| L-theanineis a natural and unique non-protein amino acid in tea.It gives tea delicious taste and unique flavor,and its proportion in tea will affect the quality and price of tea to a certain extent.Many studies have shown that theanine has many physiological effects,such as relaxing,relieving stress and so on.At the same time,it also has a significant effect on the prevention of cardiovascular and cerebrovascular diseases and other health care.In addition,it is often used as a food additive in the food industry because of its special flavor and health effects.In recent years,with the increasing market demand for theanine,it is important to increase the titer and yield of theanine and in order to reduce its production cost.Some bacterial y-glutamylmethylamine synthetases can synthesize theanine by biocatalysis using L-glutamate and ethylamine in the presence of ATP,but the reaction cost is expensive.Microbial fermentation is a simple,efficient,green and low-cost synthesis technology,which has been applied to the industrial production of numbers of amino acids.In order to improve the production efficiency of theanine and reduce production cost,we developed a method of producing theanine by microbial fermentation using recombinant Escherichia coli in this study.Through systematic metabolic engineering,an engineered Escherichia coli strain was constructed,which produced theanine by fermentation with glucose and ethylamine.First,the gene gmas encoding y-glutamylformamide synthetase from Paracoccus aminovorans was integrated into the genome of E.coli.A xylose-induced T7 RNA polymerase-PT7 promoter system was used to control the gmas gene expression,whichresulted in the preliminary accumulation of theanine.Next,the copy numbers of citrate synthase gene gltA in the Citric Acid Cycle was increased,which enhanced the metabolic flux flow from oxaloacetic acid to α-ketoglutaric acid.Glutamate dehydrogenase gene cgl2079 from Corynebacterium glutamicum(ATCC13032)was introduced to enhance the the metabolic flux flow from α-ketoglutarate to L-glutamate.Then,the sucCD gene encoding succinyl CoA synthetase was deleted to block the metabolism of α-ketoglutarate to succinic acid.The pyruvate carboxylase gene cgl0689 from C.glutamicumwas introduced to construct the metabolic pathway from pyruvate to oxaloacetic acid,which replenished oxaloacetic acid and significantly increased theanine production.Finally,the phosphoenolpyruvate kinase gene pckA from Mannheimia succiniciproducens was introduced to balance the supply of ATP in the process of theanine synthesis.Using the above engineering strategies,strain TH7 was obtained and produced38.41 g/L theanine in the shaking flask fermentation.Through single factor experiment,the optimal addition condition of ethylamine was determined in 5 L fermentor.200 g/L ethylamine was added at a flow rate of 30 mL/h after 6 h fermentation,and lasted for 20 hours until fermentation was finished.Under the optimized fermentation conditions,the engineering strain TH7 produced 70.6 g/L in 26 h,and the yield reached 41.5%.Compared with the reported production process of L-theanine,this engineering strain has significant advantages in yield and conversion rate,which lays a foundation for the industrial production of theanine. |
