Zeng Bo-Xuan, Yao Ming-Dong, Wang Ying, Xiao Wen-Hai, Yuan Ying-Jin
Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, China.
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, China.
Front Bioeng Biotechnol. 2020 Mar 17;8:152. doi: 10.3389/fbioe.2020.00152. eCollection 2020.
Direct bioproduction of DHAA (dihydroartemisinic acid) rather than AA (artemisinic acid), as suggested by previous work would decrease the cost of semi-biosynthesis artemisinin by eliminating the step of initial hydrogenation of AA. The major challenge in microbial production of DHAA is how to efficiently manipulate consecutive key enzymes ADH1 (artemisinic alcohol dehydrogenase), DBR2 [artemisinic aldehyde Δ11(13) reductase] and ALDH1 (aldehyde dehydrogenase) to redirect metabolic flux and elevate the ratio of DHAA to AA (artemisinic acid). Herein, DHAA biosynthesis was achieved in by introducing a series of heterologous enzymes: ADS (amorpha-4,11-diene synthase), CYP71AV1 (amorphadiene oxidase), ADH1, DBR2 and ALDH1, obtaining initial DHAA/AA ratio at 2.53. The flux toward DHAA was enhanced by pairing fusion proteins DBR2-ADH1 and DBR2-ALDH1, leading to 1.75-fold increase in DHAA/AA ratio (to 6.97). Moreover, to promote the substrate preference of ALDH1 to dihydroartemisinic aldehyde (the intermediate for DHAA synthesis) over artemisinic aldehyde (the intermediate for AA synthesis), two rational engineering strategies, including downsizing the active pocket and enhancing the stability of enzyme/cofactor complex, were proposed to engineer ALDH1. It was found that the mutant H194R, which showed better stability of the enzyme/NAD complex, obtained the highest DHAA to AA ratio at 3.73 among all the mutations. Then the mutant H194R was incorporated into above rebuilt fusion proteins, resulting in the highest ratio of DHAA to AA (10.05). Subsequently, the highest DHAA reported titer of 1.70 g/L (DHAA/AA ratio of 9.84) was achieved through 5 L bioreactor fermentation. The study highlights the synergy of metabolic engineering and protein engineering in metabolic flux redirection to get the most efficient product to the chemical process, and simplified downstream conversion process.
与先前研究表明的生产青蒿酸（AA）不同，直接生物生产二氢青蒿酸（DHAA）可通过省去AA初始氢化步骤来降低半生物合成青蒿素的成本。微生物生产DHAA的主要挑战在于如何有效调控连续的关键酶——青蒿醇脱氢酶（ADH1）、青蒿醛Δ11(13)还原酶（DBR2）和醛脱氢酶（ALDH1），以重定向代谢通量并提高DHAA与AA（青蒿酸）的比例。在此，通过引入一系列异源酶：紫穗槐-4,11-二烯合酶（ADS）、紫穗槐二烯氧化酶（CYP71AV1）、ADH1、DBR2和ALDH1，在 中实现了DHAA的生物合成，初始DHAA/AA比例为2.53。通过将融合蛋白DBR2-ADH1和DBR2-ALDH1配对，增强了通往DHAA的通量，使DHAA/AA比例提高了1.75倍（达到6.97）。此外，为了促进ALDH1对二氢青蒿醛（DHAA合成的中间体）的底物偏好超过青蒿醛（AA合成的中间体），提出了两种合理的工程策略，包括缩小活性口袋和增强酶/辅因子复合物的稳定性，以对ALDH1进行工程改造。结果发现，突变体H194R表现出更好的酶/NAD复合物稳定性，在所有突变体中获得了最高的DHAA与AA比例，为3.73。然后将突变体H194R整合到上述重组融合蛋白中，得到了最高的DHAA与AA比例（10.05）。随后，通过5 L生物反应器发酵，实现了报道的最高DHAA产量1.70 g/L（DHAA/AA比例为9.84）。该研究突出了代谢工程和蛋白质工程在重定向代谢通量以获得化学过程中最有效产物以及简化下游转化过程方面的协同作用。
