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Engineering cellular dephosphorylation boosts (+)-borneol production in yeast
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Haiyan Zhanga, b, c, Peng Caib, Juan Guoa, Jiaoqi Gaob, Linfeng Xieb, Ping Sua, Xiaoxin Zhaib, Baolong Jina, Guanghong Cuia, *, Yongjin J. Zhoub, *, Luqi Huanga, c, *
Acta Pharmaceutica Sinica B | 2025, 15(2) : 1171 - 1182
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Acta Pharmaceutica Sinica B | 2025, 15(2): 1171-1182
• ORIGINAL ARTICLE •
Engineering cellular dephosphorylation boosts (+)-borneol production in yeast
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Haiyan Zhanga, b, c, Peng Caib, Juan Guoa, Jiaoqi Gaob, Linfeng Xieb, Ping Sua, Xiaoxin Zhaib, Baolong Jina, Guanghong Cuia, *, Yongjin J. Zhoub, *, Luqi Huanga, c, *
Affiliations
  • aState Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, National Resource Centre for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
  • bDivision of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
  • cInstitute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
About Author:

E-mail addresses: (Guanghong Cui)

(Yongjin J. Zhou)

(Luqi Huang).

Author contributions

Haiyan Zhang: Writing – original draft, Visualization, Investigation, Data curation. Peng Cai: Methodology. Juan Guo: Supervision. Jiaoqi Gao: Methodology. Linfeng Xie: Methodology. Ping Su: Methodology. Xiaoxin Zhai: Software. Baolong Jin: Methodology. Guanghong Cui: Writing – review & editing, Validation. Yongjin J. Zhou: Writing – review & editing. Luqi Huang: Project administration.

doi: 10.1016/j.apsb.2024.12.039
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(+)-Borneol, the main component of "Natural Borneol" in the Chinese Pharmacopoeia, is a high-end spice and precious medicine. Plant extraction cannot meet the increasing demand for (+)-borneol, while microbial biosynthesis offers a sustainable supply route. However, its production was extremely low compared with other monoterpenes, even with extensively optimizing the mevalonate pathway. We found that the key challenge is the complex and unusual dephosphorylation reaction of bornyl diphosphate (BPP), which suffers the side-reaction and the competition from the cellular dephosphorylation process, especially lipid metabolism, thus limiting (+)-borneol synthesis. Here, we systematically optimized the dephosphorylation process by identifying, characterizing phosphatases, and balancing cellular dephosphorylation metabolism. For the first time, we identified two endogenous phosphatases and seven heterologous phosphatases, which significantly increased (+)-borneol production by up to 152%. By engineering BPP dephosphorylation and optimizing the MVA pathway, the production of (+)-borneol was increased by 33.8-fold, which enabled the production of 753 mg/L under fed-batch fermentation in shake flasks, so far the highest reported in the literature. This study showed that rewiring dephosphorylation metabolism was essential for high-level production of (+)-borneol in Saccharomyces cerevisiae, and balancing cellular dephosphorylation is also helpful for efficient biosynthesis of other terpenoids since all whose biosynthesis involves the dephosphorylation procedure.

Borneol  /  Monoterpene  /  LPP1  /  DPP1  /  Nudix hydrolase  /  Phosphatase
Haiyan Zhang, Peng Cai, Juan Guo, Jiaoqi Gao, Linfeng Xie, Ping Su, Xiaoxin Zhai, Baolong Jin, Guanghong Cui, Yongjin J. Zhou, Luqi Huang. Engineering cellular dephosphorylation boosts (+)-borneol production in yeast[J]. Acta Pharmaceutica Sinica B, 2025 , 15 (2) : 1171 -1182 . DOI: 10.1016/j.apsb.2024.12.039
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1.　Introduction
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(+)-Borneol, the main compound in Natural Borneol, is a valuable fragrant monoterpene used as a food flavoring and as a pharmaceutical1-3 having analgesic, anti-inflammatory, and antimicrobial activities4-7. As one of the valuable and indispensable medical materials, (+)-borneol has been extensively used in traditional Chinese, Japanese, and Indian medicine for a long time1,2. In addition, the US Food and Drug Administration (FDA) has approved borneol as a flavoring agent. (+)-Borneol is prepared mainly by steam distillation extraction of fresh leaves of the Cinnamomum tree, which suffers from low yields (32.7%–81.8%)8,9 and poses a threat to the sustainability of the plant species. Chemical synthesis is fraught with a high content (36%) of iso-borneol by-product4, which is toxic and has other side effects10.
