Metabolic Engineering Of Escherichia Coli For The Production Of ?-alanine

?-Alanine,the only kind of ? amino acid which exists naturally,has important applications in medicine,food and chemical industry as one of the 12 future global most development potential of the three carbon chemical products.At present,?-alanine commonly used chemical method for industrial production.But this method has low conversion rate,more by-products,and is easy to produce industrial waste,thus causes the environment pollution.By contrast,microbial fermentation is a environment-friendly method.This study investigated the possibility of metabolic engineering of Escherichia coli for the production of ?-alanine in anaerobic fermentation and aerobic fermentation,respectively.Through gene knockouts of the competing routes,overexpression of the key genes,addition of asistant agents and the optimization of fermentation conditions,production of ?-alanine was significantly improved which provide the basis for industrial production.The main research contents and results are as follows:?1?In order to obtain anaerobic ?-alanine production strains,the Escherichia coli B0016-050?ackA-pta pflB adhE frdA ldhA?strain with deletions on synthetic pathways of acetic acid,formic acid,ethanol,succinic acid and lactic acid,was used as the starting strain.Effects of gene knockouts of lysC?encoding lysine-sensitive aspArtokinase III?,panC?encoding pantothenate synthetase?,ptsG?encoding the major glucose transporter IICB?Glc??and aspA?encoding aspartic acid ammonia lyase?were investigated in B0016-050 using the Red recombination.Through anaeroblic fermentation,the yield of ?-alanine of strain B0016-092B?ackA-pta pflB adhE frdA ldhA lysC panC aspA ptsG?reached 0.92 mg·L^-1.Overexpression of the Corynebacterium glutamicum panD gene?encoding L-aspartate-?-decarboxylase?,pseudomonas aeruginosa aspDH?encoding L-aspartate dehydrogenase?,Actinobacillus pck?encoding phosphoenolpyruvate carboxylase?and Lactobacillus pyc?encoding pyruvate carboxylase?in strain B0016-092 B,the synthesis of ?-alanine was 33.0 mg·L^-1,13.0 mg·L^-1,13.4 mg·L^-1 and 5.4 mg·L^-1,respectively.Glycerol and NaHCO3 were used to supply the reducing power and CO2,anaerobic yield were increased by 2.3% and 45.0%,respectively.After the fermentation optimization,the anaerobic production of strain B0016-092B/pPL-panD reached 0.1 g·L^-1.?2?In order to obtain aerobic ?-alanine production strains,we constructed strain B0016-080BB?ackA-pta pflB adhE frdA ldhA lysC panC ptsG?with ?-alanine yield of 49.3 mg·L^-1 under aerobic conditions.Overexpression of the C.glutamicum panD gene significantly improved aerobic ?-alanine production to 4.3 g·L^-1.We further integrated the panD on chromosome of strain B0016-080 BB to produce strain B0016-080BB-PL-panD.Then the aspDH,pck and pyc genes were expressed in B0016-080BB-PL-panD,respectively.The highest ?-alanine yield reached 5.4 g·L^-1 in B0016-080BB-PL-panD/pPL-aspDH.Fermentation optimization indicated that the optimal shaking speed was 200 r·min^-1,the best cell mass for thermal induction was OD600 of 2.5 and the best fermentation period was 42 h with the highest ?-alanine yield of 5.0 g·L^-1 in recombinant strain B0016-080BB/pPL-panD.When tested in 5 L bioreactor,the recombinant strain B0016-080BB/pPL-panD could produce 18.4 g·L^-1 ?-alanine with productivity of 0.61 g·L^-1·h^-1.