One-Pot Biosynthesis Of D-psicose From Glycerol

D-psicose is a rare monosaccharide that naturally exists in various foods with the property of low calorie but high sweetness.It can be used as an ideal substitute for sucrose,and has a wide range of applications in food,medicine,and health care areas.Currently the most studied method for D-psicose synthesis is to isomerize D-fructose to D-psicose by D-psicose-3-epimerase(DPEase).However,this method suffers from low conversion rate caused by reversible reaction.To obtain other efficient methods for D-psicose synthesis,this study explored the pathway depending on the dihydroxyacetone phosphate(DHAP)-dependent aldolase.The main results of this study are summarized as follow:(1)The D-psicose synthetic pathway was divided into three modules.Module I:synthesis of D-psicose from DHAP and D-glyceraldehyde;Module II: synthesis of DHAP from glycerol;Module III: synthesis of D-glyceraldehyde from glycerol.In module I,we analyzed L-rhamnoose-1-phosphate aldolase(Rha D)and L-fucose-1-phosphate aldolase(Fuc A)for their efficiency of D-psicose synthesis.The results showed that Fuc A has a higher efficiency than Rha D.Moreover,Fuc A has a better product specificity,whose product is D-psicose but not D-sorbose;while Rha D produced both D-psicose and D-sorbose.In module II,we analyzed two ways from glycerol to DHAP.Pathway I is composed of glycerol kinase(Glp K)and glycerol 3-phosphate dehydrogenase(Glp D),while pathway II is consists of glycerol dehydrogenase(gld A)and three dihydroxyacetone kinases(DAK1,DAK2,and dha Mdha K-dha L).The results showed that pathway I is more efficient than pathway II.In module III,we analyzed three alcohol dehydrogenases(Adh A,Yah K and Adh P)and an alditol oxidase(Ald O)for their activities catalyzing D-glyceraldehyde synthesis.The results showed that only Ald O showed D-glyceraldehyde synthesis activity.(2)An in vivo cascade cell factory(named as KDABO)containing Glp K,Glp D,Fuc A,fructose-1-phosphatase(Yqa B)and Ald O was constructed.As a whole-cell catalyst,the engineered E.coli KDABO can directly convert glycerol to D-psicose,and without D-sorbose production.The rate-limiting steps were analyzed by stepwise increasing gene copy number.The results showed that Ald O is the key rate-limiting step,and increasing ald O copy number from one to two improved D-psicose titer by115.29%.(3)Response surface methodology was used to optimize the conversion process of D-psicose.The optimal IPTG concentration,induction temperature,induction time,reaction temperature,p H,and substrate concentration were screened out by singlefactor experiments.Significance of these factors were analyzed via Plackett-Burman design.The D-psicose synthesis process conditions were further optimized using the Box-Behnken design.The model predicted that the titer of D-psicose was 3.14 g/L.The verification test result was 3.04 g/L,which is close to the predicted value.These results validated the fitness of the model.Finally,the reaction was scaled up in a 5-L fermentor,and 5.67 g/L D-psicose was achieved with a conversion rate of 11.34%.In summary,a whole-cell catalyst for D-psicose production from glycerol was developed.This catalyst showed very high product specificity(D-psicose is the solo product).The rate-limiting steps were analyzed and optimized.The process conditions were also optimized by response surface methodology.This study provides an alternative route for the efficient biosynthesis of D-psicose.