Synthetic biology provides a sustainable answer for (+)-borneol production by engineering the biosynthesis pathways in microbes. The budding yeast Saccharomyces cerevisiae, a generally recognized as safe (GRAS) organism, has gained prominence as an ideal host for the production of high-value chemicals, such as artemisinic acid11, cannabinoids12, and alkaloids13,14. The strategies for efficient terpenoid synthesis in S. cerevisiae have been extensively studied, but the titer and yield of monoterpenes have always been inferior to that of diterpenes and sesquiterpenes15-17, which are often attributed to the cytotoxicity and in vivo biotransformation of monoterpenes18. Although some acyclic or monocyclic monoterpenes, such as geraniol19,20, citronellol21 and ®-(+)-limonene19, have been overproduced in S. cerevisiae at gram/liter levels, the production of (+)-borneol, a more complex bicyclic monoterpene, has remained low22-24 (Table 1), even with optimizing the isoprenoid biosynthetic pathway and enhancing the supply of precursors and cofactors. As a result, effective strategies for boosting (+)-borneol production have puzzled researchers for a long time.
Engineering intracellular prenyl diphosphate may play an important role in terpene production, such as in the biosynthesis of α-santalene25,26, geranylgeraniol27, farnesol28, and 11,20-dihydroxyferruginol29, the endogenous phosphatases were deleted or overexpressed to increase their productions. Moreover, in the biosynthesis of borneol, bornyl diphosphate synthases (BPPS), which catalyzes the conversion of geranyl diphosphate (GPP) to bornyl diphosphate (BPP), exhibits an unusual characteristic compared with typical terpene synthase reactions (Supporting Information Fig. S1). Most terpene synthase reactions are terminated by deprotonation, resulting in the formation of alkenes or water-trapping and leading to alcohol formation30-34. However, in the catalytic cascade reaction of BPPS, bornyl cation is quenched by re-addition of inorganic diphosphate, resulting in BPP as the main product with minor by-products of (+)-α-pinene, (+)-camphene, (+)-limonene, terpinolene, and β-myrcene31,35,36 Finally, phosphatases hydrolyze BPP to borneol. Although the endogenous phosphatase of yeast may mediate the dephosphorylation of BPP to borneol23,24, this unusual dephosphorylation reaction may be a major obstacle to heterologous biosynthesis of borneol because the reaction would likely compete with cellular phosphorylation metabolism. Furthermore, the mechanism of endogenous phosphatase-mediated dephosphorylation of BPP remains unclear. Recent studies have shown that EcNudJ22 and WvNUDX2437 may be involved in the dephosphorylation of BPP, but whether they can effectively promote the production of (+)-borneol in yeast needs further evaluation. Thus, a comprehensive analysis of the mechanism of de novo synthesis of (+)-borneol in S. cerevisiae and identifying, characterizing phosphatases, and balancing cellular dephosphorylation metabolism is essential for enhancing (+)-borneol production.
Here, we first confirmed that BPP dephosphorization is the key factor in enhancing (+)-borneol production and reducing by-products. Thus, the endogenous phosphatases were systematically validated, and two phosphatases involved in (+)-borneol production were identified. We found that the Golgi apparatus serves as the primary site for (+)-borneol generation, where its membrane protein Lpp1 facilitates the conversion of BPP into (+)-borneol. Additionally, the mechanism of endogenous phosphatase competition between (+)-borneol synthesis and lipid metabolism was elucidated via lipidomic analysis and co-localization. To balance cellular dephosphorylation and boost (+)-borneol production, we identified and validated seven plant genetic elements through a combination of bioinformatic analysis and synthetic biology validation. Finally, by engineering BPP dephosphorylation and optimizing the MVA pathway, we achieved a remarkable production of (+)-borneol (Fig. 1). This research not only fills a significant knowledge gap regarding the mechanism of de novo synthesis of (+)-borneol in S. cerevisiae but also lays a foundation for further exploration of efficient phosphatase with specific preference for BPP. The metabolic engineering strategies we used also provide a valuable understanding of the biosynthesis of other compounds that involve diphosphate precursors.
