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.
WANG Xiaojing
,
GU Zhenghua
,
LI Moying
,
ZHU Rui
,
XIN Yu
,
GUO Zitao
,
GUO Zhongpeng
,
ZHANG Liang
. Construction of Escherichia coli strains for whole-cell catalytic synthesis of Rebaudioside M and fermentation process[J]. Food and Fermentation Industries, 2025
, 51(21)
: 1
-9
.
DOI: 10.13995/j.cnki.11-1802/ts.041873
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