| The depletion of fossil energy and environmental pollution have been the serious problems that to be focused urgently since the last century.It is of strategic importance for development and utilization of renewable resources to relieve the fossil energy crisis.Metabolic engineering of microorganisms commonly aims tosynthesise compounds which are not produced by common strains.Those metabolic engineering strategies also aim at improduce the product yields and conversion efficiencies.Synthetic biology have widely brought into focus because of the use ofrenewable energy for the production of a series of high value-added platform compounds.In particular,lignocellulosic biomass has been considered as a potential substitutes for fossil resources such as petroleum.D-xylose mainly exists in lignocellulose.Depending on the biorefinery method,the xylose fraction can account for 70%-80%in the hemicellulosic fraction.However,only a small fraction of microorganisms can metabolize xylose.Therefore,the scope of its application is very limited.Ethylene glycol(EG)is an important platform chemical with huge commercial value.It has also been applied in many fields.According to statistics,the global demands of EG had reached 30 million tons in 2016 and its consumption will increase uninterruptedly.At present,the coal-based traditional methods for ethylene glycol synthesis gained a low production and yield.Therefore,it is worth to explore some new pathways converting D-xylose to EG.In E.coli,D-xylose can be isomerization to xylulose through the XI pathway without the coenzyme participation.The xylulose can be converted into the 5-p-xylulose and the D-xylulose-5-P then enters into pentose phosphate pathway for catabolism.The XDH and XylC were recruited from C.crescentus to convert D-xylose to D-xylonate which on the basis of E.coli natural xylose metabolic rounte.n view of the greater intracellular prevalence of NADH than NADPH,an aldehyde reductase FucO using NADH was employed to convert glycoaldehyde into EG,in replacement of NADPH-dependent reductase YqhD.Finally,we designed a biological way to synthesize EG in Escherichia coli:D-xylose→ D-xylonate →2-keto-3-deoxy-D-xylonate→ glycoaldehyde→EG.Comparatively speaking,the designed pathway was redox neutrality and energy free.All these genes were cloned and overexpressed in E.coli BL21(DE3),generating the engineered strain Q2766.The engineered strain Q2766 was tested on the level of shake flask.After 48h fermentation,1.3±0.05g/L of EG was accumulated by Q2766 which mean that the synthetic pathway was workable.In shaking flask cultivation,the undesirable by-products such as acetate and glycolate were also detected.Acetate is one of the main by-products of microbial metabolic process which mainly accumulated in the cell logarithmic growth period.To repress the production of acetate,genes encoding AckA(acetate kinase),IclR(isocitrate lysate regulator),and ArcA(global transcriptional regulator)were deleted respectively,and the arcA mutant presented the lowest acetate production.To control the accumulation of glycolate,aldA gene encoding aldehyde dehydrogenase was deleted in E.xoliAarcA genome which gained the double-deleted strain Q2843.After optimizations of fermentation,the engineered strain Q2843 can produced 72 g EG 1-1 and 1.38 g 1-1 h-1 EG productivity which is the highest reported titer and productivity for EG to date.This study paved the way for high-level microbial production of EG from renewable resources in the future. |
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