Microbial engineering high-yield Drimane-type chiral blocks facilitate efficient synthesis of natural heteriterpene products

01 Abstract

Drimane-type heteropterpene (DMT) has an important application prospect in the field of drug and pesticide development due to its remarkable biological activity.

Among them, pyripyropene C showed a molar level of cholesterol acyltransferase inhibitory activity (IC50 = 53 nM), Zonarol and Pelorol showed excellent neuroprotective and antifungal activities, respectively. ent-(+) -Chromazonarol showed excellent application potential in the control of important plant pathogens in agriculture.

Professor Dong Liaobin's research group from China Pharmaceutical University and Professor Li Fu 琸's team from Fudan University have made an important breakthrough in this field.

Through the innovative development of an efficient terpene production platform, rational design strategies have been used to significantly increase the output of key chiral synthetic blocks drimenol and albicanol.

The research results are published in the journal Microbe Engineering to Provide Drimane-Type Building Blocks for Chiral Pool Synthesis Meroterpenoids was published in Angewandte Chemie.

02 Content

At present, the synthesis of DMT compounds mainly relies on enantioselective polycyclization and chiral pool, but both of these methods have significant limitations.

The enantioselective polycyclization strategy has some problems, such as long synthesis route and difficult control of stereoselectivity in key bionic cyclization steps.

The chiral cell strategy with perillolactone and perillyl alcohol as starting materials requires 3-6 step side chain degradation process to obtain the required C15 skeleton, which seriously affects the atomic economy and total yield.

To overcome these challenges, the research team selected drimenol and albicanol as alternative chiral synthetic blocks.

These two compounds themselves have C15 scaffolders, which can be directly coupled with aryl halides without carbon chain degradation steps, providing a more concise and efficient synthesis path for DMT diversity synthesis.

These two key chiral synthetic blocks are rare in nature, and reported biosynthesis methods are inefficient.

For example, the drimenol yield obtained by expressing PhDS gene in tobacco leaves was only 392 μg/g fresh weight.

The production of albicanol in yeast requires 4L medium and 96 hours of fermentation to obtain 8.3 mg of product.

This yield bottleneck severely restricts the development and application of drimane type heteropterpene functional molecules, and also reveals that there is a huge optimization room for fermentation production of drimane synthetic blocks.

production system of Escherichia coli was constructed and effect of exogenous Nudix hydrolase on production was investigated

In order to increase the yield of synthetic blocks, the research team first constructed an initial screening system using drimenol as the template molecule, and obtained an initial yield of 15 mg/L.

Considering that the drimenol synthesis process may have a potential impact on yield, the researchers compared the effects of drimenol synthases from three different bacterial sources on yield, however, no significant yield differences were observed.

Based on previous studies on the catalytic mechanism of type II drimenol synthase SsDMS, it was found that the pyrophosphate group in the product needed to be hydrolyzed by endogenous hydrolase to generate the final drimenol.

The team speculated that the insufficient catalytic efficiency of endogenous hydrolase may be one of the key factors limiting the yield.

By analyzing the drimenol biosynthesis gene cluster, it was found that Nudix hydrolase was prevalent in the upstream of Drimenol, and by constructing exogenous Nudix hydrolase SsNDH, the yield was increased to 63 mg/L, which verified the hypothesis that the catalytic efficiency of endogenous hydrolase was insufficient.

In order to further increase the yield, the research team found that the fusion expression of SsDMS and SsNDH can achieve spatial optimization of the catalytic sites of both.

The research team introduced Gly-Ser-Gly flexible connection linker and systematically investigated the effect of different length linker on the fusion protein.

Combined with experimental verification, it was found that when the number of linker repeats was 3, the yield reached the highest value of 111 mg/L.

Notably, when the number of linker repeats was 4, the yield decreased significantly, suggesting that longer linkers may negatively affect the spatial arrangement and catalytic efficiency of fusion proteins.

