Enhancement Of 5-aminolevulinic Acid Production By Engineering The C4 Pathway In Escherichia Coli

5-aminolevulinic acid(ALA)is a common precursor of tetrapyrrole-like compounds in nature.It is a high value-added bio-based chemical with great development prospect in medicine and agriculture.In this study,we described a metabolic strategy to produce ALA directly from glucose in recombinant Escherichia coli via the C4 pathway.We first studied the combinations of different heterologous hemA genes(coding ALA synthase)and plasmid vectors on ALA production to comfirm the expression plasmid.In addition,the random mutation of ribosome binding site region,optimization of biosynthesis pathways towards coenzyme A and precursor(glycine and succinyl-CoA),and downregulation of hemB expression were performed to obtain higher ALA production.During the study,we also presented a DNA assembly method sDATEL to construct the plasmids related to coenzyme A.The main results were described as follows:(1)Selection and optimization of ALA synthase expression in E.coli.In this research,we studied the expression of pRSFDuet-1 and pET28a(+)in combination with ALA synthase from Rhodobacter sphaeroides(RS.hemA)and Rhodobacter capsulatus(RC.hemA),and the strain containing pRSFDuet-1-RC.hemA had the highest ALA yield of 375 mg·L-1.Then,the RBS sequence of RC.hemA gene was mutated by degenerate sequence,a RBS mutant E.coli M3 with 689 mg·L-1 of ALA production which was 83.73% higher than the parent strain was obtained after the high-throughput screening in 96-well plate and shake culture in flask.After sequencing,the RBS region of pRSFDuet-1-RC.mhemA(from E.coli M3)has changed to GGAAGGAAGGAAAC;(2)In vitro scarless method of assembling multiple unphosphorylated fragments.In order to facilitate the follow-up plasmid construction experiments,the sDATEL was presented based on the simplification and optimization of the original assembly method DATEL.sDATEL can assemble multiple DNA fragments with homologous arms in a single reaction by means of thermal cycling reactions using the 5'-3' exonuclease activity of Taq DNA polymerase and Taq DNA ligase activity.The reaction buffer component(10×,200 mmol·L-1 Tris-HCl,250 mmol·L-1 potassium acetate,50 mmol·L-1 magnesium acetate,15 mmol·L-1 NAD +,1% Triton X-100,pH 7.2),which is the best fit for the two enzymes,was determined by optimizing the reaction buffer pH,concentration of NAD+ and Mg2+.In the optimized buffer,the assembly efficiency of the two fragments reached 12450 CFUs·?g DNA-1 with an accuracy rate of 100%,the assembly efficiency of the three fragments reached 830 CFUs·?g DNA-1 with an accuracy rate of 82%,the assembly of the four fragments efficiency of 190 CFUs·?g DNA-1 with the accuracy rate of 75%.The cheap simple sDATEL method can be used for assembly of 2-4 fragments as a laboratory tool;(3)Expression of CoA pathway enzymes for enhancing ALA synthesis using the sDATEL.In this study,the key enzyme coaA which was first enzyme in the CoA pathway was mutated in R106 to eliminate the feedback inhibition of the final product.The ALA yield of E.coli AAM was 798 mg·L-1,which was higher than that of the strain E.coli M3 without the expression coaAM.Strain E.coli AAMd,E.coli AAMD and E.coli AAME were obtained from the co-expression of coaAM with dfp,coaD and coaE respectively,and the expression of dfp and coaD could contribute to the improvement of ALA production according to the measurement result.Further,the high copy plasmid pUC19 and copy plasmid pETDuet-1 containing coaAM,dfp and coaD were construced to test the concentration of ALA,and eventually the highest ALA production was 1101 mg·L-1 in the strain E.coli AAMDd-2.(4)Enhancement of C4 pathways for ALA accumulation in E.coli.ALA production via C4 pathway needs two precursor materials(glycine and succinyl-CoA).To strengthen the glycine pathway,this study replaced the original promoter of serA with the strong promoter P23119 and deleted the C-terminal last four amino acid coding sequence to prevent from the feedback inhibition by L-serine and its analogues,resulting in an ALA yield of 1138 mg·L-1.To strengthen the succinic acid pathway,the sucCD gene in the tricarboxylic acid cycle was deleted to promote the accumulation of succinyl-CoA,and the ALA accumulation of E.coli AAGS-2 was further increased to 1208 mg·L-1.To prevent the consumption of ALA and weaken the feedback inhibition of ALA synthase by heme,the downstream ALA dehydrogenase gene hemB was downregulated by replacing weaker initiation codons,and the yield of ALA in the strain E.coli AAGSB-1 in shake flask culture reached 1762 mg·L-1.Finally,the recombinant strain E.coli AAGSB-1 was fermented in the 3 L fermentor,and the yield of ALA reached 2811 mg·L-1 after 32 hours of fermentation.