipid droplet synthesis, including DGA1, ARE2, ARE3, YEH1, and YEH2 (Fig. 3B). The results showed that the expression of ARE1 could significantly increase the accumulation of α-amyrin, and ARE2 could assist ARE1 in increasing the accumulation of α-amyrin (Fig. 3C). ROX1 is a transcriptional repressor of hypoxic genes, and its deletion can enhance the metabolic capacity of the MVA pathway [34]. The optimal combination of ARE1 and ARE2 was further integrated into the ROX1 locus of S. cerevisiae, and the accumulation of α-amyrin in the A25 strain reached 222.7 mg/L, which was 39.5 % higher than that of the A17 strain. Because high-copy genome integration can improve the expression level of genes, it was found that when the copy number of ARE1 and ARE2 increased to 4, the accumulation of α-amyrin in the A28 strain reached 310.2 mg/L. At this point, the α-amyrin accumulation began to plateau and cell growth was retarded, so no further copies were added (Fig. 3D).Fig. 3. Lipid droplet engineering modification to enhance the titer of α-amyrin.A: Raman spectroscopy was used to explore the intracellular distribution of α-amyrin. B: Enhanced expression of lipid droplet-related gene accumulation α-amyrin metabolic pathway. C: Screening of the combination of optimal enhanced lipid droplets for the accumulation of α-amyrin. D: Increased copy number accumulation of lipid drop genes α-amyrin. Results from three independent experiments are presented as averages and standard deviations. E: Raman spectroscopy was used to analyze the accumulation of α-amyrin in lipid droplets in high-yield engineered strains.Fig. 3
To investigate the impact of dynamically regulating lipid droplets on the accumulation of α-amyrin, Raman spectroscopy was used to split and compare the A17 strain and A28 strain before and after the lipid droplet expansion (Fig. 3E). It was found that α-amyrin accumulated in lipid droplets, whereas the modified strain had larger lipid droplets and more lipid droplets, and the accumulation of α-amyrin was also significantly improved. Only trace amounts of β-amyrin were produced. This is also the first time that Raman spectroscopy has been used to explore the distribution of α-amyrin in cells, which provides a solid foundation for the subsequent study of terpenoids.
Optimizing catalytic efficiency of key P450 to promote ursolic acid synthesis
3.3
To achieve de novo biosynthesis of ursolic acid, the CaCYP716A83 from C. asiatica and the AtCPR1 from Arabidopsis thaliana were introduced into Strain A28, resulting in ursolic acid accumulation reaching 111.95 mg/L (Fig. 4A). Subsequently, the heme degradation gene HMX1 was knocked out, and key enzymes in the heme biosynthesis pathway (HEM12, HEM13, HEM14, HEM15) were overexpressed to enhance heme availability (Fig. 4B). Among these modifications, the overexpression of HEM13 in the HMX1 knockout background resulted in the best performance, with Strain UA9 achieving an ursolic acid titer of 140.45 mg/L (Fig. 4C). To further improve the conversion efficiency, a protein fusion strategy between CaCYP716A83 and AtCPR1 was implemented. Two fusion approaches were tested based on their flexibility and length: direct fusion of the two proteins, and fusion via six flexible linkers (GSG1, GSG2, GGGS1, GGGS2, GGGGS1, GGGGS2) attached to the N-terminus of AtCPR1. These linkers consist of glycine and serine residues, which provide conformational flexibility and reduce steric hindrance, allowing the P450 and CPR domains to adopt orientations conducive to electron transfer [35] (Fig. 4D). The fused expression significantly enhanced ursolic acid production. The optimal construct, CaCYP716A83-GSG1-AtCPR1, was integrated into the TY4 multicopy sites, producing Strain UA15, which accumulated ursolic acid at a titer of 212.2 mg/L, which was a 51.1 % increase compared with UA9 (Fig. 4F). qPCR results confirmed that four copies of the CaCYP716A83-GSG1-AtCPR1 genes had been integrated into the Ty4 sites of the UA15 strain (Fig. S1).Fig. 4. Designer P450 enzymes enable high-yield microbial production of ursolic acid.A: Ursolic acid biosynthesis pathway. B: Improved supply pathway of heme. C: Effect of increasing the supply of heme on ursolic acid accumulation. D: Schematic diagram of fusion protein granule construction. E: Effect of fusion protein on ursolic acid accumulation. F: High-copy integrated expression enhances ursolic acid accumulation. Results from three independent experiments are presented as averages and standard deviations.Fig. 4
Synergistic engineering of mitochondria and cofactors: a dual drive for the efficiency of metabolic pathways
3.4
