Research On Exploiting Enzyme Promiscuity For The Biosynthesis Of 1-alkanols And 1,n-Alkanediols From Polyhydroxy Compounds

Alkanols and l,n-alkanediols(C4-C6),as important chemical compounds,have been widely used in the fields of energy,chemicals and materials.However,most of them can't be synthesized spontaneously by microorganisms,which makes the development of 1-alkanol and 1,n-alkanediol artificial pathway of important significance.Polyhydroxy compounds are widely spread in nature with similar structures to 1-alkanols and 1,n-alkanediols,which inspired us to exploit enzymes that reduce the number of hydroxyl groups,so as to covert polyhydroxy compounds to 1-alkanols and 1,n-alkanediols.Since glycerol dehydratase and diol dehydratase play essential role in the production of 1-propanol and 1,3-propanediol,respectively,our thesis started from the dehydratases and exploited their substrate promiscuity through protein engineering.This enabled the biotransformation of long-carbon chain polyols to 1-alkanols and 1,n-alkanediols.The main contents and conclusions of this work were introduced as follows:Firstly,the potential of glycerol dehydratase and diol dehydratase towards four-carbon polyols were investigated.Four dehydratases from different strains were heterologously expressed,and the results from enzymatic experiments and product detection confirmed that the dehydratases couldn't effectively catalyze erythritol.Two of the dehydratases were subsequently mutated,resulting in the mutants with significantly enhanced activities towards 1,2,4-butanetriol.In addition,the feasibility of transformation of 1,2,4-butanetriol to 1,4-butanediol was validated by whole-cell biocatalysis.Secondly,the potential of glycerol dehydratase and diol dehydratase towards 1,2-butanediol,1,2,4-butanetriol,erythritol,1,2-pentanediol,1,2,5-pentanetriol and 1,2,6-hexanetriol were investigated.The results from in vitro enzymatic experiments and product detection confirmed that glycerol dehydratase could catalyze 1,2,5-pentanetriol,and diol dehydratase could catalyze 1,2-pentanediol and 1,2,5-pentanetriol.After site-directed mutagenesis of these two dehydratases,the abilities of the mutants towards the above polyols enhanced obviously.Thirdly,the 1,4-butanediol pathway from erythritol,1-pentanol pathway from 1,2-pentanediol and 1,4-butanediol pathway from xylose were constructed in E.coli.These three pathways containing glycerol dehydratase mutant produced 16.1 mg/L 1,4-butanediol,12.8 mg/L 1-pentanol,and 48.9 mg/L 1,4-butanediol,respectively;those containing diol dehydratase mutant produced 11.9 mg/L 1,4-butanediol,137.8 mg/L 1-pentanol,and 51.8 mg/L 1,4-butanediol,respectively.In addition,the kinetic parameters of 1,2,4-butanetriol catalyzed by glycerol dehydratase and diol dehydratase were determined.The Km value of glycerol dehydratase mutant and diol dehydratase mutant were 34.14 mM and 1.04 mM,respectively;The kcat value of them were 0.21 s-1 and 0.39 s-1,respectively.Finally,the property of glycerol dehydratase towards four-carbon polyols was further studied.Firstly,the effects of cell concentration,temperature,substrate concentration and pH on the conversion of erythritol to 1,4-butanediol by whole-cell catalysis were studied.The results showed that the concentration of 1,4-butanediol increased firstly and then decreased with the increase of cell density and substrate concentration.Low temperature and weak alkaline were favorable for the synthesis of 1,4-butanediol.Under the optimal conditions,the 1,4-butanediol concentration was 34.5 mg/L,which increased nearly 6-fold compared to the unoptimized conditions.The activities of glycerol dehydratase towards erythritol isomers and its kinetic properties towards 1,2,4-butanetriol were subsequently investigated.The results indicated that activities of the mutants towards L-threitol were significantly higher than that towards erythritol and D-threitol,the mutants showed similar catalytic rates for the three 1,2,4-butanetriols isomers.However,the Km value of(R)-1,2,4-butanetriol was obviously higher than(S)-1,2,4-butanetriol and 1,2,4-butanetriol.This will provide experimental data for the subsequent computer modeling.Due to the lack of related enzymes,many important compounds are unable to produce via biological pathways,so mining functional genes is a key step for the establishment of bioprocesses,and exploiting enzyme promiscuity is an effective strategy.This project focused on the study of enzyme promiscuity of glycerol dehydratase and diol dehydratase,and improved their activities towards long-chain polyols by protein engineering.This would provide more genetic elements for synthetic biology and offer more pathways for 1-alkanols and 1,n-alkanediols biosynthesis.