Engineering Of The Non-PTS Glucose Transport System For Enhancing L-serine Prodution By Corynebacterium Glutamicum

Bio-medicine is the mark and pillar of modern pharmaceuticals industries,and amino acid fermentation is an important part of Bio-medicine.L-serine is widely used in the pharmaceuticals industry as an vital medical intermediate.Therefore L-serine fermentation gets increasing attention for its high market value.In our previous study,a strain C.glutamicum ?SSAAI,which could produce L-serine with high yield from sucrose,was achieved through random mutagenesis and metabolic engineering.However,low yield and productivity of strain Corynebacterium glutamicum ?SSAAI on glucose were observed.Glucose is the most widely-used substrate for amino acids industrial production and it is cheaper than sucrose.In industrial production,feed stock costs affect to a large extent the overall fermentation costs.Improving L-serine yield from glucose by C.glutamicum ?SSAAI become attractive and meaningful.In this study,in order to enhance L-serine yield on glucose,engineering of non-PTS glucose utilization pathwaywas applied.In the meantime,glucose utilization pathway was reinforced further,series recombinant strains were constructed,those recombinant strains fermentation performances was evaluated.The main results were described as follows:Investigate the effect of glucose and sucrose on the growth and L-serine in C.glutamicum ?SSAAI.The lower L-serine yield was achieved on glucose(21.17 g·L-1),which was decreased by 18.7% than cultivated on sucrose(25.98 g·L-1).Meanwhile,?SSAAI showed slower substrate consumption rate during fermentation process on glucose than that of on sucrose.In order to L-serine production on glucose,the new glucose utilization pathway was constructed.The pts H encoding the general component of PTS was deleted to result a PTS-defective strain S?pts H via homologous recombination.Strain S?pts H showed severe growth retardation and achieved maximum biomass of OD562 was only 7.89.The strain S?pts H only produced 3.2 g·L-1 of L-serine.To improve the cell growth and L-serine yield,three individual strategies were employed in strain S1:(A)Reinforce glucose uptake by overexpression of iol T1.The recombinant strain achieved maximum biomass of OD562 34.18 and produced 19.60 g·L-1 L-serine.(B)Accelerate glucose phosphorylation by overexpression of ppg K.The recombinant strain achieved maximum biomass of OD562 28.20 and produced 16.61 g·L-1 L-serine.(C)Derepress iol genes transcription by deletion of regulator of Iol R.The recombinant strain achieved maximum biomass of OD562 was 38.98 and produced 23.27 g·L-1 L-serine.Subsequently,these three strategies were integrated.Co-overpression of iol T1,ppg K and deletion of iol R was performed in strain S?pts H.The recombinant strain achieved maximum biomass of O D562 44.31 and glucose consumption rate 0.83 g·L-1·h-1.Meanwhile,recombinant strain produced 26.40 g·L-1 L-serine,L-serine yield and productivity was 0.27 g·g-1·L-1 and 0.22 g·L-1·h-1,respectively.These results indicated that it would be necessary and powerful to realize practical application of non-PTS glucose transport system by integration strategy.In order to reinforce glucose utilization further,a plasmid carrying pgi,pfk A and gap A genes was transformed into non-PTS strain.The resulting recombinant strain achieved maximum L-serine production of 29.68 g·L-1.L-serine yield and productivity was 0.30 g·g-1·L-1 and 0.27 g·L-1·h-1,increasing by 40.2% and 50.0% compared with the non-PTS stain ?SSAAI on glucose,respectively.