基于代谢工程改造大肠杆菌的人工途径合成冰片研究

Metabolic Engineering of Escherichia coli for Borneol Production Using an Artificial Pathway

摘要:

目的 构建以异戊二烯醇为底物的人工简短异戊烯醇利用途径（isopentenol utilization pathway，IUP），解决传统MEP/MVA途径合成冰片存在的途径长、辅因子需求大等问题，提高冰片生物合成效率。方法 以大肠杆菌（Escherichia coli， E.coli）BL21为底盘细胞，通过设计以异戊二烯醇为底物的两步酶促催化法，构建前体IPP/DMAPP的IUP，接着通过改善冰片二磷酸合酶CbTPS1的可溶性表达水平、筛选不同来源异戊二烯基转移酶改善前体IPP/DMAPP合成GPP的通量、适配性优化冰片合成途径关键酶的表达水平、筛选高活性磷酸酶等策略，同时探索不同金属离子对冰片生物合成的影响。结果 人工简短IUP合成冰片的效率较MEP途径提高了12.70%。通过酶工程和途径优化，冰片产量提升至2 642.8 μg/L。添加Mg2?后，产量进一步提高至5 912.4 μg/L。结论 利用人工简短IUP实现了冰片的生物合成，可为其他萜类化合物的生物合成提供参考。

Abstract:

Objective This study aims to construct an artificial concise isopentenol utilization pathway (IUP) with isoprenol as the substrate, thus addressing the limitations (such as excessive pathway length and high cofactor demand) of the conventional MEP/MVA pathways and enhancing the biosynthesis efficiency of borneol.Method A two-step enzymatic catalysis pathway with isoprenol as the substrate was designed for the synthesis of precursor IPP/DMAPP in Escherichia coli (E.coli） BL21 to synthesize borneol. Subsequently, metabolic engineering strategies were employed to further improve the synthesis of borneol, such as improving the soluble expression level of the bornyl diphosphate synthase CbTPS1, screening different sources of prenyltransferases to enhance the metabolic flux of IPP/DMAPP to GPP, optimizing the expression levels of key enzymes in the borneol biosynthesis pathway, and screening highly active phosphatases. Finally, the effects of different metal ions on the biosynthesis of bornol were explored.Result The artificial IUP increased the borneol production efficiency by 12.70% compared with the MEP pathway. Through enzyme and pathway engineering, the titer of borneol reached 2 642.8 μg/L. Further supplementation with Mg²⁺ increased the borneol titer to 5 912.4 μg/L.Conclusion The biosynthesis of borneol was achieved via the artificial IUP, which can provide theoretical and methodological references for the biosynthesis of other terpenoids.