| Adipic acid,as the monomer for manufacturing nylon,fiber and plastics,has broad applications in food,pharmaceutical,plastics,and chemical industries.At present,low-level yield of adipic acid was generated from edible food feedstocks mainly due to carbon atom loss.Fatty acids（FAs）,as a kind of abundant feedstocks in nature,have a great potential for efficiently producing high-value chemicals.On the one hand,utilization of FAs avoids the competition with crop-based feedstocks,thus lowing the production costs.On the other hand,FAs catabolism can achieve 100%carbon atom recovery,which is beneficial to obtain the highest theoretical product yield.In this study,we designed and constructed an artificial adipic acid biosynthesis pathway with FAs as feedstock by integrating engineeredβ-oxidation pathway withω-oxidation pathway.Then,multiple metabolic engineering strategies were used for optimizing adipic acid biosynthesis pathway,which not only enhanced cell utilization for FAs,but also improved biosynthesis efficiency of adipic acid from palmitic acid.The main results were as follows:1.Construction and strengthen of endogenousβ-oxidation pathway.Firstly,fad L,fad D,fad E,fad B,fad A and ydi I genes were overexpressed for enhancingβ-oxidation pathway,and Escherichia coli AA0108 produced 0.67 g·L-1 hexanoic acid from palmitic acid,owing to inefficient degradation process for palmitic acid in wild-type E.coli strain.Secondly,effect of FAD concentration on hexanoic acid production was tested and analyzed,which demonstrated that intracellular FAD concentration was one of the most essential factors associated with hexanoic acid production.Finally,cofactor regeneration system was constructed by heterogeneously expressing coden-optimized formate dehydrogenase（fdh）from Candida boidinill and NADH oxidase（nox）from E.coli,and hexanoic acid production was up to 0.77g·L-1 in strain E.coli AA0109.2.Reconstruction and optimization of heterogenousω-oxidation pathway.Firstly,whole-cell bioconversion experiments showed that E.coli was naturally uncapable of oxidizing hexanoic acid into adipic acid.Then,ω-oxidation pathway was heterogeneously constructed by databases mining and literatures research,containing the alkane hydroxylase system（Alk BGT）from Pseudomonas putida and 6-hydroxyhexanoate dehydrogenase（Chn D）and aldehyde dehydrogenase（Chn E）from Acinetobactor sp.,and strain E.coli AA0201produced 0.61 g·L-1 adipic acid.Next,RBS30,RBS34 and RBS32 were screened and determined to be the high,medium and low strength of RBS with green fluorescent protein as a reporter,respectively.Finally,pathway modular engineering was performed for optimizingω-oxidation pathway by using different strength of RBSs,including the hydroxylation module（alk B,alk G and alk T genes）and oxidation module（chn D and chn E genes）,aiming at balancing heterogenous genes expression and decreasing by-products formation.The results indicated that up-regulating hydroxylation module or down-regulating oxidation module all was beneficial for adipic acid biosynthesis,among which strain E.coli AA0204 obtained the highest titer of adipic acid(0.76 g·L-1),with an increase of 24.59%compared with that of strain E.coli AA0201.3.Assembly and application of adipic acid biosynthesis pathway.Firstly,the de novo biosynthesis pathway for adipic acid was established by integrating the optimalβ-oxidation pathway andω-oxidation pathway,and strain E.coli AA0301 produced 57.00 mg·L-1 adipic acid from palmitic acid in whole-cell bioconversion.Then,fed-batch culture could enhance adipic acid production,with its titer of 0.35 g·L-1,improving 3.18-fold compared to batch fermentation.Further,several metabolic engineering strategies were employed in enhancing cell utilization for FAs.For one thing,deletion of fad R gene encoding FAs metabolism regulatory protein shortened the lag phase of cell growth.On the other hand,transportation rate of FAs was improved by optimizing expression level of fad L gene.As a result,strain E.coli AA0306 produced 0.52 g·L-1 adipic acid in shake flasks,with an increase of 32.00%in cell growth rate.Next,fermentation conditions of adipic acid production were optimized by regulating feed rate of FAs and glucose,and the results indicated that fed-batch addition of palmitic acid and low concentration of glucose could obtain the highest titer of adipic acid,reaching 1.62 g·L-1.Finally,the optimal strain E.coli AA0306 was tested in 5-L fed-batch cultivation for 96 h,and the titer and yield of adipic acid were 4.66 g·L-1 and 0.23 g·g-1,respectively. |