2.　Materials and methods
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2.1.　 Strains, medium and reagents
The yeast strains used in this study are listed in Supporting Information Table S1, and Supporting Information Fig. S2. Synthetic dropout (SD) solid medium (20 g/L glucose, 6.7 g/L yeast nitrogen base with (NH4)2SO4 and without amino acids) was used for selection of recombinant strains. SD+5-FOA plates (20 g/L glucose, 6.7 g/L yeast nitrogen base with (NH4)2SO4 and without amino acids,1 g/L 5-fluoroorotic acid, 20 mg/L histidine and uracil) were used for removing gRNA expression plasmids. Engineered strains for (+)-borneol production were cultivated in 100 mL flasks with 20 mL Delft minimal medium (pH 5.6) consisting of 5 g/L (NH4)2SO4, 14.4 g/L KH2PO4, 0.5 g/L MgSO4·7H2O, 20 g/L glucose, 2 mL/L trace metals (3.0 g/L FeSO4·7H2O, 4.5 g/L ZnSO4·7H2O, 4.5 g/L CaCl2·2H2O, 1 g/L MnCl2·4H2O, 300 mg/L CoCl2·6H2O, 300 mg/L CuSO4·5H2O, 400 mg/L Na2MoO4·2H2O, 1 g/L H3BO3, 100 mg/L KI, 19 g/L Na2EDTA·2H2O) and 1 mL/L vitamin solution (50 mg/L D-biotin, 1.0 g/L D-pantothenic acid hemicalcium salt, 1.0 g/L thiamine HCl, 1.0 g/L pyridoxin HCl, 1.0 g/L nicotinic acid, 0.2 g/L 4-aminobenzoic acid, 25 g/L m-inositol), supplemented with 20 mg/L histidine and uracil. Escherichia coli DH5α was used for plasmid amplification and cultivated in LB medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl), in which 100 μg/mL ampicillin was used for plasmid maintenance. All plasmids are listed in Supporting Information Table S2.
Super Fidelity DNA polymerase, Taq DNA polymerase, and ClonExpress II One Step Cloning Kit were purchased from Vazyme Biotech, Nanjing, China. PrimeStar DNA polymerase was purchased from Takara Bio. DNA gel purification and plasmid extraction kits were purchased from Omega Bio-Tek, US. The standard (+)-borneol (CAS:464-43-7), α-pinene (CAS:80-56-8), (+)-camphene (CAS:79-92-5), β-myrcene (CAS:123-35-3), (+)-limonene (CAS:5989-27-5), and terpinolene (CAS:586-62-9) were purchased from Macklin.
2.2.　 Genetic manipulation
Primers were synthesized by Sangon Biotech (Shanghai, China) and listed in Supporting Information Table S3. Strain XC1 is a laboratory strain having a CAS9 expression cassette integrated at the X1−5 site in the CEN.PK113–11C strain and GAL80 was deleted. Yeast DNA transformation was conducted using a chemical transformation method38. Target gene integration and deletion were performed using a CRISPR/Cas9-mediated gene editing system, and noncoding regions in the S. cerevisiae genome were chosen as integration sites to avoid affecting growth and metabolism39. All guide-RNAs (gRNAs) were designed by the CHOPCHOP web tool (http://chopchop.cbu.uib.no), and gRNA-expressing plasmids were constructed as described40,41. The donor was constructed with the one-step overlap extension PCR42, in which endogenous genes, promoter, terminator, upstream and downstream homology arm were amplified from the genomic DNA of CEN.PK113–11C. Heterologous genes, such as AvBPPS from Amomum villosum (GenBank: MG763230), LdBPPS from Lippia dulcis (GenBank: MF133333), SoBPPS from Salvia officinalis (GenBank: AF051900) and TcNudix1 from Tanacetum cinerariifolium (GenBank: MT126704) were codon-optimized for S. cerevisiae expression (Supporting Information Table S4) and synthesized by Sangon Biotech (Shanghai, China). The CbTPS1 gene from Cinnamomum burmanni (GenBank: MW196671) was amplified from plasmid pESC-LEU: CbTPS123.
2.3.　 Batch and fed-batch fermentation
The strains were first streaked on YPD plates and grown for 3–5 days. Then, single colonies were picked and precultured in 2.5 mL of YPD medium. After 16–20 h of cultivation, the strains were transferred into 20 mL of Delft minimal medium in 100 mL shake flasks for batch fermentation. The yeast cells were cultivated at 30 ℃, 220 rpm for 72 h with an initial OD600 of 0.1. In addition, 10% (v/v) isopropyl myristate (IPM) was added to the culture to capture (+)-borneol. Fed-batch fermentation was conducted in 250 mL shake flasks with 50 mL Delft minimal medium or YPD medium, with an initial inoculation of OD600 = 0.4. The yeast cells were cultivated at 30 ℃, 220 rpm, with 10% (v/v) IPM added for two-phase fermentation. During the fed-batch cultivation, residual glucose was quantified to determine the feeding rates. Cells were fed with 1–2 mL medium A (minimal medium) or medium B (modified YPD medium) when glucose was depleted. Medium A contained 500 g/L glucose solution, 25 g/L (NH4)2SO4, 72 g/L KH2PO4, 2.5 g/L MgSO4·7H2O, 10 × trace metal and 5 × vitamin solutions. Medium B contained 500 g/L glucose solution containing 50 g/L yeast extract and 100 g/L peptone. In addition, the pH was adjusted to 5.6 with the addition of 4 M KOH once a day. During the fed-batch process, samples were taken every 24 h to measure product titers and OD600.
