Metabolic Engineering Of Escherichia Coli For Efficient Production Of Succinic Acid Using Waste Biomass

Production of biochemicals and biofuels from lignocellulosic biomass has the potential to enhance the overall economic view of the lignocellulosic biorefinery.Succinic acid is a platform chemical for the production of some important polyester,alkyl resins,and it is also widely used in the pharmaceutical,medicine,food and beverage industry.Production of succinic acid from lignocellulosic biomass is laced with challenges including,the cost of biomass pretreatment,genetic engineering and utilization of cost-effective medium.Here,we investigated the biosynthesis of succinic acid from Palmaria palmata,Strophanthus preussii,Cocos nucifera water and elephant grass stalk.Firstly,microbial production of succinic acid from Palmaria palmata was investigated for the first time.In engineered Escherichia coli KLPPP,lactate dehydrogenase A(ldhA),phosphotransacetylase acetate kinase A(pta-ackA),pyruvate formate lyase B(pflB)and pyruvate oxidase B(poxB)genes were deleted while phosphoenolpyruvate carboxykinase was overexpressed.The recombinant exhibited higher molar yield of succinic acid on galactose(1.20±0.02 mol/mol)than glucose(0.48±0.03 mol/mol).The concentration and molar yield of succinic acid were 22.40±0.12 g/L and 1.13±0.02 mol/mol total sugars respectively,after 72 h dual-phase fermentation from P.palmata hydrolysate,which composed of glucose(12.57± 0.17 g/L)and galactose(18.03±0.10 g/L).The results demonstrate that P.palmata red macroalgae biomass represents a novel and alternative feedstock for biochemical production.Secondly,a biorefinery process for high yield production of succinic acid from biomass sugars was investigated using recombinant E.coli.In this study,a novel biomass methanol extracts of Strophanthus preussii was used for the synthesis of succinic acid and optimization of the process parameters was carried out for pretreatment of biomass.Glucose(9.00±0.02 g/L),galactose(4.00±0.02 g/L),xylose(6.00±0.02 g/L)and the arabinose(0.50±0.02 g/L)were the major sugars present in methanol extracts of S.preussii.E.coli K30S used methanol extracts of S.preussii effectively.E.coli K30S with overexpression of soluble nucleotide pyridine transhydrogenase(sthA)and mutation of ldhA,pla-ackA,pplB and poxB,produced a final succinic acid concentration of 14.39±0.02 g/L and yield of 1.10 ± 0.01 mol/mol total sugars after 72 h dual-phase fermentation in M9 medium.This value was 64%of the maximum theoretical yield using methanol extracts of S.preussii.Thirdly,the genetically engineered strain with heterologous overexpression of pyc from Bacillus subtilis and ldhA,pflB,pta-ackA,poxB,pgi,murein cluster C(mreC)mutations and downregulation of phosphoenolpyruvate carboxylase(ppc)in E.coli were constructed and the mixed sugar fermentations was carried out with the best stains.C.nucifera water containing 5.00 ± 0.02 g/L glucose,6.10±0.01 g/L fructose and 6.70±0.02 g/L sucrose was utilized for the production of succinic acid.Fermentation of C.nucifera water with E.coli M6PM produced a final succinic acid concentration of 11.78±0.02 g/L and yield of 1.23±0.01 mol/mol total sugars after 72 h dual-phase fermentation in M9 medium while modeled C.nucifera water was 0.38± 0.02 mol/mol total sugars.The novel substrate of C.nucifera water resulted in 72%maximum theoretical yield of succinic acid.These investigations show the importance of C.nucifera water as a substrate for the production of biochemicals.Fourthly,efficient production of succinic acid from elephant grass stalk hydrolysate comprising of 11.60±0.04 g/L glucose,27.22±0.04 g/L xylose and 0.65±0.04 g/L arabinose was investigated.Fermentation of elephant grass stalk hydrolysate by engineered E.coli M6PM,involve the use of lactate dehydrogenase A,pyruvate formate lyase B,phosphotransacetylase-acetate kinase A,pyruvate oxidase B,phospho glucose isomerase,murein cluster genes mutations,down regulation of phosphoenolpyruvate carboxylase and overexpression of B.subtilis pyruvate carboxylase produced a final succinate concentration of 30.03±0.02 g/L and a yield of 1.09 mol/mol during 72 h dual-phase fermentation.The high succinate yield from elephant grass stalk demonstrated possible application of renewable biomass as feedstock for the synthesis of succinic acid using recombinant E.coli.Conclusively,these results revealed that biomass represents an alternative feedstock for the production of biochemicals.The implementation of lignocellulosic biomass in biorefinery will enhance utilization of biomass for the production of biochemical and consequently improving succinic acid production in an integrated biorefinery.