莱鲍迪苷M(rebaudioside M, Reb M)是一种天然的稀有甜味剂,以其安全性、高甜度和较少的回苦味而受到关注。从甜叶菊中提取莱鲍迪苷M的传统方法成本高且产量低。该研究采用大肠杆菌进行全细胞催化合成Reb M,避免了复杂的酶纯化步骤,并适合工业化规模生产。该研究通过构建大肠杆菌底盘菌株并优化其发酵过程,实现了Reb M的高效合成。通过使用优化后的酶UGT76G1(突变T284S M88L L200A)和UGT91C1(突变F208M F379A),并引入蔗糖合酶atuSUS1以增强二磷酸尿苷-葡萄糖循环。同时,通过敲除araA(L-阿拉伯糖异构酶)和ushA(UDP糖水解酶)、过表达galU(葡萄糖-1-磷酸尿苷转移酶)并添加促溶标签XXA改造代谢途径,构建了出发菌株。通过发酵优化,确定最佳发酵条件为:在37 ℃下培养4 h后,加入0.1 mmol/L IPTG和15 g/L L-阿拉伯糖,在16 ℃下诱导16 h。在此条件下,出发菌株在42 ℃下反应24 h后,Reb M产量达到24.318 g/L,底物转化率达到91%,较初始条件提高了7.56倍。该研究成果为利用大肠杆菌生产莱鲍迪苷M提供了重要的指导。
Rebaudioside M (Reb M), a natural and scarce sweetener, has garnered significant attention owing to its safety profile, high level of sweetness, and relatively subdued aftertaste bitterness.The conventional approach of extracting Reb M from Stevia is marred by high costs and low yields.In the present study, a whole-cell catalytic strategy was employed using Escherichia coli for the synthesis of Reb M.This approach obviated the complex and laborious steps associated with enzyme purification and holds great promise for industrial-scale production.Through the meticulous construction of an E.coli chassis strain and the optimization of its fermentation regimen, an efficient synthesis of Reb M was realized.Specifically, the optimized enzymes UGT76G1 (bearing mutations T284S, M88L, and L200A) and UGT91C1 (with mutations F208M and F379A) were utilized. Simultaneously, to construct the initial strain, the metabolic pathway underwent modification.This involved the knockout of araA and ushA(encoding L-arabinose isomerase and UDP glycoside hydrolase), overexpress galU(encoding Glucose-1-phosphate uridylyltransferase) and the addition of the solubilizing tag XXA.Through an elaborate fermentation optimization process, the optimal fermentation conditions were ascertained as follows:following a 4-hour incubation at 37 ℃, 0.1 mmol/L IPTG and 15 g/L L-arabinose were added, followed by an induction period of 16 hours at 16 ℃.Under these precisely defined conditions, after a 24-hour reaction at 42 ℃, the Reb M yield of the chassis strain reached 24.318 g/L, and the substrate conversion rate attained 91%, representing an 7.56-fold enhancement over the initial conditions.The outcomes of this research offer crucial and valuable guidance for the large-scale production of Reb M using E.coli.
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