PhoN engineering transformation guided by rational design

To further increase the yield of drimenol, the research team used structural analysis and rational design to optimize the catalytic efficiency of rate-limiting enzyme PhoN in the "artificial two-step" pathway.

First, the team used AlphaFold2 to predict the structure of PhoN and found a structurally similar acid phosphatase 1D2T (RMSD 0.333A) through homologous modeling.

Sequence alignment analysis combined with alanine scanning assay confirmed the catalytic active sites, including K133, R140, S166, G167, H168, R201, H207 and D211.

Given that the phosphorylation and hydrolysis reactions catalyzed by PhoN are reversible, the research team hypothesized that by increasing the positive charge near the active pocket, the enzyme-phosphate interaction could be enhanced, thereby increasing the phosphorylation efficiency.

Based on the PhoN-DMAP molecular docking results, the team selected four residues (E122, G125, L158, and I171) in the substrate 4A range for positively charged amino acid replacement.

Structural prediction showed that E122R/K mutation could introduce effective positive charge at key substrate entry sites.

Although G125R/K increases the positive charge, it causes steric hindrance.

Mutations in the internal residues L158 and I171 have limited effect on the surface charge.

The experimental results were highly consistent with the prediction: compared with the wild-type strain (111 mg/L), drimenol production of E122R mutant increased by 1.6 times to 178 mg/L, supporting the role of positive charge in enhancing phosphorylation efficiency.

The yield of G125R/K mutants was significantly reduced (17 and 9 mg/L, respectively), which may be related to pocket steric resistance.

L158 and I171 mutations also resulted in yields below 10 mg/L in both cases.

These results demonstrate the positive effect of directional introduction of positively charged residues on improving the catalytic efficiency of PhoN.

In addition, the research team also hopes to further increase production by improving the overall stability of PhoN phosphatase.

Coevolutionary analysis of protein families can reveal the conserved tendency of amino acids, and modification based on coevolutionary design can improve the overall stability and enzyme activity of proteins.

Given that PhoN belongs to a large family of highly similar acid phosphatases, the team analyzed its evolutionary pattern to guide the transformation of the enzyme on this basis.

The research team selected 500 acid phosphatase sequences with more than 80% similarity to PhoN, used EMBL-MUSCLE for multi-sequence comparison, and used ConSurf Web Server for conservative analysis, and divided amino acid residues into nine grades according to their degree of conservation.

With in the active pocket 12A range, the team identified six less conserved amino acid residues: S90, Y135, K153, T157, R160, and I222.

Based on these findings, the research team designed ten PhoN variants to replace these residues with more conserved amino acids, including S90A, S90G, Y135K, Y135H, K153T, T157K, R160K, R160T, I222V, and I222A.

Fermentation experiments showed that drimenol yield of S90G, T157K and R160K mutants increased by about 1.2 times compared with wild-type strains.

These results indicate that this family of enzymes tends to evolve towards higher stability and catalytic efficiency during the long evolutionary process.

Combining dominant mutants to increase yield and explore mechanism of high yield

After that, the research team combined the dominant mutants obtained by the two rational design strategies. First, the mutants S90G, T157K and R160K obtained by co-evolutionary modification were pin-two combinations and three-mutation combinations.

The drimenol yields of the dual variants S90G/T157K, S90G/R160K and T157K/R160K were 162 mg/L, 150 mg/L and 163 mg/L, respectively, which was about 1.4-1.5 times higher than that of the wild type.

The yield of three mutants, S90G/T157K/R160K, unexpectedly decreased to only 8 mg/L, suggesting that the breakdown of the co-evolutionary relationship between residues may affect the stability of the enzyme.

The team combined the dominant mutant E122R obtained by charge modification with the dominant mutant obtained by conservative modification, in which the drimenol yield of the E122R/T157K/R160K mutant was significantly increased, reaching 398 mg/L, which was 27 times higher than the initial yield.

