背景:衣康酸,不饱和C5二碳酸,具有显著的市场需求和前景。它具有许多生物学功能,比如抗癌,抗炎,和医学中的抗氧化作用,是工业中必不可少的可再生平台化学品。然而,土曲霉工业生产衣康酸的发展,当前的标准生产菌株,受到该物种不可避免的缺点的阻碍。开发高效的细胞工厂对于衣康酸的可持续和绿色生产至关重要。
结果:本研究采用组合工程策略构建大肠杆菌细胞以有效生产衣康酸。两个必需基因(顺式-aconitate脱羧酶(CAD)编码基因cadA和乌头酸酶(ACO)编码基因acn)采用各种遗传构建体和质粒组合来产生12个待筛选的重组大肠杆菌菌株。其中,以柠檬酸盐为底物的大肠杆菌BL-CAC显示出最高的滴度,并进一步系统优化了诱导和反应条件。随后,利用酶进化来优化限速酶CAD和合成蛋白质支架来共定位ACO和CAD,以提高衣康酸的生物合成效率。在优化的反应条件下结合进料控制策略,衣康酸滴度达到398.07mM(51.79g/L)的工程大肠杆菌BL-CAR470E-DS/A-CS细胞作为催化剂,在异源宿主中的最高比产量为9.42g/g(DCW)在48小时。
结论:每单位生物质的优异催化性能显示出高效生产衣康酸和有效降低催化细胞消耗的潜力。这项研究表明,有必要不断探索工程策略来发展高性能的细胞工厂,以突破现有的瓶颈,实现经济的商业化生产衣康酸。
BACKGROUND: Itaconic acid, an unsaturated C5 dicarbonic acid, has significant market demand and prospects. It has numerous biological functions, such as anti-cancer, anti-inflammatory, and anti-oxidative in medicine, and is an essential renewable platform chemical in industry. However, the development of industrial itaconic acid production by Aspergillus terreus, the current standard production strain, is hampered by the unavoidable drawbacks of that species. Developing a highly efficient cell factory is essential for the sustainable and green production of itaconic acid.
RESULTS: This study employed combinatorial engineering strategies to construct Escherichia coli cells to produce itaconic acid efficiently. Two essential genes (cis-aconitate decarboxylase (CAD) encoding gene cadA and aconitase (ACO) encoding gene acn) employed various genetic constructs and plasmid combinations to create 12 recombination E. coli strains to be screened. Among them, E. coli BL-CAC exhibited the highest titer with citrate as substrate, and the induction and reaction conditions were further systematically optimized. Subsequently, employing enzyme evolution to optimize rate-limiting enzyme CAD and synthesizing protein scaffolds to co-localize ACO and CAD were used to improve itaconic acid biosynthesis efficiency. Under the optimized reaction conditions combined with the feeding control strategy, itaconic acid titer reached 398.07 mM (51.79 g/L) of engineered E. coli BL-CAR470E-DS/A-CS cells as a catalyst with the highest specific production of 9.42 g/g(DCW) among heterologous hosts at 48 h.
CONCLUSIONS: The excellent catalytic performance per unit biomass shows the potential for high-efficiency production of itaconic acid and effective reduction of catalytic cell consumption. This study indicates that it is necessary to continuously explore engineering strategies to develop high-performance cell factories to break through the existing bottleneck and achieve the economical commercial production of itaconic acid.