Production Of Isobutanol And L-phenylalanine By Recombinant Escherichia Coli

In recent years,with the depletion of fossil fuel resources and the increasingly serious environmental pollution problems,more and more researches have been devoted to the production of biofuels from renewable substrates to replace traditional fossil fuels.With the advantage of clean,flammable,renewable and can be mixed with gasoline,alcohols become an ideal biofuel.Among them,isobutanol has higher octane number and energy density,lower hygroscopicity and is more soluble in water.Accordingly,isobutanol can be used directly without being mixed with gasoline,and can be transported by relying on existing gasoline pipelines.In summary,isobutanol can become a new generation of biofuel with great development potential to replace gasoline.Lignocellulose is the most abundant resource in nature,mainly derived from plant cell walls,which is renewable and sustainable.Natural lignocellulose has a complex structure and is difficult to decompose,making it difficult to directly utilized as a substrate for microorganisms.Therefore,monosaccharides such as glucose,xylose,and arabinose obtained by physical,chemical or biological degradation of lignocellulose can be used as important sources of microbial production.Due to the slow utilization rate of xylose in Escherichia coli,little attention has been paid on isobutanol production from xylose.In wild-type E.coli,assimilation of xylose is mainly through the isomerase pathway,and then it enters the pentose phosphate pathway to be further metabolized.However,the utilization of xylose in this pathway is relatively slow,and long pathways are needed for generation of pyruvate and acetyl-Co A from xylose.As a result,growth of E.coli is often interfered when xylose is selected as carbon source.To solve this problem,we introduced the Dahms pathway into E.coli,expecting to obtain pyruvate through a shorter enzymatic reaction step and thus improving cell growth of recombinant E.coli.Firstly,xylose isomerase Xyl A and xylulokinase Xyl B were inactivated and xylose dehydrogenase Xdh,lignoate dehydratase Yag F,2-dehydro-3-deoxy-D-valeric acid Aldolase Yag E,glutamate pyruvate transaminase Ald A,isocitrate lyase Ace A,and isocitrate dehydrogenase kinase/phosphatase Ace K in the Dahms pathway were overexpressed in E.coli.After that,we introduced the exogenous Ehrlich pathway into E.coli to realize the synthesis of isobutanol from pyruvate.So far,we have successfully established a complete pathway for isobutanol production from xylose.After batch fermentation,the recombinant strain BWL2 could accumulate 64.8 mg/L of isobutanol.Considering that more reducing forces are required in the synthesis pathway of isobutanol,reducing the activity of electron transport chain in recombinant E.coli may effectively increase the levels of NADPH and NADH Accordingly,we knocked out the cbd gene encoding ubiquinol terminal oxidase III,replaced the cyo gene encoding ubiquinol terminal oxidase I with xdh encoding xylose dehydrogenase,and replaced nuo encoding NADH enzyme I with xyl FGH encoding xylose transport ABC protein.The final strain BWL4 could synthesize 224.5 mg/L isobutanol from 20g/L xylose.In order to alleviate the conflicts of cell growth and isobutanol profduction.in recombinant strain,we next tried to synthesize isobutanol by using mixture of glucose and xylose,and expecting E.coli could maintain normal growth by xylose and synthesize isobutanol from glucose.Considering carbon catabolite repression（CCR）would hinder the simultaneous utilization of glucose and xylose in E.coli,we first knocked out the pts G gene encoding the IIBC component of the PTS system.After that,the ED pathway of E.coli was modified to directly produce pyruvate from glucose,comprising replacing pgi encoding glucose phosphate isomerase with zwf encoding 6-phosphate glucose dehydrogenase,replacing gnd encoding 6-phosphogluconate dehydrogenase with pgl encoding glucose 6-dehydrogenase,replacing pta encoding phosphoacetyltransferase with edd encoding 6-phosphogluconate dehydratase,replacing ack A encoding acetate kinase with eda encoding 2-keto-3-deoxygluconate 6-phosphate aldolase).Finally,batch fermentation was carried out for recombinant strain WED6and isobutanol production could reach 352.19 mg/L.As one of the three aromatic amino acids,L-phenylalanine is widely used in food additives and animal feed.In particular,it can be used in the biosynthesis of low-calorie sweetener aspartame.Due to the advantages of short production period,cheap raw materials,and environmentally friendly process,microbial production of L-phenylalanine has become the main production method.In E.coli,the key factors affecting L-phenylalanine production are intracellular levels of two precursors,phosphoenolpyruvate（PEP）and erythrose 4-phosphate（E4P）.In E.coli,the glucose transport system PTS consumes about 50%of the PEP in the cells.Therefore,we first knocked out the pts I gene encoding the PTS enzyme IIA.However,after knocking out this gene,the glucose utilization rate in E.coli was obviously interfered.Accordingly,glf encoding glucose-promoting diffusion protein from Pseudomonas mobilis was introduced into E.coli.Afterwards,we reduced acetate secretion by knocking out pox B encoding pyruvate dehydrogenase,and pta encoding phosphoacetyltransferase.The intracellular levels of both precursors were then increased by overexpressing pps A encoding phosphoenolpyruvate synthase and tkt A encoding transketolase.The carbon flux into L-phenylalanine was further increased by overexpressing feedback inhibition resistant 3-deoxy-D-arabino-heptulonic acid-7-phosphate synthase aro G^fbr and the chorismate mutase phe A^fbr.Finally,L-phenylalanine transporter Ydd G was overexpressed to increase the secretion of L-phenylalanine.The final recombinant E.coli obtained could synthesize 1.69 g/L of L-phenylalanine.