US 12,065,682 B2
Method for producing L-theanine via fermentation by a genetically engineered bacterium and the application thereof
Xiaoguang Fan, Tianjin (CN); Xiaodong Liu, Henan (CN); Jing Li, Henan (CN); Ning Chen, Tianjin (CN); Bochao Liu, Henan (CN); Shuai Liu, Henan (CN); Chaochao Sun, Henan (CN); Yongchao Liu, Henan (CN); Jiajia Teng, Henan (CN); Mengtao Zhang, Henan (CN); Yuanqing Ji, Tianjin (CN); Yuhang Zhou, Tianjin (CN); and Qingyang Xu, Tianjin (CN)
Assigned to Henan Julong Biological Engineering Co., Ltd, Ruzhou (CN); and Tianjin University of Science and Technology, Tianjin (CN)
Filed by Henan Julong Biological Engineering Co., Ltd, Henan (CN); and Tianjin University of Science and Technology, Tianjin (CN)
Filed on Aug. 17, 2022, as Appl. No. 17/889,397.
Application 17/889,397 is a division of application No. 17/566,702, filed on Dec. 31, 2021, granted, now 11,453,898.
Claims priority of application No. 202111159099.6 (CN), filed on Sep. 30, 2021.
Prior Publication US 2023/0109256 A1, Apr. 6, 2023
Int. Cl. C12P 13/14 (2006.01); C12N 1/00 (2006.01); C12N 15/70 (2006.01)
CPC C12P 13/14 (2013.01) [C12N 15/70 (2013.01); C12N 1/00 (2013.01); C12N 2510/00 (2013.01)] 5 Claims
 
1. A method for producing L-theanine via a fermentation by a genetically engineered bacterium, wherein the method is as follows: inoculating a seed solution into a fermentation medium by 10-15% inoculum size, wherein a pH value is controlled within 6.7-7.2 during the fermentation, a temperature is maintained within 28-36° C., and a dissolved oxygen is within 10-30%; adding a glucose solution by a fed-batch way to maintain a glucose concentration in the fermentation medium less than 1 g/L after glucose in the fermentation medium is completely consumed;
wherein, an optical density-linked (OD-linked) ethylamine supplementary strategy is taken in the fermentation; when OD60onm is above 8-12, addition of ethylamine is started, and an ethylamine fed-batch rate (g-L−1-h−1) is adjusted for once every 0.8-1.2h; the ethylamine fed-batch rate (g-L−1.h−1)=0.5×OD600m value/(fermentation volume (L) x fermentation time (h)); and
the genetically engineered bacterium is obtained by serving a strain using a strain as an original strain, wherein the original strain is obtained after integrating a single copy of a RNA polymerase gene T7RNAP, a dual copy of a γ-glutamylmethylamide synthetase gene gmas, and a knockout of a xylose operon transcription factor gene xyIR and a knockout of a succinyl-CoA synthetase gene sucCD on a genome of Escherichia coli W3110, and then by integrating an exogenous fructose 6-phosphate phosphoketolase gene xfp, an exogenous phosphoacetyl transferase gene pta, an exogenous acetyl-CoA synthetase gene acs, an exogenous citrate synthase gene gltA, and an exogenous phosphoenolpyruvate carboxylase gene ppc on the genome, and knocking out an acetokinase gene ackA.