Tian Yongqing, Yang Yige, Ni Minmin, Wo Jing
Institute of Engineering Biology and Health, Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, 310014, China.
Jiangsu Key Laboratory of Advanced Catalytic Materials and Technology, School of Petrochemical Engineering, Changzhou University, Changzhou, 213164, China.
Microb Cell Fact. 2025 Mar 7;24(1):54. doi: 10.1186/s12934-025-02680-6.
Vanillin is a widely utilized flavor compound of significant value in the food and pharmaceutical sectors, which can be obtained through natural extraction, chemical synthesis, or biotechnological processes. However, the yield from vanilla pods is insufficient to meet market demand, and chemically synthesized vanillin not only encounters limitations in its application within the food and pharmaceutical industries but also needs to address environmental concerns and unsustainable raw material sources. Hence, it is imperative to explore alternative approaches to develop an efficient and cost-effective green vanillin. To address the challenges encountered in vanillin biosynthesis, such as substrate uptake limitations and product-induced inhibition of cell growth,we leveraged the advantages of surface display technology and artificial multi-enzyme scaffolds to construct a hybrid surface-display biocatalytic system by assembling Eugenol oxidase (EUGO) and dioxygenase (NOV1), which can convert lignin biowaste 4-n-propylguaiacol (4-PG) into vanillin on the surface of Escherichia coli BL21(DE3).
To assemble bioactive macromolecules of EUGO and NOV1 on the surface of E. coli BL21(DE3), we utilized Lpp-OmpA-SpyCatcher (LOAS) as an anchoring motif and displayed EUGO-linker-NOV1-SpyTag (ELNS) by covalent interaction between SpyTag andSpyCatcher to allow their spatial proximity. After optimization of the reaction system, our self-assembly display system exhibited highly efficiency in converting 4-PG into vanillin and reached a final concentration of vanillin at 12.58 g/L, 2.5 times higher than that achieved by thewhole-cell biocatalytic system. The LOAS-ELNS display system was applied to the sustainable biosynthesis of vanillin from lignin-derived 4-n-propylguaiacol at least 10 times.
This work provided a generalized approach to co-expressing proteins and offered an efficient, eco-friendly, and renewable method for the biosynthesis of vanillin from 4-PG.
香草醛是一种在食品和制药行业具有重要价值的广泛应用的风味化合物，可通过天然提取、化学合成或生物技术方法获得。然而，香草豆荚的产量不足以满足市场需求，化学合成的香草醛不仅在食品和制药行业的应用中存在局限性，还需要解决环境问题和不可持续的原材料来源。因此，探索替代方法来开发高效且经济高效的绿色香草醛势在必行。为了解决香草醛生物合成中遇到的挑战，如底物摄取限制和产物对细胞生长的抑制，我们利用表面展示技术和人工多酶支架的优势，通过组装丁香酚氧化酶（EUGO）和双加氧酶（NOV1）构建了一种混合表面展示生物催化系统，该系统可在大肠杆菌BL21(DE3)表面将木质素生物废料4-正丙基愈创木酚（4-PG）转化为香草醛。
为了在大肠杆菌BL21(DE3)表面组装EUGO和NOV1的生物活性大分子，我们利用Lpp-OmpA-SpyCatcher（LOAS）作为锚定基序，并通过SpyTag和SpyCatcher之间的共价相互作用展示EUGO-接头-NOV1-SpyTag（ELNS），以使它们在空间上接近。经过反应体系优化后，我们的自组装展示系统在将4-PG转化为香草醛方面表现出高效性，香草醛最终浓度达到12.58 g/L，比全细胞生物催化系统高出2.5倍。LOAS-ELNS展示系统至少10次应用于从木质素衍生的4-正丙基愈创木酚可持续生物合成香草醛。
这项工作提供了一种共表达蛋白质的通用方法，并为从4-PG生物合成香草醛提供了一种高效、环保和可再生的方法。