The introduction of heterologous pathways can easily lead to an imbalance in cell redox homeostasis and limit the accumulation of products. Therefore, the supply of NADPH cofactors is critical. Among microorganisms, NADH kinase (POS5), isocitrate dehydrogenase (IDP2), and malate dehydrogenase (MAE1) are the main sources of NADPH in S. cerevisiae. To enhance the supply of cytosolic NADPH, the UA13-UA15 strain was constructed by enhancing the expression of POS5, IDP2, and MAE1 (Fig. 5A). It is worth noting that the POS5 gene encoding NADH kinase reduced the production of ursolic acid, and the titer of ursolic acid reached 241.3 mg/L when MAE1 was enhanced. Mitochondria are the site of cellular respiration, the center of cellular metabolism, and the key to energy production (Fig. 5B). To improve cellular respiration and promote carbon source utilization, the respiratory chain in the inner membrane of mitochondria was modified. Among them, NADH dehydrogenases NDI1 and NDE1/2 are located in respiratory complex I, ubiquinol-cytochrome c reductases; QCR2, QCR6, CYT1, RIP1, HAP4, and COR1 are located in respiratory complex III; and cytochrome c oxidase CYC1, CYC3, COX5, and COX11 are located in respiratory complex IV [36,37] (Fig. 5A). By enhancing the expression of these genes, it was found that ursolic acid production could be significantly increased. In particular, the effect of CYC1 was the most significant, and the titer of UA20 ursolic acid reached 283.3 mg/L (Fig. 5C).Fig. 5. Modification of mitochondrial engineering to maintain carbon metabolism balance.A: Schematic diagram of the enhanced NADPH cofactor in the mitochondrial matrix and the transport of the respiratory chain in the inner mitochondrial membrane. B: The optimal combination of NADPH cofactors was screened to enhance the accumulation of ursolic acid. C: Screening enzymes in the inner mitochondrial membrane respiratory chain to promote cellular aerobic respiration and enhance ursolic acid accumulation. Results from three independent experiments are presented as averages and standard deviations.Fig. 5
Engineering of a high-yielding microbial strain for asiatic acid production
3.5
Asiatic acid is synthesized from ursolic acid through hydroxylation at the C-23 and C-2α positions. Therefore, the previously modified ursolic acid strain UA20 was used as the chassis to express CaCYP714E19 and CaCYP716C11 (Fig. 6A). To further optimize the compatibility of CYP and CPR, different plant-derived CPRs were screened, including HtCPR from Helianthus tuberosus, MtCPR from Medicago truncatula, and LjCPR from Lotus japonicus. The experimental results showed that the CPR derived from MtCPR was highly compatible with CYP, and the asiatic acid titer reached 15.1 mg/L (Fig. 6B). It is important to illustrate the conjugation of CPRs from different sources to cytochrome P450s. Subsequently, to reduce the spatial distance between different P450 enzymes and improve the catalytic efficiency of enzymes, AtMSBP1 and AtMSBP2 from A. thaliana and SvMSBP1 and SvMSBP2 from Vaccariae semen were integrated into AS2 strains to obtain strains AS5–AS8 (Fig. 6C). The results showed that when AtMSBP1 was intensified, the asiatic acid titer of the AS5 strain reached 35.8 mg/L, which was 137 % higher than that of the AS2 strain (Fig. 6D). Subsequently, a shaker bottle fermentation optimization was carried out for AS5 strains, and it was found that when the glucose addition was 50 g/L, the asiatic acid titer reached 46.2 mg/L, which was 29 % higher than that of the AS5 strain (Fig. S4A).Fig. 6. Metabolic engineering for asiatic acid biosynthesis.A: Asiatic acid synthesis path. B: Effects of different sources of CPR on asiatic acid synthesis. C: Mechanism of action of MSBP protein. D: Effects of different sources of MSBP proteins on asiatic acid synthesis. Results from three independent experiments are presented as averages and standard deviations.Fig. 6
Fed-batch fermentation in a 5 L bioreactor
3.6
To evaluate the production potential of asiatic acid, a fed-batch fermentation process was conducted in a 5 L bioreactor. The fermentation was carried out in two distinct phases: a cell growth phase followed by an asiatic acid production phase. In the initial phase, glucose was used as the carbon source to support robust cellular growth, after which the fermentation conditions were adjusted to promote asiatic acid synthesis. The initial glucose concentration was 40 g/L, and DO was maintained at 15 % saturation by adjusting the agitation rate and aeration. No additional nitrogen source was supplemented throughout the fermentation. Excess ethanol exerts stress on strain growth and impairs its production capacity. Therefore, the glucose feeding rate was dynamically adjusted according to the ethanol accumulation level of the strain. When fermentation reached 12 h, the initial glucose was depleted, but ethanol concentration remained high. At this point, high concentration glucose was supplemented slowly at a rate of 7 mL/h. Once the ethanol concentration decreased to 2–6 g/L, the