研究目标和重要性
本文的研究目标是开发一种优化的微生物工程平台,高效生产drimane型杂萜类化合物(DMT)的关键手性砌块——drimenol和albicanol。DMT是一类具有广泛生物活性的天然产物,在药物和农业领域有很大的应用潜力,如pyripyropene C可以强效抑制胆固醇的酰基辅酶A,ent-(+)-chromazonarol可用于防治农作物病原菌。
DMT, characterized by a drimane terpenoid moiety fused with a non-terpenoid component, comprise over 500 compounds with remarkable structural diversity and a wide range of biological activity (Figure 1A). For instance, pyripyropene C exhibits potent acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitory activity with an IC50 of 53 nM. ent-(+)-Chromazonarol (2) is a promising candidate against Sclerotinia scleotiorum, an agriculturally important plant pathogen.
然而,传统的DMT合成方法存在原子利用率低、步骤繁琐等问题。drimenol和albicanol作为关键的手性合成砌块,可以大大简化合成路线,但它们在自然界产量很低,微生物发酵的产量也不高,限制了其应用。
Previous investigations have primarily relied on two strategies to access DMT: the enantioselective polycyclization approach and the chiral pool approach. However, both strategies face significant challenges. The former approach is linear and sometimes exhibits variable enantioselectivity in the biomimetic cyclization step. In the latter approach starting from sclareolide or sclareol, a 3 to 6-step side chain degradation is required to achieve the desired C15 drimane skeleton, which reduces atom economy and overall efficiency (Figure 1B).
因此,开发高效生产这两种关键合成砌块的微生物工程平台,对于推动DMT及其衍生药物的研发和产业化具有重要意义。
创新思路和方法
本文提出了几个优化PhoN-IPK系统来提高drimenol和albicanol产量的创新思路(图1C):
图1
引入外源Nudix水解酶SsNDH,并将其与drimenol合酶SsDMS融合,提高焦磷酸水解效率 通过理性设计和共进化分析,筛选出提高PhoN催化效率的关键突变位点(E122R、T157K和R160K) 优化发酵工艺参数,最终使albicanol产量达到3.5 g/L
To overcome the low titer issues, we aim to improve the efficiency of this PhoN-IPK system. It has been reported that knockout of the acid phosphatase gene, phoN, caused a 17-fold decrease in lycopene production, while overexpression of phoN resulted in 4-fold higher production compared to wild-type diacylglycerol kinase (DGK). Additionally, the conversion of intermediate drimenyl diphosphate (DPP) to drimenol relies on diphosphate hydrolysis, which might be insufficiently supported by endogenous hydrolase in E. coli.
Herein, we developed a comprehensive strategy to improve drimenol and albicanol production, significantly advancing the PhoN-IPK system. We incorporated a Nudix hydrolase (SsNDH) and optimized its fusion with the terpene synthase, enhancing the hydrolysis of DPP. We also engineered PhoN through rational design and consensus approaches, identifying key mutations (E122R, T157K, and R160K) that improved its activity.
与以往的研究相比,本文从融合水解酶、改造磷酸化酶、改进发酵工艺等多个层面系统优化了整个生物合成途径,显著提高了目标产物的产量。同时,对PhoN突变体的晶体结构解析和分子动力学模拟,揭示了氨基酸突变提高酶活的内在机制。这些方法为工业化生产 drimenol、albicanol等关键中间体提供了新思路。
引入Nudix水解酶SsNDH显著提高drimenol产量
文章首先研究了提高drimenol产量的策略。通过引入来自 S. showdoensis的 Nudix 水解酶 SsNDH,drimenol 产量从最初的 15 mg/L 提高到 63 mg/L,提高了4.2倍。
Co-expression of the gene of ssndh with ipk, ispA, idi, and phoN in E. coli increased the drimenol yield to 63 mg/L, representing a 4.2-fold improvement compared to the original yield of 15 mg/L. This result confirmed our hypothesis about the insufficient catalytic efficiency of E. coli's inherent hydrolases.
进一步将SsNDH与drimenol合酶SsDMS融合表达,产量再次提高到100 mg/L。优化linker长度后,最高达到111 mg/L(图2C,D)。
Subsequently, we constructed and co-expressed the SsNDH-SsDMS fusion protein, which increased the drimenol yield to 100 mg/L, representing a 1.6-fold improvement over the non-fused system (Figures 2B and 2C).
图2
这些结果表明,引入外源水解酶及其与合酶的融合表达策略可以显著提高drimenol的合成效率,突破了宿主内源性水解酶活性不足的限制。
理性设计与进化共识分析指导PhoN突变提高酶活
接下来,作者通过理性设计和氨基酸保守性分析,优化了PhoN的催化效率。在PhoN的活性口袋附近引入正电荷(如E122R突变),可以增强酶与底物的静电相互作用,使drimenol产量提高1.6倍(178 mg/L)(图3C,D)。
The E122R mutant strain exhibited a 1.6-fold increase in drimenol production (178 mg/L, Figure 3D) compared to the original strain (111 mg/L), supporting the role of positive charge in enhancing phosphorylation efficiency.
图3
基于500个PhoN同源酶的多序列比对,筛选出6个非保守性突变位点(S90, Y135, K153, T157, R160和I222)。将其替换为更保守的氨基酸后,发现S90G、T157K和R160K突变分别使产量提高约1.2倍(图3F,G)。
Fermentation of the strains revealed that S90G, T157K, and R160K yielded around 1.2-fold increases in drimenol production compared to the wild-type strain (Figure 3G).
