Molecular Modification Of Cellobiose 2-epimerase And Construction Of Escherichia Coli Cell Factory For Promoting Efficient Synthesis Of Lactulose

Lactulose is a non-digestible disaccharide with strong prebiotic properties,which is widely used in the fields of health food and clinical medicine.Synthesis of lactulose based on biocatalysts is considered to be a greener,safer,and more efficient pathway.Currently,Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase（Cs CE）is regarded as the most promising enzyme for producing lactulose.However,there are several constraints such as low isomerization activity,poor substrate affinity,and the accumulation of byproduct-epilactose.Molecular modification is an effective strategy for improving the catalytic performance of enzymes.This study is dedicated to exploring the key factors that affect the isomerization activity.The flexible loop and substrate binding site of Cs CE were modified to enhance the isomerization activity and substrate affinity through semi-rational design strategy,thereby improving the conversion rate of lactulose.Based on the research of enzymology,a multi-enzyme cascade catalytic cell factory for Escherichia coli was constructed and used to explore its application in the study of whey powder value-added,promoting the industrial application process of biological preparation of lactulose.Main contents and results are as following:Firstly,in response to the issues of poor isomerization activity and unclear key factors of Cs CE,the sequence and crystal structure of enzymes in AGE superfamily with different catalytic directions were compared.The key influence regions affecting the catalytic direction of isomerization and epimerization were discovered,and verified through molecular modification.According to the crystal structure comparison of AGEs,it was found that they have the same（α/α）₆-barrel scaffold.However,there were significant differences in the length and shape of the flexible loop located at A and B,and the conjecture that“the position（A or B）and shape of the flexible loop affect its catalytic direction”was further proposed based on the the substrate specificity and catalytic mechanism of AGEs.Cs CE and Marinomonas mediterranead mannose isomerase（Mm MI）were selected as templates,and the positions with significant differences in the shape of the A/B flexible loop have been chosen for replacement.A total of seven mutants:Cs-AS,Cs-AS+BL,Cs-BL,Cs-AP,Mm-BS,Mm-BS+AL,and Mm-AL were successfully constructed and the catalytic direction of the mutants was explored to verify the hypothesis.The results showed that the A-flexible loop of Cs CE with two catalytic direction significantly affect its isomerization activity,while had little impact on its epimerization activity.Besides,the existence of a B-flexible loop with one catalytic direction of Mm MI directly affect its isomerization activity.In summary,it could be concluded that the position,length,and shape of the flexible loop of the AGEs were closely related to the catalytic direction of the enzyme.Based on the above results,the effect of flexible loop on the catalytic activity of Cs CE was investigated.Four mutants were constructed by replacing the portion of the Cs CE flexible loop（"lower part"）with the corresponding flexible loop of CEs derived from Bacillus thermoamylovorans（Bt CE）、Rhodothermus marinus（Rm CE）、Ruminococcus albus（Ra CE）and Spirochaeta thermophila（St CE）through flexible loop exchange.Compared to the wild type Cs CE,the isomerization activity of the recombinant enzyme Rm C enhanced by 2.2 times,and the catalytic efficiency increased by 1.34-fold.Molecular dynamics（MD）simulation results exhibited that the flexible loop of Rm C was farther away from the active center and had a larger substrate entrance,which was conducive to substrate entry and stabilization of the enzyme-substrate conformation.In conclusion,the shape and composition of the flexible loop at the entrance of the active center were key factors affecting the catalytic activity of the enzyme,which could be regulated by influencing substrate binding and proton transfer.The catalytic efficiency was limited by the poor substrate affinity of Cs CE（K_m,417.5 m M）.Considering that the modification of flexible loop could promote the interaction between the enzyme and the non-reducing end of the substrate,it was further speculated that the binding action of the non-reducing end of the substrate may directly affect the substrate affinity of the enzyme.Therefore,a semi rational design strategy was adopted to reshape the substrate binding site of Cs CE.According to the crystal structure of the enzyme-ligand complex,Cs CE/W308and Cs CE/W372 were located on the pyran ring at the reducing and non-reducing ends of the substrate,respectively.Cs CE/W308 was conserved in the entire AGE superfamily,while Cs CE/W372 was only conserved in the CEs.The site-saturation mutation of Cs CE/W372confirmed that this residue was responsible for recognizing the non-reducing end of disaccharides and formingπ-πinteractions,which contributed to the stability and correct conformation of the substrate.The substrate binding region was further divided into three sub regions:region A,the substrate-binding subsection that interacts with the reducing end of the substrate,wherein Cs CE/W308 was located and two adjacent residues Cs CE/I306 and Cs CE/W307 were selected;region B,the substrate-binding subsection that interacts with the non-reducing end of the substrate,wherein Cs CE/W372 was located and the adjacent residue of Cs CE/Q371 was selected;and region C,the entrance of the catalytic site,wherein the Cs CE/W355 residue was located.The optimal mutant Cs CE/Q371E had 3.3-fold increased isomerization activity and significantly enhanced substrate affinity（K_m,269.65 m M vs K_m,417.5 m M）.Molecular dynamics simulation further revealed that the Cs CE/Q371E active center had a rich hydrogen bond network,making the substrate conformation more stable,and the enhanced interaction between catalytic residue His188 and substrate conduced to improve the isomerization activity.Besides,the distance between the catalytic residue His377 and the C2/O1 atoms was changed in Q371E,thereby prompting the catalytic process to proceed in the direction of isomerization.To sum up,the results indicated that remodeling of substrate binding sites could significantly improve the affinity between enzymes and substrates,ultimately increasing lactulose production.Due to the disadvantages of the single enzyme catalytic system such as presence of by-product epilactose,high cost of enzyme purification,and the need for high-temperature（75℃）,the restriction was removed by constructing a dual enzyme cascade catalytic cell factory,and it was further applied to the study of biocatalytic preparation of lactulose from whey powder.Firstly,an isomerase with the ability to convert epilactose to lactulose was introduced into the CE catalytic system to construct a CE-MI cascade system,which increased the yield of lactulose from 3.55 g/L to 4.0 g/L.The results showed that the cascade system promoted the conversion of the by-product epilactose.Molecular modification was carried out for CE and MI respectively,and a mutant（Mm MI/F242N）with a 43%increase in lactulose production was successfully obtained.The optimized catalytic modules Cs CE/Q371E and Mm MI/F242N were constructed on the p ET28b plasmid,and the expression levels of the two enzymes were regulated through a RBS strategy,the co-expression strain with the highest yield of lactulose was obtained:E.coli p ET28b-_T7-_RBSW-Cs CE/Q371E-_T7-_RBS29-Mm MI/F242N.Applying this cell factory to the research on value-added utilization of whey powder,the conversion rate of lactulose reached 91.3%,and the production of by-product epilactose decreased to 3.5%,significantly superior to the single enzyme catalyzed system（lactulose,63%;epilactose,12%）.In summary,results indicated that the constructed Escherichia coli cascade cell factory could promote the conversion of the by-product epilactose into the target product lactulose,overcome the adverse thermodynamic balance effects of single enzyme catalyzed isomerization reactions,and did not require enzyme isolation and purification.It had good application potential in the preparation of high yield and high purity lactulose.