Synthesis Of 1,2,4-Butantriol From Cellulose Hydrolysate By Recombinant Escherichia Coli

1,2,4-butanetriol（BT）is an unnatural 4-carbon polyol with high added value,which is used in medicine,military industry,polymer and other fields.It is a type of chemical with great potential.With the increasing attention to environmental problems,more and more research focuses on the use of carbon neutral cellulose resources.The previous group constructed a recombinant Escherichia coli（E.coli）that can produce BT efficiently by co-using glucose and xylose.On this basis,using cheap and renewable corn cob as raw material,this study explored the process of producing BT from cellulose hydrolysate by simplifying the hydrolysis production process and improving the strain tolerance through genetic engineering.To explore a simple and low-cost cellulose pretreatment process suitable for BT fermentation,we treated the hydrolysate with different detoxification methods.The results showed that the hydrolysate prepared by Ca（OH）₂,activated carbon and ionic resin in sequence could be used for E.coli KXW3009 fermentation,compared with the strain in the acid hydrolysate which could not grow normally.However,the hydrolysate production process was complicated and the cost and sugar loss rate were high.The study also showed that the hydrolysate prepared by simple Ca（OH）₂ could make the strain with BT pathway grow to a certain degree and about 0.4 g·L^-1 BT was synthesized.Therefore,in the next study,the strain was modified to adapt the hydrolysate treated by Ca（OH）₂as the substrate to produce BT.In order to improve the tolerance of the strain to hydrolysate,11 kinds of resistance genes were overexpressed in the strain with BT pathway.Using furfural as the representative substance,the tolerant recombinant strains adapted to hydrolysate fermentation were preliminarily screened.The results showed that the growth of all recombinant strain was improved to varying degrees with the furfural.Except for the thy A,mdt JI and nad E genes,the overexpression of other genes increased BT yield,and the strains overexpressing aldehyde reductase Ucp A,chaperone protein Gro ESL and NAD（P）transhydrogenase Pnt AB showed better fermentation performance.The above results indicated that the modified strain had preliminary ability to produce BT in toxic hydrolysate.In order to improve the ability of the strain to synthesize BT from simple detoxification hydrolysate,the fermentation conditions were optimized in shaker flasks,and the control strain E.coli KXW3009R1 was fermented with hydrolysate to obtain 3.0 g·L^-1 BT.The BT strain modified with furfural tolerance was subjected to tolerance verification and fermentation analysis in the hydrolysate,the highest BT yield of 6.2 g·L^-1 was obtained by the resistant recombinant E.coli KXW3009R3 harbouring the ucp A gene,which is a 14.5-fold increase compared to the yield of the original strain under initial fermentation conditions.E.coli KXW3009R3 was fermented in a 5 L fermenter to produce 15.3 g·L^-1 BT,which the conversion rate is 55%.No public reports of higher yields of BT in hydrolysate was found(the maximum yield is currently reported to be less than 5 g·L^-1).A production method of BT synthesis using cellulose hydrolysate was obtained.