Metabolic Modification Of L-serine Competition Pathway In Corynobacterium Glutamicum

L-serine is a nonessential amino acid and takes part in the biosynthesis of many important metabolites, which is widely used in pharmaceuticals, cosmetics and food additives industries. However, L-serine is one of few amino acids for which high-production yields have not yet been achieved by fermentation directly from cheap sugar. In our previous study, a strain C. glutamicum SYPS-062, which could directly produce L-serine from sugar, was isolated. Through random mutagenesis and metabolic engineering, a mutant C. glutamicum SYPS-062 33 a ?SS(33a ?SS) was obtained, which could accumulate 21.27 g·L-1 L-serine from sucrose. However, superfluous by-products accumulation, low L-serine production and productivity limited its industrial production. In regard to these problems, this study aimed to investigate metabolic engineering and process optimization strategies towards eliminating by-products synthesis as well as increasing L-serine production and productivity. The main results were described as follows:(1) L-alanine was one of the main by-products in L-serine synthesis by C. glutamicum 33 a ΔSS. Transaminases Ala T and Avt A is the principal L-alanine supplying enzyme. Inactivation of Ala T reduced L-alanine by 28 %, whereas inactivation of Avt A could reduce by 63 %. When transaminases Ala T and Avt A were inactivated simultaneously, L-alanine accumulation was further reduced by 84 %(1.52 g·L-1), whereas L-serine titer was reached to 25.37 g·L-1, which was improved by 19 % when compared with the control 33 a ?SS(21.27 g·L-1).(2) L-valine was another main by-product in L-serine synthesis by C. glutamicum 33 a ΔSS ?ala T ?avt A. Acetohydroxyacid synthase(AHAS) is the key enzyme during L-valine synthesis in C. glutamicum. The deletion of C-terminal 249 bp of the regulatory subunit ilv N of the AHAS could decrease the activity of AHAS effectively, reduce the L-valine accumulation significantly and further improve L-serine production. The resulting strain could produce 26.23 g·L-1 L-serine, 1.23 g·L-1 L-alanine, and 2.63 g·L-1 L-valine in shake-flask fermentations. Compared with the control 33 a ?SS ?ala T ?avt A, the by-products L-valine and L-alanine were reduced by 64 % and 19 %, respectively.(3) Medium optimization and fed-batch experiments further improved L-serine production and productivity. Corn steep liquor and beef extract significantly stimulated cell growth and increased sucrose consumption rate as well as L-serine productivity. Compared to beef extract, Corn steep liquor showed more advantageous for L-serine production. With addition of 2 g·L-1 Corn steep liquor, L-serine productivity was enhanced to 0.44 g·L-1·h-1, an increase of 67 % compared to the control. Within 56 h of the fermentation, L-serine production was reached to 25.11 g·L-1 and the fermentation time was shortened by 40 h. Subsequently, batch fermentation in 5 L stirred-tank bioreactor was carried out. 21.84 g·L-1 of L-serine accumulation was achieved with a productivity of 0.39 g·L-1·h-1 and yield of 0.21 g·g-1 sucrose. Based on this result, fed-batch experiment further improved L-serine production. Within 96 h of the fermentation, L-serine titer was reached to 42.62 g·L-1, which was the highest production of L-serine from sugars to date.