| D-Mannitol,a natural six-carbon sugar alcohol and an isomer with sorbitol,has been proved to an important precursor of antitumor drugs and immune stimulants.It has been widely used in food,medical and chemical industries due to its special physiological function.The whole cell catalysis is a kind of catalysis technology between enzyme and fermentation.It has the advantages of mild conditions,few by-products and high yield.Mannitol dehydrogenase（MDH）,as a key enzyme,can directly catalyze the conversion of the substrate fructose into D-Mannitol with the participation of NADH required.In this study,a whole-cell catalytic system for the efficient synthesis of D-Mannitol based on multi-enzyme cascade reaction was constructed,which solved the problems of low efficiency of cofactor NADH regeneration enzyme and unbalanced conversion rate of multi-enzyme cascade catalytic system.This study also provides provides a green and efficient biotransformation method for future large-scale production of D-Mannitol while great importance is attached to the production of other sugar alcohols.（1）Screening of mannitol dehydrogenases from different sources.The gene fragments of Lpmdh from L.pseudomesenteroides,Lmmdh from L.mesenteroides,and Pbmdh from P.bacterium 1109 were cloned and expressed respectively.The enzymatic properties of mannitol dehydrogenases from three sources were studied.The optimal reaction temperature of Lp MDH and Lm MDH was 30℃and the optimal p H was 6.5.The optimal reaction temperature of Pb MDH was 60℃and the optimal p H was 8.0.Lm MDH acid resistance is stronger than Pb MDH,so it is suitable for the biotransformation of weak acid conditions.Comparing the D-fructose conversion ability of the three sources of MDH under the condition of sufficient coenzyme,Lp MDH has the highest efficiency in catalyzing fructose to D-Mannitol,and the molar conversion rate of mannitol reaches 82.3%.The results of the transformation ability study identified Lp MDH as the best key enzyme for subsequent transformation.（2）Construction and screening of coenzyme circulation system.The formate dehydrogenase gene Cbfdh from Candida boidinii and the glucose dehydrogenase gene Bagdh from Bacillus amyleliquefaciens DSM7 were cloned respectively.Comparing the effects of adding formate dehydrogenase and glucose dehydrogenase on the coenzyme recycling system,the reaction rate of adding Ba GDH is faster and the conversion rate of D-Mannitol is 2.1 times of that adding Cb FDH.Therefore,Ba GDH was used for the synthesis of D-Mannitol as an enzyme for coenzyme regeneration.On the basis of screening the coenzyme system,Lp MDH and Ba GDH crude enzyme solutions were added to the in vitro reaction system according to different ratios,and the conversion ability of D-Mannitol was preliminarily tested.The rate was the highest at 82.1%when the ratio of Ba GDH to Lp MDH was 4:1.（3）Construction of a recombinant single-cell factory with tunable double-enzyme synergistic expression.Lpmdh and Bagdh were expressed in tandem in E.coli,and a single-cell factory was constructed to initially realize the whole-cell conversion of D-fructose to D-Mannitol.The molar conversion rate of D-Mannitol was 59.7%.In order to solve the problem of low efficiency of the whole-cell catalytic system,the coenzyme regeneration ability is improved by increasing the copy amount of Bagdh and balancing the double-enzyme catalytic rate on the basis of the optimal adaptation ratio of the enzyme amount in vitro to achieve the efficient conversion of D-Mannitol.The molar conversion of alcohol increased to70.5%.（4）Optimization of the conditions of the whole-cell catalytic system.The whole-cell catalytic system was optimized from four aspects:temperature,p H,cell mass and co-substrate concentration.The optimum conditions were determined as reaction temperature 30℃,initial p H value 6.5,cell mass OD600 30.The molar ratio of cosubstrate glucose to substrate is 1:1.（5）Whole-cells were transformed into D-Mannitol at the level of 5 L fermenter.The engineered strain E.coli BL21/p ETDuet-Lpmdh-Bagdh-Bagdh was transformed in a 5 L fermenter for 24 h under optimal reaction conditions,the highest yield of D-Mannitol was81.9 g·L-1,and the molar conversion rate was 81.9%,reaching the domestic leading level. |
