Metabolic Modification Of Glycometabolism For Sucrose To Increase L-Serine Production By Corynobacterium Glutamicum

L-Serine（L-Serine, L-Ser） is a non-essential amino acid and widely used in food, cosmetics, medicine and other industries, which takes part in metabolites and synthesis. Recently, the direct fermentative production of L-serine from sugars attracts increasing attention. In our previous study, Corynebacterium glutamicum SYPS-062 33 a ?SSA（33a ?SSA）, which could directly produce L-serine from sugar, was mutagenesis and metabolic engineering could accumulate 19.75 g·L^-1 L-serine from sucrose. The main purpose of this work is to study the effect of carbon source on C. glutamicum 33 a ?SSA. Furthermore, this study was aimed to engineer the metabolism of sucrose to reduce the fructose accumulation and improve the production of L-serine. The main results were described as follow:（1） The fermentation parameters of L-serine-producing C. glutamicum 33 a ?SSA were analyzed in comparative batch cultures grown on sucrose, glucose, fructose. Our previous study indicated that the L-serine production added up to 19.75 g·L^-1 on sucrose, which proved that sucrose was the most suitable carbon for L-serine production. In the early stage of the fermentation, the extracellular fructose continues to increase, which accumulated to 21.96 g·L^-1.（2） The scrK gene from Clostridium acetobutylicum ATCC 824（encoding fructokinase） was overexpressed in C. glutamicum 33 a ΔSSA to promote the utilization of fructose. Furthermore, the fructokinase activity was 1.21 U·mg^-1 and the fructose was 12.93 g·L^-1 which reduced by 41.12%. The concentration of fructose-6-phosphate and glucose-6-phosphate which were the metabolic intermediates of glycolysis were improved by 6.20-fold and 29.44%, respectively.（3） The pfkA gene was overexpressed in C. glutamicum 33 a ΔSSA/pDXW^-10-scrK to structure the strain ΔSSA/pDXW^-10-pfkA-scrK for promoting the utilization of fructose-6-phosphate and glucose-6-phosphate. The activity of phosphofructokinase was increased by 13.42-fold and the activity of fructokinase was up to 1.14 U·mg^-1. L-serine accumulation was increased to 25.31 g·L^-1 in shake-flask fermentations, L-serine productivity was increased to 0.21 g·L^-1·h^-1 and yield was 0.25 g·g^-1.（4） Medium optimization was used for the recombinant strain 33 a ?SSA/pDXW^-10-pfkA-scrK to improve L-serine production and growth. With molasses as carbon source, the biomass of strain increased effectively. But molaaes was not benefit for the accumulation of L-serine. With addition of 0.24% of total sugar molasses, biomass（OD562） was 76.37. Subsequently, L-serine production reached to 32.76 g·L^-1 with a productivity of 0.27 g·L^-1·h^-1 and yield of 0.33 g·g^-1 sucrose. In 5 L batch, L-serine was reached to 30.58 g·L^-1 and biomass（OD562） was 67.63. 5 L fed-batch experiment strategy was carried out in this study which makes L-serine titer reached to 36.96 g·L^-1 with a productivity of 0·39 g·L^-1·h^-1 and yield of 0.26 g·g^-1 sucrose.