Metabolic Engineering Of Escherichia Coli For Efficient Synthesis Of L-theanine

L-theanine is one of the amide compounds,which is the most prevalent free amino acid in tea.In addition to tea polyphenols,L-theanine also provide the tea’s characteristic taste,which have become the markers used to measure tea quality.L-theanine showed many positive results in clinical trials,including lowering blood pressure,improving T cell proliferation,inhibiting lung cancer cell invasion and migration,enhancing the anticancer effect of anti-tumor drugs,and lowering the side effects of anti-tumor drugs used to inhibit lung cancer cell invasion and migration.As a result,L-theanine is extensively utilized as a dietary supplement in food,health care items,and medication all over the world.In 2014,China formally approved the use of L-theanine as a new food raw ingredient.In 2020,China produced 682.9 tons of L-theanine,accounting for 77.89%of global output.With the rapid growth of market demand,it is the primary problem to find innovative production methods to improve the production capacity and production economy of L-theanine.Currently,L-theanine can be biosynthesized usingγ-glutamylmethylamide synthetase coupled with an ATP regeneration system,achieved a maximum yield of 104 g/L.However,there are several issues with pure enzyme catalysis,including a complicated catalytic system,high substrate costs,additional stages in enzyme synthesis and reaction,and higher equipment occupancy.As a result,it is important to improve the production route through microbial fermentation,simplify the operating stages,and cut production costs even more.This study developed a method for effective L-theanine synthesis by recombinant E.coli fermentation,based on synthetic biology and metabolic engineering strategies.An engineering strain capable of effectively utilizing glucose and ethylamine to produce L-theanine was created by systematic bioprocess modification.To promote L-theanine synthesis efficiency,the copy number of theγ-glutamylmethylamide synthetase gene gmas from Paracoccusa minovorans was doubled.The fructose-6-phosphate phosphoketolase gene xfp（from Bifidobacterium adolescentis）was introduced to create a route from fructose 6-phosphate to acetyl phosphate and xylulose 5-phosphate to acetyl phosphate,hence skipping the carbon loss incurred by the pyruvate decarboxylation step in the glycolysis pathway.The expression levels of citrate synthase gene glt A and phosphoenolpyruvate carboxylase gene ppc were raised to help Acety L-Co A turn to citric acid,and supply adequate oxaloacetic acid for the catalytic first reaction of TCA.Following that,the ack A（acetate kinase gene）was knocked out.The acs（acety L-Co A synthetase gene）and the pta（phosphate acetyltransferase gene）was strengthened,coupled with rise in the carbohydrate flow of acetyl phosphate to acety L-Co A and a decrease at the growth of by-product acetic acid.Finally,the appropriate ethylamine feeding strategy in a 5 L fermentor was established by optimizing the fermentation medium recipe and adjusting the ethylamine feeding rate.After the bacteria’s OD₆₀₀ value reached 15,260 g/L ethylamine solution was injected,the rea L-time flow rate of ethylamine was regulated response to changes of OD₆₀₀.The engineered strain THEA8 produced 94 g/L L-theanine after 24 h fermentation under the conditions described above.The glucose conversion rate was 56.9%,and the acetic acid was less than 1 g/L.Ethylamine is a substrate to L-theanine biosynthesis,although it is highly flammable and toxic.A biosynthesis pathway from acety L-Co A to ethylamine was created related to engineering bacteria THEA3-2,to avoid the use of ethylamine in the industrial process.At the first,the transaminase gene spu C from Pseudomonas putida was cloned into the genome of E.coli to construct the tour from acetaldehyde to ethylamine.The expression of the gene eut E,which encodes acetaldehyde dehydrogenase,was then enhanced to provide much more substrate for the transaminase.After that,the gene al D from Lysinibacillus sphaericus,which encodes alanine dehydrogenase,was equipped to strengthen the metabolic stream from pyruvate to alanine.It collaborates with transaminase to compensate for the alanine shortage induced by a single reaction.In shake flask fermentation without ethylamine,the engineering strain THEB3 made 5.91 g/L of L-theanine by using above strategy.