2.4.　 Extraction and quantification of (+)-borneol
After 72 h of cultivation, the biomass was measured from the OD600, and the organic phase (10% IPM, which contains 100 mg/L (±)-camphor as an internal standard) was collected for product analysis. The organic phase was centrifuged at 13,000×g for 5 min to remove cellular debris and diluted with ethyl acetate to within an appropriate range. The (+)-borneol amounts were calculated by external standard calibration and corrected by using the internal standard (±)-camphor. GC–MS (Thermo Fisher Scientific) was used for qualitative analysis. Data were acquired in full-scan mode (m/z 50–650). The injection port temperature was 280 ℃, and the oven temperature program was as follows: started at 80 ℃ for 2 min, ramped to 138 ℃ at a rate of 30 ℃/min for 5 min, ramped to 150 ℃ at a rate of 5 ℃/min for 2 min, and finally ramped to 320 ℃ at a rate of 50 ℃/min and held for 5 min.
2.5.　 Fluorescence microscopy
To confirm tSoBpps subcellular localization, tSoBpps was fused to a green fluorescent protein (eGFP) at the N terminus or C terminus with a flexible linker GGGS; similarly, a red fluorescent protein (mPLUM) was fused to the N terminus or C terminus of Sec743 and Lpp1. The fusion cassettes were transformed into strain ZHY2. Colonies were cultivated in YPD medium for 18 h at 30 ℃ and 220 rpm. Cell cultures (5 μl) were dropped onto microscope slides and viewed with an EVOS M5000 microscope (Thermo Fisher Scientific). To confirm whether tSoBpps co-localized with vacuolar membrane protein Dpp1, the vacuolar membrane of strain ZHY47 was stained red with FM 4–64.
2.6.　 Lipidomic analysis
Lipidomic analysis was conducted with strains XC1 (wild-type), ZHY7 (without additional phosphatase), and ZHY73 (with additional phosphatase). Cells were cultivated in Delft minimal medium. Lipids were extracted and quantified as described44 using (UPLC)-Q Exactive HF MS (Thermo Fisher Scientific). Relative contents of lipids were calculated by the ratio of peak area between lipids and internal standard per gDCW.
3.　Results
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3.1.　 Optimizing the mevalonate pathway
The limited availability of GPP and the strong competition for GPP by the endogenous sterol biosynthesis pathway essential for growth45 makes it difficult to produce a pool of GPP sufficient for borneol synthesis. To address this issue, we overexpressed ERG20ww in the XC1 strain; ERG20ww encodes a mutant farnesyl diphosphatesynthase (FPPS) that has a high efficiency of GPP synthesis. In the same strain, we co-expressed HMG2K6R46,47, which encodes an HMG-CoA mutant that is known to stabilize the protein from protein degradation. In addition, we replaced the promoter of the endogenous ERG20 with the ERG7 promoter to reduce the GPP consumption by FPP synthesis21, resulting in strain ZHY2 (Fig. 2A). Subsequently, the truncated (+)-bornyl diphosphate synthase genes tSoBPPS, tAvBPPS, tCbTPS1, and tLdBPPSS488T were integrated into the chromosomes of ZHY2, respectively. Compared with the chassis strain ZHY2, the strains that included the (+)-bornyl diphosphate synthase genes produced (+)-borneol and also small amounts of monoterpenes (+)-α-pinene, (+)-camphene, (+)-limonene, terpinolene, and β-myrcene (Supporting Information Fig. S3A and S3B). Expression of tSoBPPS produced the highest (+)-borneol production of 2.4 mg/L compared with the other three BPPS genes (Fig. 2B, Fig. S3C).