Notably, the yield of the four-mutant E122R/S90G/T157K/R160K was lower than that of the optimal three-mutant (240 mg/L), a phenomenon similar to that of S90G/T157K/R160K, indicating that multiple mutations do not always produce synergistic effects.

In order to clarify the mechanism of yield enhancement, the team successfully analyzed the crystal structure of PhoNE122R/T157K/R160K (PDB: 8YC1).

Molecular dynamics simulations showed that the stable binding time of the mutant to the ligand was about 20 ns longer than that of the wild type, further supporting the extended protein-ligand interaction time as a key factor in the increased yield of the triple mutant.

High-yield systems were migrated to albicanol and bioreactor production

After making an important breakthrough in drimenol production, the research team began to evaluate its application in heteriterpene synthesis.

In the initial attempt of nickel-catalyzed reduction coupling of drimenol iodide and aryl iodide, mainly diene products were obtained due to the β-H elimination reaction.

This result prompted the team to turn to albicanol, whose iodide can be successfully coupled to different aryl halogens under Weix conditions.

Based on this assumption, the research team applied the optimized PhoN-IPK system to the production of albicanol.

SsDMS was replaced with AncC, a cyclase derived from Antrodia cinnamomea, and the initial yield was 281 mg/L.

Through the optimization of fermentation conditions (IPTG concentration 0.05mM, glycerol concentration 2%) and the regulation of ISO addition strategy, the flask yield was increased to 1805 mg/L.

Using a two-stage feeding strategy in a 5L fermenter (starting the feeding when OD600 is 30, adding 8 g ISO when albicanol is 1.6 g/L), the final yield reached 3.5 g/L, creating the highest record for microbial fermentation production of albicanol.

Albicanol was used as a synthetic block to synthesize different DMT

To demonstrate the potential application of albicanol as a synthetic block, the research team completed synthesis studies of four representative DMT using Albicanol as a starting material.

Starting from albicanol, zonarol (1) was synthesized in three steps with a total yield of 66%.

ent-(+) -Chromazonarol (2) was obtained by four-step synthesis with the total yield of 65%.

mycoleptodiscin A (3) was obtained by six-step synthesis with a total yield of 22%.

pelorol (4) was obtained by six-step synthesis with a total yield of 14%.

Compared with traditional strategies using perillactone/perillyl alcohol as starting material, these synthetic routes avoided the side chain degradation step and significantly improved the synthesis efficiency.

The results fully demonstrate the superiority of albicanol as a chiral synthetic block in the synthesis of DMT diversity, and provide a more efficient synthesis pathway for the development of DMT drugs.

Dong Liaobin Group of China Pharmaceutical University and Li Fu 琸 Group of Fudan University innovated the combination of microbial engineering and chemical synthesis to establish an efficient interdisciplinary technical system for the key bottleneck problem in the synthesis of heteriterpenoid natural products.

Through systematic enzyme engineering and structural biology research, the team not only realized the large-scale preparation of chiral synthetic blocks, but also clarified the catalytic mechanism of acid phosphatase in the "artificial two-step" system at the molecular level, providing a theoretical basis for the development of efficient terpenoid biosynthesis systems.

This study establishes an innovative research paradigm that uses microbial engineering to obtain key chiral blocks and then achieves efficient synthesis of target molecules through flexible chemical transformation.

This strategy overcomes the assembly problem of complex enzymatic cascade reaction in traditional total biosynthesis and the difficulty of obtaining chiral blocks in classical total synthesis, and promotes the discovery and drug development of heterterpenoid bioactive molecules.

Article source:

Du, W.; # Cheng, Z.; #Pan, X.; # Liu, C.; Yue, M.; Li, T.; Xiao, Z.; Li, L.-L.; Zeng, X.; Lin, X.; Li, F.*; Dong, L.-B.*, Microbe engineering to provide drimane-type building blocks for chiral pool synthesis of meroterpenoids. Angew. Chem. Int. Ed. 2025, e202419463. DOI: https://doi.org/10.1002/anie.202419463.