Metabolic Engineering Of Escherichia Coli Heme Biosynthesis Pathway For Production Of 5-aminolevulinic Acid

5-Aminolevulinic acid(ALA) is a natural non-amino acid with five carbons. In organisms, ALA is an important intermediate for biosynthesis of tetrapyrrole compounds, such as heme, chlorophyll, cytochrome and vitamin B12. As an important photodynamic therapy, plant growth regulator, herbicide and insecticide, ALA has been widely used in medicine and agriculture industry.Recently, microbial production of ALA has become a significant research and gradually replaced the traditional chemical synthesis due to metabolic engineering and synthetic biology. At present, the main microbial production of ALA is biotransformation. However, the cost is high due to the addition of precursors, glycine and succinic acid. This dissertation chose the microbial production of ALA by regulating heme biosynthesis pathway in Escherichia coli as the objective. Firstly, the regulatory mechanisms of heme biosynthesis pathway were preliminarily investigated. Based on this, through a series of strategies and modifications at transcriptional level, translational level and protein level, including enzyme mutation, construction of RBS mutation libraries of key genes by high throughput screening, regulation of ALA dehydratase expression and pyridoxal 5′-phosphate biosynthesis at genome level, ALA production was improved due to the enhancement of upstream metabolic pathway and decrease of downstream metabolic pathway. The main results were described as follows.(1) Identification and regulation of key genes in heme biosynthesis pathway. The heme pathway genes hemB, hemC, hemD, hemE, hemF, hemG and hemH were respectively overexpressed and co-overexpressed with key genes of C5 pathway hemA and hemL to investigate the effect on cell growth and ALA production. Up-regulation hemD or hemF increased ALA accumulation while reverse results were obtained when overexpressing hemB, hemG or hemH. In contrast, no significant differences were detected after overexpression of hemC and hemE. On this basis, combined analysis of the transcriptional level of the key genes, the activity of ALA dehydratase and ALA production, the regulatory mechanism of heme biosynthesis pathway was preliminarily illustrated. It speculated that ALA dehydratase encoded by gene hemB was an important regulatory site for heme biosynthesis pathway and feedback inhibited by protoporphyrinogen IX.(2) Cell growth and ALA production were improved with the optimization of key enzymes expression based on the regulatory mechanism of heme biosynthesis pathway. Through assembly and optimization the expression of genes hemA, hem L, hemD and hemF that were beneficial for ALA biosynthesis, the recombinant strain E. coli LADF-6 produced 3.25 g·L-1 ALA in a 3 L fermenter. The key component of heme Fe2+ was added in the medium during the cultivation of recombinants that expressing of heme biosynthesis pathway genes to improve the cell growth, the production of ALA in E. coli LA decreased. However, both the cell growth and ALA production in the recombinants that expressing of hemD and hemF were improved. Compared with that of without addition of Fe2+, ALA production was increased by 35.2% and 34.1%, respectively. The recombinant E. coli LADF-6 increased to 4.05 g·L-1 in a 3 L fermenter with the optimization concentration of Fe2+ 7.5 mg·L-1.(3) The production of ALA increased with the modification and optimization of the key enzymes expression of ALA upstream pathway. The fusion of different number arginine residues with positive charge to the behind of Thr2 position at the glutamyl-tRNA reductase N-terminus affected cell growth and ALA production. It was found that ALA production increased by 76.4% compared with that expression of the wild type when one arginine residue was inserted. Based on this, the RBS sequence of hemL and hemA was randomly mutated and the mutation libraries were constructed by high-throughput screening, respectively. The highest ALA production with RBS mutation of hemL and hemA was 1.75 g·L-1 and 2.05 g·L-1, respectively. Compared with hemL, the RBS mutation of gene hemA resulted in higher ALA production. Combined analysis of RBS strength and the corresponding ALA production, it was beneficial for ALA production when RBS strength of hemA was higher than that of hemL. The optimal recombinant strain produced 2.41 g·L-1 ALA in the flask after assembling and optimizing the expression of genes with different RBS strength.(4) ALA production was improved by down-regulating the expression of ALA dehydratase. An artificial designed non-encoded sRNA was firstly used to regulate the expression of hemB. Although the ALA production was improved, it further increase was affected due to the poor cell growth. Meanwhile, different promoters were used to replace the promoter of hemB and its initial codon was mutated at genome level to regulate the expression of hemB. When the promoter of hemB was only replaced by stationary-phase attenuated promoter fliCp derived from E. coli K-12, ALA production increased to 2.68 g·L-1 in the flask, which increased by 11.1%. This indicated that the regulation of hemB was realized. Combined cell growth and the analysis of hemB transcriptional level, the transcriptional level of hemB was down-regulated after cell growth entered into the middle and later of exponential phase. This might lead to the decrease the catabolism of ALA and improved ALA production.(5) Optimization of pyridoxal 5′-phosphate(PLP) biosynthesis pathway and the recombinant strain cultivation to increase ALA production. Glutamate-1-semialdehyde aminotransferase, the key enzyme of ALA biosynthesis, catalyzes glutamate-1-semialdehyde into ALA which needs the participation of PLP. A heterogenous PLP biosynthesis pathway from Bacillus subtilis was introduced by overexpressing yaaD and yaa E with plasmid or integrating into the E. coli genome. And the native PLP synthesis pathway in E. coli was also enhanced by replacement of the key gene pdxH promoter with T7 or J23119. These strategies were used to increase the activity of GSA-AT through the improvement of PLP concentration. It was found that ALA production increased to 2.86 g·L-1 when the promoter of pdxH was replaced by T7. In addition, genes recA and endA were deleted to improve the plasmids stability. And the production of ALA in shake flask was 3.77 g·L-1 with the expression of transport protein RhtA, key genes hemA, hemL, hemD and hemF. Finally, with pH two-stage strategy, the production of ALA increased to 5.71 g·L-1 in a 3 L fermenter.