代谢工程改造热带假丝酵母高效合成1,2,4-丁三醇

Metabolic engineering of Candida tropicalis for efficient production of 1,2,4-butanetriol

摘要:

【背景】 1,2,4-丁三醇是一种手性多羟基醇，作为一种关键的平台化合物，主要用于合成高能材料1,2,4-三硝酸酯丁三醇，这种材料可作为飞机、火箭和导弹等武器的推进剂。随着合成生物学的快速发展，利用微生物细胞工厂生产1,2,4-丁三醇受到越来越多的关注。【目的】 以前期构建的产1,2,4-丁三醇的热带假丝酵母(Candida tropicalis)工程菌为出发菌株，通过代谢工程策略进一步优化人工途径，降低中间产物积累，提高1,2,4-丁三醇的发酵水平，评价工程菌利用廉价的玉米芯水解液发酵生产1,2,4-丁三醇的能力。【方法】 通过启动子工程优化木糖脱氢酶基因的表达水平，减少木糖酸中间产物的积累。利用碳酸钙作为中和剂调节发酵过程的pH，通过发酵培养基铁离子浓度优化等策略提高热带假丝酵母细胞工厂发酵生产1,2,4-丁三醇的能力。【结果】 相较于出发菌株，构建的工程菌发酵生产1,2,4-丁三醇的产量提高了42%，5 L发酵罐中产量达到6.20 g/L。同时，工程菌以玉米芯水解液作为原料能够生产出2.60 g/L的1,2,4-丁三醇。【结论】 通过代谢工厂策略和发酵过程优化，提高了热带假丝酵母合成1,2,4-丁三醇的能力，还首次评价热带假丝酵母细胞工厂利用玉米芯水解液合成1,2,4-丁三醇的可行性，为大规模地利用生物质原料发酵生产1,2,4-丁三醇挖掘出一个有潜力的宿主。

Abstract:

[Background] 1,2,4-butanetriol (BT), a chiral polyol, plays a role as a platform chemical in the synthesis of energetic materials such as 1,2,4-butanetriol trinitrate, which is used as a propellant in a variety of military applications, including aircraft, rockets, and missiles. With the rapid development of synthetic biology, building microbial cell factories for the production of 1,2,4-butanetriol has received increasing attention. [Objective] In this study, we employed metabolic engineering to optimize the exogenous 1,2,4-butanetriol synthesis pathway in a pre-constructed strain of Candida tropicalis, aiming to reduce the accumulation of intermediates and increase the production of 1,2,4-butanetriol. Furthermore, we evaluated the ability of the engineered strain to produce 1,2,4-butanetriol by fermentation with inexpensive corncob hydrolysate. [Methods] We employed promoter engineering to optimize the expression level of the xylose dehydrogenase gene and reduce the accumulation of xylonate. Furthermore, we enhanced the production of 1,2,4-butanetriol in C. tropicalis by using calcium carbonate as a neutralizer to regulate the pH and optimizing the concentration of iron ions in the fermentation medium. [Results] Through the combined efforts of metabolic engineering and fermentation process optimization, the final yield was increased by 42%, reaching 6.20 g/L in a 5 L fermenter. In addition, the engineered strain was capable of producing 2.60 g/L 1,2,4-butanetriol by using corncob hydrolysate as the substrate. [Conclusion] We improve the ability of C. tropicalis to synthesize 1,2,4-butanetriol by metabolic engineering and fermentation process optimization. Moreover, we for the first time evaluate the feasibility of C. tropicalis cell factories for the synthesis of 1,2,4-butanetriol with corncob hydrolysate, which provides support the large-scale production of 1,2,4-butanetriol by fermentation with biomass feedstocks.