| The reactive carboxyl,hydroxyl or vinyl groups in structures of D-lactate and its derivatives 3-hydroxypropionic acid(3HP)and acrylic acid have provided them with specific chemical reactivity,and finally led to their wide applications in various chemical reactions.They have also participated in material development through the formation of polymers and heterochain polymers,molecular grafting and surface modification,and have a huge application market.Currently,D-lactate and its derivatives 3HP and acrylic acid production all face problems of improving process performance including increasing titer,yield,productivity,and the development of production process based on renewable resources.Developing of highly efficient microbial cell factories for their production through metabolic engineering approaches is very important and promising.In this work,we obtained a fast growing Escherichia coli platform strain via adaptive laboratory evolution(ALE)and revealed the main reason of the fast growth phenotype.Then we developed a highly efficient D-lactate producer and finally successfully produced 3HP and acrylic acid from D-lactate intermediate through anaerobic fermentation.Through this work,we have provided new methods for producing D-lactate and derivatives 3HP and acrylic acid via E.coli cell factories.Firstly,we obtained a fast growth E.coli platform strain WE269 through ALE and investigated the genetic mechanism of this phenotype.The platform strain shew a 20%and72%increase in stationary biomass content and ?maxas compared with wild type strain under test conditions.Through whole genomic mutation analysis and reverse engineering,we found that sucD(M245I)and ilv G(?1 bp)mutation were the main reason of fast growth phenotype of platform strain.We also found that transcription of genes in PP pathway,DNA replication machinery under stress conditions and several efflux pumps were upregulated in the platform strain,while transcription of several genes in the RNA transcription machinery and lipid synthetase were downregulated.Secondly,we constructed a highly efficient D-lactate producer through metabolic engineering of the obtained platform strain.After fermentation tests and process optimization,we achieved a D-lactate titer of 98.3 g/L,productivity of 2.05 g/(L·h),yield of0.97g/g and optical purity of above 95%,which was one of the best E.coli producers using glucose as carbon sources via single stage anaerobic fermentation and have potential for application in industrial production.Thirdly,we constructed a 3HP producer through metabolic engineering of the platform strain.We have constructed and optimized the 3HP synthetic pathway via D-lactate intermediate through pathway design,gene mining,codon optimization,combinatory pathway optimization;strengthen the energy supply and key enzyme gene replacement.After fermentation tests of strains integrated with effective 3HP synthetic pathway,the recombinant strain W3110(p3HP1.0)produced 2.0 g/L 3HP in a 5 L fermenter fed-batch fermentation after subtraction of formate interference.Finally,we constructed an acrylic acid producer through metabolic engineering of the platform strain obtained.We designed and constructed an acrylic acid synthetic pathway via D-lactate intermediate and integrated it with the lactate production strain obtained in this work to get an acrylic acid producer.After fermentation tests in 25 m L scale anaerobic bottles,the engineered strain successfully produced 74 mg/L acrylic acid. |
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