Downregulated expression of ERG20 by replacing its promoter with the ERG7 promoter significantly improved (+)-borneol production by 423.6% (ZHY3-tSo vs ZHY8, Fig. 2B), suggesting that reducing the GPP consumption by FPP synthesis was essential for (+)-borneol biosynthesis in S. cerevisiae. To further increase GPP availability, we overexpressed the mevalonate kinase gene ERG12 (strain ZHY5), which resulted in a 17.3% increase in (+)-borneol production (Fig. 2B). Dusseaux et al.19 reported that EfmvaE and EfmvaS catalyze consecutive steps of mevalonate biosynthesis from acetyl-CoA. Overexpression of EfMvaS and EfMvaE (strain ZHY7) made a small improvement of (+)-borneol production (Fig. 2B). Enhancing GPP availability significantly improved (+)-borneol production but there was a decreased proportion of (+)-borneol among all monoterpenes (Fig. 2B). These results suggested that at high GPP level BPPS was less efficient in biosynthesis of (+)-borneol compared with its by-product monoterpenes. Further overexpression of key genes HMG2K6R, tHMG1, ERG8, ERG19, and IDI1 in ZHY7 resulted in a decrease in (+)-borneol production (Supporting Information Fig. S4).
3.2.　 Relieving the rate-limiting step of BPP dephosphorylation
Although the production of (+)-borneol increased when we engineered increased availability of GPP precursors, the decrease of (+)-borneol proportion among the monoterpenes suggested that BPP could not be completely converted into (+)-borneol (Fig. 2B). Unlike the other monoterpenes that can be formed spontaneously by deprotonation or water attack of carbocations in the BPPS catalytic cascade reaction, (+)-borneol biosynthesis requires the removal of a diphosphate group (Fig. S1). We tried to overexpress endogenous diacylglycerol pyrophosphate phosphatases DPP1 and LPP1, which were involved in catalyzing diacylglycerol pyrophosphate to diacylglycerol48. It was found that overexpressing LPP1 or DPP1 resulted in a 152% and 26% increase in (+)-borneol production, respectively (Fig. 3A). Furthermore, overexpressing LPP1 and DPP1 increased the percent of (+)-borneol production, suggesting that BPP dephosphorylation was enhanced. Deletion of LPP1 and DPP1 resulted in a decrease of (+)-borneol production (Fig. 3B). Overexpression of other endogenous phosphatases had marginal effects on (+)-borneol production (Supporting Information Fig. S5). These results confirmed that BPP dephosphorylation was a rate-limiting step in (+)-borneol production, and LPP1 and DPP1 were involved in (+)-borneol production in S. cerevisiae.
The results showed that LPP1 overexpression was more efficient than overexpression of DPP1 in increasing (+)-borneol production (Fig. 3A), which might have been due to the different cellular locations of these two phosphatases48. Subcellular localization analysis showed that tSoBpps predominantly co-localized with Lpp1 at the Golgi apparatus, which was identified by the Golgi marker protein Sec743 (Fig. 3C). Localization of Lpp1 at the Golgi apparatus agreed with reports that Lpp1 is a Golgi membrane protein48,49. In contrast, Dpp1 was mainly located on the vacuolar membrane and exhibited limited co-localization with tSoBpps (Fig. 3C). These results explained the superior effect of LPP1 over DPP1 in increasing (+)-borneol production and elucidated the Golgi apparatus as the primary site for (+)-borneol production, where LPP1 catalyzed BPP dephosphorylation into (+)-borneol (Fig. 3D).
Considering the different locations of tSoBpps (Golgi apparatus) and its substrate GPP (cytoplasm, endoplasmic reticulum, and vacuole)50, we tried to improve their interaction by fusing tSoBPPS with the ERG20ww gene (high-efficiency GPP synthesis); however, this approach failed to improve (+)-borneol production (ZHY20 vs ZHY67, Fig. 4A). We also tried to improve the interaction between a phosphatase and tSoBPPS to facilitate BPP dephosphorylation. A flexible linker (GSG/GGGS), affinity peptides, or cleavable 2A peptides were used as fusion agents; however, all of which failed to improve (+)-borneol production (Supporting Information Fig. S6). Chemical dynamics simulations51 and Complementary DFT-based molecular dynamics simulations52 suggested that the active site diphosphate moiety of tSoBPPS steers reaction trajectories toward BPP formation (Fig. S1), and increasing the interaction between tSoBpps and Lpp1 might disrupt the stability of the diphosphate moiety and compromised tSoBpps catalytic activity.
