Metabolic Engineering Of Corynebacterium Glutamicum For L-isoleucine Production

Amino acids are promising biochemical products and widely used in food,medicine and feed.L-Isoleucine is an essential amino acid with highly added value in pharmaceutical field.Its main method of industrial production is microbial fermentation and the main producer is Corynebacterium glutamicum.Compared with the blindness of traditional mutation breeding,metabolic engineering is more targeted.Thus,to satisfy the increasing market need,metabolic engineering of C.glutamicum for high L-isoleucine production has attracted more and more attention.In this study,genomic modification combined with the alr-based auxotrophic complementary expression system was used.Starting from a L-isoleucine producing strain,C.glutamicum WM001,multiple metabolic engineering strategies were combined to improve L-isoleucine production.Finally,a high L-isoleucine producing strain was constructed without the need of any antibiotics.The main conclusions are listed below:?1?Exchange of the native promoter of ilvA with strong promoter tac and deletion of alaT increased the supply of precursors,2-ketobutyric acid and pyruvate,thereby increasing L-isoleucine production and reducing byproducts;deletion of brnQ and blocking of the L-isoleucine uptake system,were beneficial to the cell growth and further improved L-isoleucine production.But,the stronger promoter tacM did not further increase L-isoleucine production.After genomic modification,the best mutant strain WM004 accumulated 20.0 g?L^-1 L-isoleucine in 72-h flask fermentation,which is 21.9%increase compared to the original strain WM001,and the level of by-products decreased significantly.?2?The alr-based auxotrophic complementary expression system was constructed in the mutant strain WM004,for avoiding the addition of antibiotics to maintain expression vector.Compared with pJYW-4,the vector pYCW-1 is more suitable for the mutant strain WM005 in which gene alr is deleted.?3?After changing the 47th isoleucine of AHAS to tyrosine,the mutant AHAS encoded by ilvBN2 was more beneficial for the L-isoleucine production and avoiding the much formation of L-valine.Co-expressing gene ppnK1 enhanced the supply of NADPH and further improved the L-isoleucine production.The recombinant strain WM005/pYCW-1-ilvBN2-ppnK1 could accumulate 23.7 g?L^-1 L-isoleucine in 72-h flask fermentation,which is 15.1%increase compared to the control WM005/pYCW-1.Further overexpression of the operon lrp-brnFE or genes ilvC,ilvD and ilvE was not beneficial for cell growth and the L-isoleucine production,with much formation of by-products;further overexpression of the operon hom1-thrB led to the significantly inhibited growth and was also not beneficial for the L-isoleucine production.Without addition of antibiotics,the best recombinant strain WM005/pYCW-1-ilvBN2-ppnK1could hardly lose plasmid in 72-h fermentation.?4?The best recombinant strain WM005/pYCW-1-ilvBN2-ppnK1 accumulated 32.1 g?L^-1L-isoleucine after 72-h 5-L fed-batch fermentation without addition of any antibiotics,which is34.3%increase compared to the control WM001,and produced little by-products.