Engineering The Pathway Of5-Aminolevulinic Acid Biosynthesis In Escherichia Coli And The Accompanying Metabolism Shifts

5-aminolevulinic acid (ALA), is a natural nonprotein amino acid. It is the substrate for the biosynthesis of tetrapyrrole compounds, such as heme, chlorophyll, vitamin B12, and it has various applications. Till now, chemical synthesis is the main method utilized to acquire commercial ALA. Biotechnological production of ALA, however, has emerged as very promising alternative and it is attracting many attentions. Dependent on the C4pathway, both Escherichia coli and Rhobacter sphaerbdies have been tested as host strains for the biosynthesis of ALA with supplement of succinate and glycine and the peak yield of ALA produced through this strategy reached to7.3g/L.Recently, our group has developed a novel approach for biotechnological production of ALA through the C5pathway of ALA biosynthesis. Herein, glucose is the sole carbon source and the nitrogen source is ammonium salt. The maximum yield of ALA we acquired is1.95g ALA per20g glucose. Based on these results, I have carried out further research, such as identifying ALA exporters, inhibiting the activity of ALA dehydratase (HemB), enhancing the instability of HemB, and promote the degradation of heme, to improve the yields of ALA.In the second part of my research I have found that the overexpression of hemA and hemL genes of the C5pathway in either Escherichia coli or Corynebacterium glutamicum could cause some effects on the formation some main products (acetate in Escherichia coli and glutamate in Corynebacterium glutamicum),besides influencing the formation of ALA. It was proposed that the increased production of porphyrins and heme derivates could stimulate respirationand TCA cycle in both organisms and hence leaded to decreased acate formation in Escherichia coli and increased glutamate formation in Corynebacterium glutamicum.The third part of my thesis is about the importance of HemB protein. I was deeply attracted by an unexpected finding during my endeavor to improve the production of ALA, that the increasing degradation rate of HemB protein (by attaching the SsrA protein degradation tag to the C terminal of HemB) causing decreased production of ALA. Transcriptome analysis shown global gene shifts due to the instability of HemB. Transcriptions of the genes involved in glycolysis, glyoxylate cycle, biosynthesis of acetate, acid resistance (glutamate dependent acid resistance system, especially) and stess response have been strengthened, by different scales. The averag And the mRNA levels of hemA and hemL have decreased by3-4folds. All these changes affect negatively the accumulation of our desired product ALA. Moreover, we ReALized that the C5pathway of ALA biosynthesis is much more influential to the center metabolism of carbon than we previously thought.