3.3.　 Optimizing endogenous dephosphorylation pathways
As BPP dephosphorylation was a rate-limiting step in (+)-borneol production, to further increase the production of (+)-borneol, we increased the copy number of LPP1 or DPP1 in the strain ZHY20 to give strains ZHY61 and ZHY69. However, (+)-borneol production did not increase, and ZHY61 (LPP1 copy number increase) decreased slightly in growth (Fig. 4B). We then tried to overexpress LPP1 and DPP1 simultaneously in strain ZHY20; the resulting strain ZHY73 had a 49.2% higher (+)-borneol production with a slight decrease in growth (Fig. 4A). Similarly, overexpressing LPP1 and DPP1 in ZHY67 that possessed the ERG20ww-tSoBPPS fusion improved (+)-borneol production by 21.4% (ZHY71), with a concomitant decrease in cell growth (Fig. 4A) and cell aggregation (Supporting Information Fig. S7). The compromised cell growth accompanying phosphatase overexpression led us to question whether there was a competitive relationship between (+)-borneol synthesis and cell growth.
In yeast, Lpp1 and Dpp1 are involved in regulating concentrations of diacylglycerol diphosphate, phosphatidate, and diacylglycerol (Fig. 4C), molecules associated with phospholipid metabolism53-56. Considering the potential competitive interactions between (+)-borneol synthesis and lipid metabolism, we surmised that the accumulation of (+)-borneol may disrupt lipid metabolism, and, at the same time, lipid metabolism may limit (+)-borneol production (Fig. 4C). Thus, we conducted a lipidomic analysis of strain ZHY7 (borneol production without overexpression of LPP1/DPP1) and ZHY73 (borneol production with overexpression of LPP1/DPP1). Engineering the mevalonate pathway for borneol production in strain ZHY7 led to a decrease in lipid content compared with the wild-type strain (Fig. 4D), which suggested that high-level BPP biosynthesis competed with endogenous phosphatase and perturbed lipid metabolism. Expression of additional endogenous phosphatases in ZHY73 not only improved (+)-borneol production but also additional endogenous phosphatases restored the lipid content to near wild-type level (Fig. 4D). These results suggested that the biosynthesis of (+)-borneol mediated by endogenous yeast phosphatase occurs at the expense of lipid production, which may explain the limited catalytic efficiency of endogenous phosphatase towards BPP.
3.4.　 Heterologous phosphatases boost (+)-borneol production
Because the catalytic efficiency of endogenous phosphatase toward BPP was limited by lipid metabolism, an increase in endogenous phosphatase copy number posed a risk of retarding cell growth. Therefore, it was necessary to screen for effective heterologous phosphatases. WvNUDX24 has been reported to be involved in borneol biosynthesis in Wurfbainia villosa37, which unfortunately failed to increase (+)-borneol production in S. cerevisiae (Supporting Information Fig. S8). We also tried overexpressing E. coli Nudix hydrolase, including EcNudJ22, but that approach resulted in a decrease in (+)-borneol production (Supporting Information Fig. S9). Conversely, overexpressing tTcNudix1 from Tanacetum cineriifolium57 contributed to an increase in (+)-borneol production without compromising the final cell biomass (Fig. 5A and B). This result showed that a heterologous phosphatase was superior to elevated expression of endogenous phosphatases LPP1 and DPP1 (ZHY72 vs ZHY73, Fig. 5B). Co-overexpression of tTcNudix1 and endogenous LPP1/DPP1 further boosted (+)-borneol production to 27.3 mg/L (ZHY74, Fig. 5B).
To search for more alternative phosphatases, we analyzed the transcriptome data of two borneol-rich plants, Cinnamomum camphora and Blumea balsamifera. By evolutionary tree analysis (Fig. 5C), we identified and tested CcNudix1 and BbNudix1.3. It was glad to find that these two genes increased (+)-borneol production by 69% and 31%, respectively (Fig. 5D and E). Notably, sequence comparisons of these Nudix1 genes revealed three short conserved sequences in the center of the protein chains(Supporting Information Fig. S10A), a feature not possessed in other Nudix genes, including EcNudJ22 and WvNUDX2437. Although other putative Nudix family genes contain the NUDIX domain (Gx5Ex5[UA]xREx2EExGU, where U is an aliphatic, hydrophobic residue58), these genes failed to increase (+)-borneol production (Fig. S10B). These results suggested that Nudix1 hydrolase may have high specificity for BPP in S. cerevisiae.
Additionally, we found nine lipid phosphatases grouped in three branches (Fig. 5F). Similar to yeast's LPP1, these enzymes exhibited the phosphatase sequence motif present in a superfamily of phosphatase enzymes, comprising three domains: KX6RP (domain 1), PSGH (domain 2) and SRX5HX3D (domain 3)59-61 (Supporting Information Fig. S11). Overexpressing C. camphora CcLPPE2, CcLPPG, and B. balsamifera BbLPPE2 and BbLPPG improved (+)-borneol production (Fig. 5G and H), whereas other putative phosphatases failed to promote (+)-borneol biosynthesis in yeast (Supporting Information Fig. S12).
