Metabolic Engineering Of Corynebacterium Glutamicum For Acetoin Production

Acetoin is an important platform compound, widely used in food and otherindustries. Corynebacterium glutamicum is fast-growing Gram-positive soil bacteriumand achieves high cell densities under aerobic condition, which could obtain desiredproductivity. Focusing on the acetoin biosynthetic reactions, C. glutamicum ATCC13032was engineered as the starting strain, aiming at enhancing the acetoin yield andproductivity with C. glutamicum ATCC13032by metabolic engineering.The alsSD operon for acetoin production from Bacillus subtilis168wasexpressed in C. glutamicum ATCC13032. Additional inactivation of aceE geneencoding the E1p enzyme of the pyruvate dehydrogenase complex, ldhA geneencoding NAD+-dependent L-lactate dehydrogenase and butA gene encoding2,3-butanediol dehydrogenase increased acetion production. Subsequently, the effectof rotation speed on acetoin production was explored. At last, the fermentation ofstrain CGL3was carried out in rich medium to increase acetion productivity.The alsSD operon for acetoin production from Bacillus subtilis168was insertedin C. glutamicum-Escherichia coli shuttle plasmid pEC-XK99E, the resulting plasmidpEC-XK99E-alsSD was transformed into C. glutamicum ATCC13032and got strainCGL1. The acetolactate synthase activity was performed, and the acetolactatesynthase activity of CGL1increased by17fold compared with wild-type strain. StrainCGL1produced2.14g/L acetoin.Additional inactivation of aceE and ldhA gene increased the supply of acetoinprecursor and decreased the production of lactate. The aceE-ldhA double-knockoutstrain CGL2generated5.09g/L acetoin. Lactate was not detected in strain CGL2.However CGL2produced little2,3-butanediol in culture. ButA gene encoding2,3-butanediol dehydrogenase was deleted to decrease the depletion of acetoin, andthe production of acetoin increased to5.88g/L.Succinic was detected in strain CGL4. In order to decrease the production ofby-product, the effect of rotation speed on acetoin production was explored. The finalstrain CGL4accumulated up to8.33g/L acetoin under optimum fermentationconditions, and the acetoin yield reached51.1%of the theoretical yield. To elevate the acetoin productivity of the engineered strain CGL4, fermentationwas carried out in rich medium puls65g glucose/L at150rpm. Strain CGL4generated22.44g/L acetoin. Acetoin reached70.5%of the theoretical yield andacetoin productivity was improved to0.44g/(h L). The productivity of acetionimproved by1.16fold compared with minimal medium. These results demonstratethat acetoin production by C. glutamicum ATCC13032could be successfullyachieved by metabolic engineering, providing some valuable information for otherstudies.