这些结果说明,合理引入正电荷以增强PhoN与底物的结合,以及基于进化保守性分析优化关键位点,是提高PhoN催化效率的有效策略。
有利突变的组合进一步提高drimenol和albicanol产量
在前期突变的基础上,作者将有利突变进行组合,进一步提高了drimenol和albicanol的产量。其中,PhoN的三重突变体E122R/T157K/R160K使drimenol产量达到398 mg/L,比野生型提高了3.6倍(图4A)。
Notably, the triple mutant E122R/T157K/R160K achieved an impressive yield of 398 mg/L (Figure 4A).
图4
晶体结构分析和分子动力学模拟表明,突变延长了PhoN与底物的稳定结合时间,这可能是提高催化效率的关键因素(图4C,D)。
Analysis of the RMSD between the ligand and protein revealed significant changes in ligand-enzyme interactions. Notably, in contrast to the wild-type protein, PhoNE122R/T157K/R160K maintained stable interactions with the ligand for up to 40 ns before ligand dissociation from the substrate pocket (Figure 4D).
将优化的PhoN-IPK系统应用于albicanol生产,在摇瓶发酵中产量达到1805 mg/L,在5L生物反应器中进一步提高到3.5 g/L(图4E),实现了albicanol的高效生物合成。
Maximum yield (1805 mg/L) was achieved at 96 hours, followed by a decline in productivity. Further optimization in bioreactor conditions, including a two-stage feeding approach initiated when the OD600 reached 30, led to improved yields. At 34 hours, when the albicanol titer reached 1.6 g/L, an additional 8 g of ISO was added to ensure sufficient precursor supply. This strategy, with a total of 16 g ISO addition, resulted in a peak albicanol titer of 3.5 g/L at 58 hours (Figure 4E), representing the highest reported microbial production of albicanol to date.
这些结果表明,系统整合有利突变可以显著提升关键酶的催化性能,是优化复杂生物合成途径的重要策略。优化的细胞工厂为drimenol和albicanol的工业化生产奠定了基础。
以albicanol为关键中间体实现多种DMT的高效合成
最后,作者以albicanol为手性合成子,通过Appel碘代、Weix偶联等化学转化策略,高效合成了zonarol、ent-(+)-chromazonarol、mycoleptodiscin A和pelorol等多种DMT(图5),所需步骤较传统路线大大减少。这些结果证明了drimenol和albicanol作为关键中间体在DMT全合成中的广泛适用性。
图5
To showcase the synthetic potential of drimenol (5) and albicanol (6) as chiral building blocks, we aimed to synthesize several DMT, including zonarol (1), ent-(+)-chromazonarol (2), mycoleptodiscin A (3), and pelorol (4), with different aryl-ring modes for divergent synthesis. The synthesis commenced with iodination of these two chiral compounds under Appel's conditions, resulting in the corresponding alkyl iodide on gram scale. Fortunately, alkyl iodide (7) obtained from albicanol successfully produced coupled products 9 in moderate to good yields with various aryl halides using Weix's condition (Figure 5A).
此外,与经典的以sclareolide为起始原料的合成路线相比(图1B),基于albicanol的策略可以显著缩短合成步骤,提高原子经济性。
Classical synthetic strategies, which rely on plant-derived chiral pool building blocks including sclareolide and sclareol, are atom-inefficient as they require the multi-step side chain degradations. While alternative building blocks like drimenol and albicanol offer promising solutions, their limited natural availability and inefficient microbial production severely hinder their applications.
综上所述,本研究的核心发现可总结为:
引入外源Nudix水解酶SsNDH及其与萜类合酶的融合,可显著提高DMT合成砌块的合成效率; 基于结构分析和进化保守性,优化磷酸激酶PhoN的关键氨基酸,可有效提高其催化活性; 在改造的宿主中整合优势突变,drimenol和albicanol的产量分别达到398 mg/L和3.5 g/L,实现了关键合成子的高效生物制造; 以albicanol为起始原料,可高效合成多种具有药用潜力的DMT天然产物,为开发新型药物先导化合物开辟了途径。
对业界的影响与潜在商机
本研究建立的高效微生物细胞工厂及后续化学合成策略,为drimenol、albicanol等关键中间体及DMT类天然产物的工业化生产提供了可行方案,有望加速其在药物、农药等领域的研发和应用。
作为学术界的科研人员,可以在本文的基础上,进一步拓展以下方向:
基于结构生物学分析,理性设计和定向进化改造PhoN、terpene合酶等关键酶,进一步提高催化效率和产量; 鉴定和开发新的氧化水平多样的倍半萜合酶或氧化酶,拓展可生物合成的drimane型中间体库; 探索生物合成与化学合成的耦合策略,从中间体出发多样性导向合成DMT类天然产物及其类似物。
研究方向的进一步探索
本研究虽然在drimenol和albicanol的微生物合成方面取得了重要突破,但离实现DMT的完全生物合成还有较大差距。未来可以在以下几个方向开展深入研究:
在产酶菌中整合萜类骨架与非萜类片段的偶联途径,实现DMT的一步发酵生产;
Building on previously reported drimane-type sesquiterpene synthases with diverse oxidation patterns, this system can be adapted to generate an expanded array of drimane-type building blocks, providing synthetic chemists with versatile starting materials for complex DTM synthesis.
采用生物工程和代谢工程策略,进一步提高关键中间体的产量,降低发酵成本; 开发高通量筛选技术,从天然酶资源中发掘和创制新型drimane合酶,构建中间体的生物合成工具箱；
