Central Carbon Flux Regulation Of Escherichia Coli For L-threonine Production Using A Thermal Switch

The unbalanced carbon distribution in microbial central metabolism has always been an urgent problem in the field of metabolic engineering,which hinders the further improvement of the titer and yield of metabolite biosynthesis.Dynamic regulation strategies have been widely used in the construction of microbial cell factories to solve the contradiction between cell growth and product synthesis.In this study,L-threonine-producing Escherichia coli TWF001was used as the starting strain.FabV resistance-free expression plasmids and thermal switch plasmids were constructed using genetic engineering tools and metabolic engineering strategies,and the central metabolic pathway was reconstructed by a thermal-controlled expression strategy.A thermal switch system was designed to regulate the central carbon flux and close the central metabolic bypass pathway,which effectively improved the production performance of the L-threonine producing strain.The main results are as follows:1.The protein sequences,highly homologous to Vibrio cholerae FabV,were obtained in Pseudomonas putida KT2442 and Pseudomonas aeruginosa POA1 through protein homology comparison,and the mfabI on the p F2 plasmid was replaced with two genes encoding the proteins,resulting in pFpp and pFpa,and pFpa has obvious growth advantages in triclosan supplemented medium.FabV was used as a selection marker to assemble with the temperature-sensitive circuit cI^ts-p _R-p _L and the regulatory circuit tetR-P_LtetO-1,yielding thermal switch plasmids pFT22 and pFT24.The reliability and sensitivity of the thermal switch plasmid were respectively evaluated at the protein level and the transcription level by using reporter genes lacZ and gfp.The thermal switch plasmid pFT24 with the Ssr A protein degradation tag can inhibit lacZ expression at 37℃ or less,and turn off gfp expression at 40℃ or above.The higher expression level of the gene lacZ takes 60 minutes by transferring from 30℃ to 42℃,and the GFP protein can degraded completely after 80 to 100 minutes.2.Multiple genes related to pyruvate metabolism were deleted in E.coli.The thermal-controlled expression plasmid pFT26 was used to express pyruvate oxidase PoxB and acetyl-Co A synthase in combination to construct an acetate bypass pathway to replace E.coli pyruvate dehydrogenase in central metabolism.This enables TWF115△ace E to continue to synthesize L-threonine in the presence of 2-oxobutyrate,and its yield reaches 12.51 g/L.Then,a variant PoxB^F112W of the E.coli pyruvate oxidase PoxB was obtained by directed evolution,and its activity ratio of pyruvate/2-oxobutyrate in substrate selectivity was increased to 20.01.This variant was employed to replace the wild-type PoxB to reconstruct the acetate bypass pathway,which further improves the strain’s tolerance to 2-oxobutyrate,and its L-threonine titer in shake flask reachs 13.98 g/L.3.Up-shift temperature to 42℃ has no adverse effect on the L-threonine production of most E.coli by comparing 37℃ constant temperature fermentation mode and up-shift temperature fermentation mode.Based on the decarboxylation of oxaloacetate and the thermal-controlled expression of pyruvate carboxylase PYC,a thermal switch system was developed and constructed to dynamically regulate the carbon flux of the central metabolic pathway in E.coli,which automatically adjust the relative levels of pyruvate and oxaloacetate in the cell.This system was used to express rht C and pycmt in combination,resulting in 23.29 g/L L-threonine,and the yield of L-threonine was increased by 43.68%.The thermal switch plasmid was further used to shut down the expression of the genes pta and glt A,as well as the L-alanine synthesis pathway,respectively.After shutting down the L-alanine synthesis pathway,the L-threonine titer was further improved to 25.85 g/L,and the molar yield of L-threonine reaches 124.03%.4.The themal switch system was comprehensively analyzed and evaluated by adjusting time points of temperature shift of the shaking flask fermentation and adopting supplemental glucose fermentation in flask and fed-batch fermentation in benchtop bioreactor.Pyruvate was used as the substrate to biosynthesize L-threonine and the changes of the intracellular metabolites of strains in the temperature-shift fermentation were analyzed by omics.Changing time points of temperature shift dramatically affected the L-threonine production of the strains harboring the thermal switch system.The best time point of temperature shift in shake flask fermentation is 6 h for the production of L-threonine,which can achieve higher yield than others.The premature temperature shift inhibits cell growth and glucose consumption.The L-threonine production of strain TWF106/pFT24rp reached 46.6 g/L in shake flask with glucose supplement,and its molar yield was 118.39%,while the glucose consumption capacity of the strain in the middle and late logarithmic periods of temperature-shift fermentation was obviously suppressed after closing L-alanine synthesis pathway,and its yield was not as good as TWF106/pFT24rp.The temperature shift at 15 h in fed-batch fermentation made L-threonine yield of strain TWF106/pFT24rp higher.The external addition of pyruvate further shows that the temperature shift can efficiently utilize the pyruvate substrate to synthesize L-threonine.Through metabonomics analysis,the high temperature of 42℃ in up-shift temperature fermentation has a great impact on the intermediate metabolites in many metabolic pathways of L-threonine-producing E.coli,including glucose uptake,pentose phosphate pathway,glycolysis pathway,TCA cycle and amino acid synthesis pathway.