Regulation Of L-Threonine Anabolism Based On Metabolic Pathway Analysis

L-threonine is involved in protein synthesis and in the metabolic activities of life,and is widely used in food,feed and medicine.Currently,industrial production of L-threonine is based on direct fermentation with microorganisms such as Escherichia coli,which has the characteristics of low cost and high production intensity.In order to elucidate the key metabolic features of the L-threonine synthesis pathway in E.coli for metabolic regulation or metabolic engineering of the strain,this study was carried out on a high L-threonine producing strain,in terms of media and fermentation conditions optimisation and metabolic pathway analysis,with the following results.（1）The effect of medium formulation and fermentation conditions on L-threonine synthesis was examined.The medium was screened for carbon and nitrogen sources,single-factor tests for other nutritional factors,and Plackett-Burman（PB）experiments to screen for two significant factors,betaine HCl and phosphate.The shake flask batch fermentation yield was 0.7 g·L^-1 at 20%loading,37°C,p H 7.0,inoculum OD₆₀₀ value of 0.5 and 1%inoculum.batch replenishment culture of phosphate on a 5L fermenter yielded 71 g·L^-1of L-threonine at an added concentration of 9.8 g·L^-1 phosphate.（2）Metabolic flow analysis and metabolic control analysis were carried out for the effect of phosphate on L-threonine production by E.coli.A metabolic network diagram of L-threonine was constructed based on the flow balance principle to estimate the metabolic fluxes at different phosphate concentrations,and metabolic flow analysis was performed for three key nodes,glucose-6-phosphate,phosphoenolpyruvate andα-ketoglutarate.Metabolic control analyses based on principal component analysis were used to obtain phosphoenolpyruvate carboxylase(CCP_PEPC=0.058),pyruvate kinase(CCP_PK=0.068),glucose-6-phosphate dehydrogenase(CCP_G6PD=0.11),fructose-1,6-bisphosphate aldolase(CCP_FBA=-0.00523),malate dehydrogenase(CCP_MDH=0.048),and hexokinase(CCP_HD=0.05)control coefficients,elucidating the effects and contributions of several enzymes to threonine metabolic fluxes.（3）At the metabolite level,differential metabolic pathway analysis and screening of key potential biomarkers based on metabolomics techniques were performed.GC-MS analysis was performed on the experimental bacterium YQ001 and the control bacterium W3110.The results showed relatively low concentrations of by-product amino acids and relatively high yields of L-threonine in the test bacteria.Principal component analysis showed significant differences in metabolism levels between the two strains.Three major potential biomarkers,inositol,glutamic acid and citric acid,were obtained by screening based on differential metabolite score marker maps.（4）Based on the results of metabolic flow analysis,metabolic control analysis and metabolomics analysis,metabolic regulation of certain key pathways,key enzymes and biomarkers was performed.The results showed that the addition of glycine,manganese sulphate and sodium nitrate all contributed to an increase in L-threonine production,especially when inositol(0.1 g·L^-1)and sodium glutamate(5 g·L^-1)were added to increase L-threonine production by 1.86-4.9 times compared to the control.During batch replenishment fermentation with the optimised medium formulation,L-threonine yields reached 77 g·L^-1,a 10%increase in L-threonine production compared to using the pre-optimised fermentation medium.