3.5.　 Fed-batch fermentation
To evaluate production potential, we conducted fed-batch fermentation in shake flasks to maximize (+)-borneol production. To avoid supplementation of histidine and uracil during fermentation, we used the URA3 and HIS3 prototrophic strain ZHY74UH62. Strain ZHY74UH was cultivated in YPD or Delft minimal medium with 20 g/L glucose in the initial batch stage, and YPD or Delft minimal medium was added after all the glucose had been consumed. After 168 h of cultivation in YPD medium, the highest (+)-borneol titer of 753 mg/L with a final OD600 of 66 was obtained (Fig. 6A), the highest (+)-borneol titer reported in the literature (Table 1). Growth in Delft minimal medium produced a (+)-borneol titer of 337 mg/L with a final OD600 of 59 (Fig. 6B). After 168 h of cultivation, the (+)-borneol proportion reached 84.7 ± 0.5% and 82.4 ± 0.7% in YPD and Delft minimal medium (Fig. 6C).
4.　Discussion
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(+)-Borneol is a high-end spice and precious medicine that has been widely used in Chinese medicinal formulae. Here, by metabolic engineering the yeast, we constructed a yeast strain for (+)-borneol production. Enhancing mevalonate flux improved (+)-borneol production and other monoterpenes. We found that BPP dephosphorylation was a bottleneck for (+)-borneol production. We demonstrated that endogenous phosphatases Lpp1 and Dpp1 mediated BPP dephosphorylation to form (+)-borneol, especially, we found that overexpression of LPP1 has the highest production and Lpp1 co-localized with tSoBpps to the Golgi apparatus. To the best of our knowledge, this finding is the first in which the Golgi apparatus was identified as the primary site for terpenoid synthesis. Although the reason for the predominant localization of tSoBpps in the Golgi apparatus is unknown, the phenomenon may be associated with minimizing cell pressure. It was well known that the accumulation of monoterpenes has the potential to cause cell toxicity, whereas using different organelle compartmentalization strategies, such as peroxisome, lipid droplet, and mitochondria63, can not only improve the catalytic efficiency of enzymes but also avoid the harmful effects of toxic substances on cells. This observation that tSoBpps is located in the Golgi apparatus provides a good case for Golgi apparatus compartmentalization for the production of terpenoids. It will facilitate further investigation on developing the Golgi apparatus as a new organelle for other terpenoid production.
Balancing intracellular dephosphorylation levels is crucial for the synthesis of terpenoids, and LPP1/DPP1 plays a crucial role in regulating levels of prenyl diphosphate64. Previous studies have shown that knocking out LPP1/DPP1 promotes α-santalene production25,26. However, LPP1 and DPP1 deletion failed to significantly increase the production of other terpenoids, including monoterpenoids, sesquiterpene, and tetraterpenoids, such as sabinene47, α-terpinol65, linalool66, valencene67 and α-farnesene68, β-carotene69 and lycopene70. Interestingly, the effects of LPP1 and DPP1 deletion on β-elemene and D-limonene production were opposite in different strains71,72. This ambiguous success has misled many researchers to directly knock out LPP1/DPP1 in the production of terpenoids without sufficient evidence. The Hmg2 degradation rate depended on the level of cellular FPP and the FPP-derived molecule farnesol, thereby mediating early product inhibition of the mevalonate pathway73. Therefore, we agree with the statement that deletion of LPP1 and/or LPP1 positively affects terpenoid synthesis only above a certain threshold level of flux toward FPP67. On the one hand, we reduced the flux of FPP by replacing the native ERG20 promoter with the ERG7 promoter. It can be conceived that low levels of FPP would not cause feedback inhibition of Hmg2, so the strategies of LPP1 and DPP1 deletion were not applicable to our strain. On the other hand, the intermediate BPP, as a prenyl diphosphate, lacks effective exogenous enzymes to channel it towards (+)-borneol, while LPP1 and DPP1 account for most of the isoprenoid phosphate phosphatase activities in yeast. Obviously, overexpression of LPP1/DPP1 rather than knockout can effectively balance cell dephosphorylation and promote (+)-borneol synthesis. By analogy to the formation of geranylgeraniol or farnesol via the hydrolysis of GGPP or FPP by LPP1/DPP164, we attributed the production of (+)-borneol to the hydrolysis of BPP by these endogenous phosphatases.
