Construction And Optimization Of Efficient Cell Factories For Cis,Cis-muconic Acid Production

cis,cis-Muconic acid(MA)is a dicarboxylic acid with two conjugated double bonds,and has a good UV absorption properties at 260nm.MA can be used not only for UV protection agent and special ordnance supplies such as stealth coatings,but also can be potentially used as a raw material for new functional resins,pharmaceuticals and agrichemicals.It mainly can be used to synthesize adipic acid which is a large amount of chemicals.Recently,microbial production of MA was paid greatly attention to due to sustainability.However,losts of issues for microbial oroduction of MA must be addressed for future industrial application.In terms of host strain,it is a pathogenic bacterium with unclear genetic background for Klebsiella pneumonia,and it is difficult to genetically manipulate for Saccharomyces cerevisiae.In terms of substrates,the benzoate or catechol are not suitable and sustainable substrate due to petroleum feedstocks.In addition,many engineered strains were developed to produce MA by shunting shikimate synthesis and introducing plasmid-based synthetic pathway.These manipulations were also not ideal.Either the fermentative media have to be supplemented some nutrient indegredient such as aromatic amino acids,or the engineered strains with plasmids were unstable.These would increase the production cost of MA.To address the above issues,new synthetic cell factories for efficient production of MA were developed with Escherichia coli as host strain and glucose as renewable substrate.In this study,according to the rational design,we constructed 3 E.coli cell factories to produce protocatechuic acid,9 E.coli cell factories to produce catechol,27 E.coli cell factories to produce MA by successively investigating the combinatorial regulation of aroZ gene,aroY gene and catA gene for MA biosynthesis with different synthetic promoters in WJ060 which overproduced 3-dehydroshikimic acid.The best engineered strain MA30 was able to produce 1.7 g/L of MA from glucose.Fed-batch fermentation further improved the production of MA and the titer reached 10.3 g/L in 78 hours.We could produce safely and stably in minimal salts medium,and it will be possible to achieve the lower production cost and large scale production of MA.Subsequently,in order to further improve the performance of the E.coli cell factory,we firstly constructed a high throughput screening method of MA according to the UV absorption property of MA in 260 nm.Then we used the new technology of evolutionary engineering(Genome replication engineering assisted continuous evolution)to construct the mutant library of engineering strain for MA production.After two rounds of screening,we had successfully screened the strain of E.coli MA30-G2,of which the yield of MA improved 8%.Fed-batch fermentation further improved the production of MA and the titer reached the highest 11.5 g/L in 72 hours,and the yield of MA improved 11.6%comparing with the parent strain of MA30.With this irrational design,we successfully increased the performance of cell factory.By genome resequencing and comparative analysis between the two evolved strains and the parent strain,7 genes which might affect the biosynthesis of MA were identified.These genes could be as potential targets for further engineering the strain to improve the production of MA.Combination the rational design with the irrational design,we constructed and optimizated efficient cell factories for MA production,it not only laid foundation for MA biosynthesis,but also provided reference for biosynthesis of other biobased products.