Metabolic Pathway Modification For High Efficient Synthesis Of 3-HP By Escherichia Coli

With the continuous consumption of petrochemical resources and the constant deterioration of the climate,people are gradually changing from the petrochemical economy to a biological economy.Using renewable resources to produce high value-added biological products is an inevitable choice to achieve sustainable social development and circular economy.3-Hydroxypropionic acid(3-HP)has been selected by the US Department of Energy twice as one of the developmental chemicals with top priority.Using 3-HP as a precursor,it can synthesize a variety of commercially valuable compounds and is widely used in materials,chemicals,medicine,etc.At present,3-HP is mainly based on the synthesis of genetically engineered bacteria in the biological method,which has the advantages of high synthesis yield and easily relates to the pathway to enhance efficiency.In this paper,glucose is used as the sole carbon source,and Escherichia coli with bright genetic background,cheap and easy to obtain,was selected as the host strain to construct the malonyl-CoA pathway.The efficient synthesis of 3-HP was achieved through the modification of the metabolic pathway.The main contents are as follows:(1)Three pairs of primers containing homologous fragments were designed using the plasmids preserved in the laboratory as the template.PCR amplified the target fragments,and recombinant ligase was used to construct the recombinant plasmid pA2c-mcr containing the malonyl-CoA reductase mcr gene.The recombinant plasmid was verified by bacterial PCR,enzyme digestion,and sequencing,and the expression of the target protein was preliminarily verified.(2)In order to improve the activity of malonyl-CoA reductase(MCR),two pairs of mutant primers were designed by site-directed mutation technology,and the recombinant plasmid p A2c-mcr*containing multiple mutation sites(N940V/K1106W/S114R)was constructed.PCR and sequencing verification showed that the site had been mutated successfully,and the activity of MCR enzyme was significantly increased after mutation.Then the mutant plasmid pA2c-mcr*and the recombinant plasmid pBbE7a-accDABC containing acetyl-CoA carboxylase(ACC)were transferred into E.coli BL21(DE3)to obtain the recombinant strain ZYL5,which was verified by preliminary fermentation.It could synthesize 0.29 g/L 3-HP,which is about 1.8 times higher than the unmutated recombinant strain ZYL4.(3)In order to introduce the FapR transcription factor with the characteristic of responding to the concentration of malonyl-CoA into this pathway,bioinformatics analysis was performed on the FapR transcription factor in Bacillus subtilis.The amino acid of the transcription factor was obtained,and the number of bases was 188.The relative molecular weight is 21400.34,the fatty acid coefficient is 102.66,and the isoelectric point of the protein is 5.53,which is hydrophilic and stable.In terms of structure and function,it mainly plays a role in the nucleus.It is a non-secretory protein and cannot conduct transmembrane transformation.Besides,there are two conserved structural domains HTH and thiolipase family,with different functions in vivo,which can effectively combine with the DNA sequence and intermediate metabolites.Verification of the secondary and tertiary structure of the protein revealed that the protein is mainly ?-helix,accounting for 48.94%of the whole structure.In contrast,the ?-turn structure is less,accounting for only 7.45%.Finally,through homologous sequence alignment or phylogenetic tree construction,it is concluded that they have higher similarity with the same genus Bacillus cereus in terms of homology ratio and kinship,and the degree of evolution is related to kinship.The relationship is relatively close.(4)In order to reduce the toxic effects generated in the metabolic pathway,the synthetic plasmid pBFR1k-lacI-FapR containing the FapR transcription factor was introduced into this pathway to construct a metabolite biosensor.The recombinant plasmid was verified by bacterial solution PCR,enzyme digestion and sequencing,and was transferred into E.coli BL21(DE3)together with pBbE7a-accDABC and pA2c-mcr*to obtain recombinant strain ZYL8.The recombinant strain ZYL8 can synthesize 0.43 g/L 3-HP,the yield is increased by about 1.6 times compared with the strain without biosensor,and increased by about 14 times compared with the initial strain ZYL3.