Rational Design And Modification Of Key Enzymes In Adipic Acid Biosynthesis Pathway

Adipic Acid is an important platform compound in industry,which is mainly used in synthesis of nylon 66,whose market is expected to reach$30.7 billion by 2021.It is also widely used in food,medicine,chemical industry and other industries.However,nowdays the commercial production of adipic acid still relies on chemical synthesis,which also produces large amounts of chemical contaminants especially nitrogen oxides in the whole reaction pathway.Therefore,a green and environment friendly way for producing adipic acid is needed to replace the chemical method.At present,a lot of progress has been made in biosynthesis of adipic acid,but these advances mainly focused on the improvement of microbial strain engineering and fermentation conditions,and there is little understanding and research on the enzymes.5-carboxy-2-pentenoyl-Co A reductase is the limited-enzyme in adipic acid synthesis pathway.The activity and substrate specificity of it have important influence on the synthesis of adipic acid;Adipyl-Co A synthetase is the last step in adipic acid synthesis pathway.It has direct influence on the synthesis of adipic acid.In this paper,we used the knowledge of structural biology and computational simulation to analysis the structures of the two key enzymes in the adipic acid biosynthesis pathway.The enzyme activity-increasing mutant was obtained by site-directed mutagenesis.Finally,based on the bioinformatics knowledge and enzyme design tools 5-carboxy-2-pentenoyl-Co A reductase was redesigned to obtain the enzyme with high catalytic efficiency and substrate specificity.Enzyme engineering technology will improve the catalytic efficiency and substrate-specificity of enzymes,thereby increasing the production of adipic acid,which was expected to promote the industrialization of adipic acid.The main research contents are as follows:?1?The modification of 5-carboxy-2-pentenoyl-Co A reductase in the adipic acid synthesis pathway.First,we built the model of 5-carboxy-2-pentenoyl-Co A reductase by homology modeling and docked the ligand into it.After analyzing the structure of the model and the position of the ligand,we found that Lys295 and Glu334 lied on the oral and bottom of the binding pocket,respectively.Any change of the two amino acids may make a great influence on the shape of the binding pocket.In order to make 5-carboxy-2-pentenoyl-Co A to match the binding pocket well,we carried out ten mutants of the enzyme:E334D,K295R,K295Q,K295Y,K295F,E334R,E334H,E334K,E334W and E334F.The experiment results showed that the enzyme activity and the yield ofadipic acid were improved in different degree.And the highest enzyme activity of these mutants was E334D,whose value was 95.5 U/mg.It was 2.3 times compared with in wildtype?41.4 U/mg?.The mutant E334D reached the highest value of 0.23g/L after fermented for 24 h,which was almost 1.5 times as the yield of wild type.?2?Redesign of 5-carboxy-2-pentenoyl-Co A reductase in adipic acid synthesis pathway.After analyzing we found that the side chain of 5-carboxy-2-pentenoyl-Co A was a strongly polar carboxyl group while most of the amino acids in this binding pocket were hydrophobic.It will unhelpful of binding between 5-carboxy-2-pentenoyl-Co A and the enzyme.So we redesigned the binding pocket by introducing hydrophilic amino acids on the pocket with Evo EF2 to improve the binding effect between the ligand and the enzyme.We chose the amino residuses within 5?from 5-carboxy-2-pentenoyl-Co A as the redesigned sites.After caculation and redesign,we got 10 models?Des 0?Des 9?.There formed hydrogen bonds between ligand and Q253R or I250Q in Des 0,Des 3,Des 4 and Des 9.Between Q253R and L89S?in Des 0and Des 9?or L89T?in Des3 and Des4?or Thr364 can formed hydrogen bonds.At last,molecular dynamics simulation wascarried out to check the hydrogen bonds redesigned.The simulation result showed that the hydrogen bonds in Des 0 were more stable,it was helpful on improvement the binding effect between the ligand and enzyme.The enzyme activity of Des 0was almost 1.5 times as the wildtype.?3?The modification of adipyl-Co A synthetase in adipic acid synthesis pathway.First,we built the model of adipyl-Co A synthetase by homology modeling and analyzed the structure.We found that Glu210?lied on the oral of the binding pocket.Small size of pocket mouth might affect the entry and exit of phosphate groups,then affect on the phosphorylation of ADP and the enzyme activity.Then we found that after mutating Glu210?to Arg210?,the mouth of the binding pocket would be enlarged,and the phosphate group would enter and exit the binding pocket more easily.Combined with literature reports we mutated Glu210?to Arg210?,Gln210?and Asp210?,respectively.The experiment results showed that after the mutation of Glu210?into Gln210?and Asp210?,the enzyme activity of the mutants and the yield of adipic acid were increased.However,the enzyme activity and the adipic acid yield of the mutant strain E210?R was decreased.And the maximum production of adipic acid of E210?N,E210?Q,E210?R and wildtype reached 0.32,0.30,0.13 and 0.15 g/L,respectively.?4?Study on the catalytic mechanism of adipyl-Co A synthase in adipic acid synthesis pathway.The performance of adipoyl-Co A synthetase has been improved through rational design,but some predictions do not match the actual situation.Due to the lack of specific catalytic mechanisms,it was difficult to analyze the above results.In this study,through simulation and analysis,we found that Lys228?acts as a mediator in the transmission of the phosphate group in adipoyl-Co A synthase.In one word,the phosphate group was delivered from His251?-PO₃ to Lys228?forming Lys228?-PO₃ complex,then Lys228?-PO₃ delivered phosphate group to ADP to complete the catalytic reaction.There are salt bridges and hydrogen bonds between E210?and Y122?and K228?of wild-type adipyl-Co A synthase,which restricts the transmission of phosphate group by the K228?.The loss of a salt bridge or hydrogen bond in mutants E210?N and E210?Q led to K228?flexibility increases,so the mutant's vitality was enhanced,but the mutant E210?R almost lost all hydrogen bonds and salt bridges,which made it difficult to maintain an orderly internal environment,which ultimately led to lower enzyme activity and adipic acid production capacity.