glucose feeding rate was increased to 10 mL/h and maintained until the end of fermentation, while ensuring that residual glucose remained near zero throughout the process. Throughout the fermentation process, the accumulation of asiatic acid gradually increased, reaching a titer of 99.6 mg/L at 120 h (Fig. 7A). In addition, adding 0.4 g/L of FeSO_4_·7H_2_O during inoculation increased the titer of asiatic acid to 170.4 mg/L (Figure S4B and Fig. 7B).Fig. 7De novo synthesis of asiatic acid in a 5 L fermenter.A: The glucose flow rate was controlled at 7 mL/h for 12 h, with a flow rate controlled at 10 mL/h for 72 h, while the DO was controlled at 15 %. B: The glucose flow rate was controlled at 7 mL/h for 12 h, with a flow rate controlled at 10 mL/h for 72 h, while the DO was controlled at 15 % with the addition 0.4 g/L of FeSO_4_.7H_2_O.Fig. 7
Discussion
4
Asiatic acid, a major bioactive compound in C. asiatica, is widely used in health-promoting and functional foods due to its dual pharmaceutical and nutraceutical applications. It is also a highly valued ingredient in the cosmetics industry. Zhu et al. achieved a major breakthrough in titer by systematically enhancing the catalytic microenvironment of P450 enzymes through strategies such as cofactor-coupled cytochrome P450 engineering [21]. However, challenges including cytotoxicity induced by heterologous enzyme expression, insufficient precursor supply, low catalytic efficiency of key P450 enzymes, and imbalanced metabolic flux remain to be addressed. To address these limitations, this study optimized the metabolic network for the key intermediates α-amyrin and ursolic acid. Raman spectroscopy-assisted analysis and fine-regulated lipid droplet formation were employed to alleviate cellular toxicity and enhance cell fitness. The catalytic efficiency of the critical P450 enzyme for ursolic acid synthesis was improved through heme cofactor balancing and a protein fusion strategy. Mitochondrial engineering and coordinated cofactor supply were further applied to balance metabolic flux and energy provision, thereby reducing carbon loss. Based on these strategies, a modularly engineered S. cerevisiae chassis was developed, enabling the successful establishment of an asiatic acid biosynthetic pathway. The final strain achieved a titer of 170.4 mg/L in a 5 L bioreactor.
To biosynthesize asiatic acid, efficient production of its key precursors, α-amyrin and ursolic acid, is essential. Metabolic engineering can enhance precursor supply by redirecting flux toward target pathways. Promoters function as effective regulatory tools in metabolic pathways, with varying strengths significantly influencing transcriptional levels of genes [38]. It has been demonstrated that the use of the strong promoter PGK1 substantially increased α-amyrin production. However, this intensified the competition with the native sterol pathway for the common precursor 2,3-oxidosqualene. Previous studies have indicated that these four degrons (K15, K3K15, KN119, and KN113) all possess a phenylalanine residue serving as the N-terminal rule residue, along with an additional 14-residue lysine-asparagine spacer. This structure attracts varying degrees of ubiquitination, leading to different protein degradation rates. Therefore, these degradation signal peptides, which confer distinct degradation rates, were fused to the N-terminus of the native ERG7 protein [[39], [40], [41]]. Furthermore, the overexpression of ADH2 and ALD6 not only alleviated ethanol inhibition but, more importantly, reinforced acetyl-CoA replenishment, supplying more carbon units for terpenoid backbone synthesis [42]. After these metabolic modifications, observed cellular growth retardation was mainly attributed to cytotoxicity induced by the accumulation of hydrophobic terpenoid intermediates, which also severely constrained the yeast's innate terpenoid-synthetic capacity [23]. To resolve this issue, this study systematically optimized the system by integrating Raman spectroscopy with lipid droplet engineering. Raman spectroscopy offers the unique advantage of in situ, label-free monitoring of metabolite distribution and composition in living cells, allowing precise discrimination of α-amyrin from other intracellular components based on its characteristic spectral fingerprint [33]. Using this technique, we directly visualized the specific storage of α-amyrin within lipid droplets. Further regulation of lipid droplet-related genes confirmed that expanding lipid droplet storage capacity significantly enhanced α-amyrin accumulation and effectively mitigated the associated cytotoxicity, thereby overcoming a key bottleneck for efficient terpenoid synthesis [43]. In summary, the precise application of multiple metabolic engineering strategies greatly enhanced the production of the precursor α-amyrin.
