The Accompanying5-aminolevulinic Acid Accumulation And Metabolism Shifts Of Engineering And Regulating Heme Biosynthesis Pathway In Escherichia Coli

5-aminolevulinic acid (ALA) is a kind of narural nonprotein amino acid. It is the precursor of living organism tetrapyrrole compounds biosynthesis, such as vitamin B12, heme and chlorophyll. In the field of medicine, ALA received much attention as a pharmaceutical for cancer therapy and tumour diagnosis. In agriculture, ALA can be used as a kind of selective and biodegradable herbicide, insecticide and growth-promoting factor. So ALA has important value of market development and application prospect.At present, chemical synthesis is the main method applied to produce commercial ALA. However, the procedure of chemical synthesis is very complicated, and it is difficult to separate the accompanying by-product. This strategy also causes environmente problems. Biotechnolofical production of ALA has attracted much attention for its unique advantages, such as easy operation, using low-cost reproducible resourses and environment friendly. There are two kinds of ALA biotechnolofical production methods. The first is depend on C4pathway, both Rhobacter sphaerbdies and Escherichia coli have been tried to be used as the host strains for the biosynthesis of ALA with supplement of succinate and glycine. But the using of expensive precursor, succinate and glycine, make this C4pathway syrategy not suitable for large-scale industrial production. The second method is based on C5pathway, and it uses cheap glucose as the sole carbon source, which developd by our group. However, since the ALA yield of this strain is still not high enough, we need do further research to find the factors that restrict the production of ALA to make basis for high ALA production.Based on these results, firstly, this research tried further to development the production of ALA by metabolic engineering.The attempt including:Finding more effective ALA transporters; Knocking out the gene of transcription regulater ArcA; Exploration the effect of dissolved oxygen on the produanction of ALA; Overexpression the Glutamy-tRNA synthetase (GluRS) from Acidithiobacillus ferrooxidans; Overexpression the HemB transcription repressor Lt from Bradyrhizobium japonicum. Among these attempts, knocking out the arcA gene had the most outstanding effect on the accumulation of ALA. It made the yield of ALA developed by43.59%. And we also found that, the expression of gltx down regulated by the dissolved oxygen at the later stage of fermentation, and overexpression the Glutamy-tRNA synthetase from Acidithiobacillus ferrooxidans can release this repression.sRNA RyhB participates in iron homeostasis metabolism in Escherichia coli, it activated in iron limitate conditions and inhibates iron-protein presatation to decrease the consume of iron. We constitutive expressed RyhB to regulate the heme biosynthesis pathway and develop the5-aminolevulinic acid accumulation in Escherichia coli. The small RNA ryhB, which regulates the metabolism of iron in Escherichia coli, was constitutively expressed in engineered Escherichia coli DALA. The resulting strain Escherichia coli DALRA produced16%more5-aminolevulinic acid than the parent strain Escherichia coli DALA in batch fermentation, and the accumulation of heme was decreased in Escherichia coli DALRA. Meanwhile, we found that addition of iron in the medium increased heme formation and reduced5-aminolevulinic acid yield, whereas the presence of iron chelator in the medium decreased heme concentration and increased the5-aminolevulinic acid production efficiency (5-aminolevulinic acid yield/OD600). The qRT-PCR analysis showed that the mRNA levels of hemB and hemH were also decreased besides the known RyhB target genes of acnAB, sdhAB, fumA, and cydAB in Escherichia coli DALRA. These results indicated that small RNA can be used as a tool for regulating5-aminolevulinic acid accumulation in Escherichia coli. The presence of possible targeting sites in the mRNA of hemB and hemH indicated that they could be targets of RyhB. The down regulation of hemB and hemH by ryhB reduced the metabolic flux from ALA to heme, resulting in the increased ALA production and decreased heme accumulation. Although the mechanism of the regulation of hemB and hemH by RyhB was not confirmed, at least three reasons were supposed to be responsible for increased ALA accumulation in E. coli DALRA:Firstly, RyhB up regulated gltX expression which involved in ALA biosynthesis; Secondly, RyhB down regulated the gene transcription that involved in ALA metabolism; Thirdly, less heme accumulation de-repressed the feedback inhibition of ALA synthesis genes.Finally, this research focuses on the effects of the over-expression of hemAM and hemL on the metabolism and respiration of Escherichia coli. Overexpression of C5pathway genes, hemAM and hemL, caused the increase of ALA and heme production. However, it also made the accumulation of acetate, lactate and succinate decresed observably. The results of gene micro array presented that the expression of acetate production and elimination, TCA cycle related genes all down regulated. And the compose of respiration electron transport chain changed from NDH-1(4H+/e") and Cyo to NDH-2and Cyd-I (1H+/e-). Enhancing the expression of C5pathway genes resulted in the accumulation of heme in the strain cells. For heme is the important component of aerobic respiration electron transport chain, on one hand, it affected the centre metabolism of Escherichia coli by taking part in respiration. On the other hand, the biosynthesis of heme is coupled to electron transport chains for energy generation, so the biosynthesis of heme changed the energy generation pathway and metabolic flux of Escherichia coli, decreased the accumulation of by-product, such as acetate and lactate, and developed the utilization efficiency of carbon source.In conclusion, at the basis of the strain using C5pathway to produce ALA which constructed by our lab firstly, we used multiple traditional metabolic strategies, such as gene knockout and heterologous gene expression, and a new strategy that constitutive expression sRNA RyhB to develop the production of ALA. In addition, we used transcriptomics to analyse the effect of over expression C5pathway gene on respiration metabolism in E. coli. These results provided research direction and new idea for developing ALA production, and also increase potencial new application value for C5pathway.