By lipidome analysis, we revealed that this endogenous phosphatase-mediated terpenoid synthesis comes at the expense of lipids, further explaining the importance and complexity of LPP1 and DPP1 in terpenoid synthesis and cellular metabolism. Although overexpression of LPP1 and DPP1 restored most lipids to their normal levels, this competition imposed significant limitations on the overall (+)-borneol titer and increased copies of LPP1 and DPP1 impaired cellular fitness. Therefore, it is imperative to identify heterologous and efficient phosphatases capable of hydrolyzing BPP to facilitate the formation of (+)-borneol. Although Yang et al.37 showed that WvNUDX24 is involved in the catalysis of BPP, it failed to increase (+)-borneol production in yeast. In fact, WvNUDX24 could hydrolyze BPP, GPP, NPP, and FPP to their respective monophosphate products, which may explain its inability to increase (+)-borneol production in engineered strains with high GPP content. After all, the Nudix hydrolases could hydrolyze a wide range of organic pyrophosphates with varying degrees of substrate specificity58; this may also result in EcNudJ failing to increase (+)-borneol production in yeast. Therefore, we adopted bioinformatics analysis combined with a rapid screening method for BPP-specific phosphatases based on synthetic biology validation and three plant-derived Nudix1 hydrolases (TcNudix1, CcNudix1, BbNudix1.3) and four lipid phosphatases (CcLPPE2, BbLPPE2, CcLPPG, and BbLPPG) were identified. Notably, expressing heterologous phosphatases was feasible to improve (+)-borneol titer without perturbing cellular lipid metabolism, which is superior to elevated expression of endogenous phosphatases. It is noteworthy that the Nudix1 gene is not present in S. cerevisiae, and although YPL117C (IDI1) belongs to the Nudix hydrolase superfamily58, it failed to improve (+)-borneol production in yeast. Thus, overexpressing tTcNudix1 satisfied the dual requirement for BPP dephosphorylation and increased (+)-borneol production. This finding that plant-derived Nudix1 hydrolase and lipid phosphatases improve the production of (+)-borneol lays a foundation for further exploration of efficient phosphatase with specific preference for BPP.
BPPS, which converts GPP to BPP, is unusual in that the enzyme incorporates a diphosphate group in its final product, whereas terpene synthase reactions are generally terminated by deprotonation to form alkenes or water-trapping to form alcohols30-34. In the case of SoBPPS, the anionic diphosphate moiety steers the reaction trajectories toward product formation51,52, and the final carbocation is quenched by the re-addition of inorganic diphosphate, leading to the production of BPP as the main product31,35,36 (Fig. S1). Throughout the catalytic process, two conserved motifs, DDxxD and NSE/DTE, contain multiple L-aspartate residues that bind a trinuclear cluster of divalent metal ions (Mg2+or Mn2+), thereby enabling the complexation of the anionic diphosphate moiety of the substrate35. We observed that increasing the interaction between tSoBPPS and phosphatase disrupted the stability of the diphosphate moiety and affected the catalytic activity of tSoBPPS (Supporting Information Fig. S6). Although dephosphorylation of BPP is a rate-limiting step in (+)-borneol production, it is not feasible to increase (+)-borneol production by assembly of phosphatase into tSoBPPS. This finding provides a valuable understanding of the biosynthesis of (+)-borneol, highlighting the importance of the balance between BPP formation and dephosphorylation in a consecutive manner. Therefore, further enhancement of the dephosphorylation pathway by expression of highly efficient and BPP-specific phosphatase is a feasible strategy to increase the production and proportion of (+)-borneol.
5.　Conclusions
Less
Microbes have been extensively harnessed for the high-level production of various terpenoids by engineering isoprenoid synthase, biosynthesis pathways, and cell metabolism. However, the dephosphorylation of isoprenoid synthase has largely been ignored, potentially contributing to the low production of (+)-borneol in yeast. In this study, we systematically identified and characterized phosphatases involved in BPP dephosphorylation and optimized the cellular dephosphorylation process by overexpressing endogenous phosphatase and balancing cellular dephosphorylation metabolism, which significantly improved (+)-borneol production. In summary, we successfully reprogrammed the metabolism of S. cerevisiae to achieve high-level production of (+)-borneol by engineering BPP dephosphorylation and the mevalonate pathway. This novel dephosphorylation engineering strategy should pave a new way for enhancing isoprenoid biosynthesis in microbial cell factories.
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Year 2025 volume 15 Issue 2
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doi: 10.1016/j.apsb.2024.12.039
  • Receive Date：2024-10-15
  • Online Date：2026-09-17
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  • Received：2024-10-15
  • Revised：2024-12-20
  • Accepted：2024-12-24
Affiliations
    aState Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, National Resource Centre for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
    bDivision of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
    cInstitute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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