The heterologous expression of P450 enzymes in microbial hosts remains challenging due to their low catalytic efficiency, strict substrate specificity, and limited activity toward non-native substrates [44]. Establishing an optimal microenvironment for P450 expression is therefore crucial [45]. As heme constitutes the active center of P450s, enhancing the expression of rate-limiting enzymes in the endogenous heme biosynthesis pathway can improve P450 activity [46]. Zhu et al. increased the production of asiatic acid in Saccharomyces cerevisiae by knocking out the heme degradation gene HMX1 and enhancing the expression of key genes such as HEM2 and HEM3 [21]. Optimization of CYPs and CPRs as redox chaperones using protein fusion engineering is an effective strategy for improving their catalytic efficiency. Therefore, by screening the position and type of linker peptides, the production of ursolic acid was further increased [32,47,48]. The ursolic acid biosynthesis pathway also demands a substantial supply of the NADPH cofactor [24]. Overexpression of POS5 was applied to elevate NADPH levels, reinforcing earlier findings that cofactor balancing significantly boosts production [49]. An observation that warrants further exploration is that enhanced expression of the respiratory chain component CYC1 significantly increased ursolic acid production. The underlying mechanism likely involves mitochondrial engineering acting as a global metabolic coordinator, and these modifications are expected to help minimize carbon loss and alleviate oxidative stress associated with multi-step P450 catalysis [[50], [51], [52], [53]].Collectively, these integrated strategies were demonstrated to substantially improve P450 functionality and enable efficient heterologous synthesis of ursolic acid in yeast.
Building upon an engineered high-yielding ursolic acid platform strain, the introduction of key enzyme genes enabled the successful biosynthesis of asiatic acid (Fig. S5). Specifically, Zhao et al. achieved asiatic acid production by incorporating CaCYP714E19 and CaCYP716C11, both derived from C. asiatica [20]. The choice of cytochrome P450 reductase (CPR) source has been shown to considerably enhance electron transfer during catalysis, thereby promoting product accumulation [8]. For instance, M. truncatula CPR (MtCPR) has been demonstrated to effectively improve asiatic acid synthesis. Furthermore, Yang et al. reported that modulating the spatial proximity between different P450 enzymes can significantly facilitate the production of quillaic acid [54]. This suggests that MSBP protein shortens the distance between different P450 enzymes and promotes enzyme expression. Finally, these strategies culminated in an asiatic acid titer of 46.2 mg/L in shake-flask cultures. To evaluate the genetic stability of the strain, subculturing fermentation was performed. The results showed that the asiatic acid production of the passaged strain did not exhibit a significant decreasing trend, indicating favorable genetic stability (Fig. S6).
In conclusion, this study established an efficient yeast cell factory for asiatic acid by employing a multi-pronged engineering strategy: enhancing precursor supply through pathway engineering, resolving cytotoxicity and improving cellular fitness via integrated lipid droplet engineering and Raman spectroscopy-assisted characterization, implementing multi-level optimization of P450 function, and achieving global metabolic coordination through mitochondrial engineering. Following systematic reprogramming of the yeast chassis, an asiatic acid titer of 170.4 mg/L was achieved in a 5 L bioreactor. The integrated strategies developed here provide a systematic and transferrable engineering-biology framework for the microbial synthesis of other cytotoxic, high-value terpenoids. Nevertheless, industrial-scale production remains a formidable challenge. Future efforts should focus on applying systems metabolic engineering and synthetic biology tools—such as rational and semi-rational design as well as dynamic regulation of key P450 enzymes—to further enhance the platform's capacity for high-level asiatic acid synthesis and ultimately enable its industrial application.
CRediT authorship contribution statement
Chen Sang: Writing – review & editing, Writing – original draft, Validation, Investigation. Wenqian Wei: Methodology, Investigation, Formal analysis. Xinran Yin: Validation, Software, Investigation. Qihang Chen: Investigation. Xinglong Wang: Investigation. Weizhu Zeng: Writing – review & editing. Jingwen Zhou: Writing – review & editing, Supervision, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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