Metabolic Engineering Of Escherichia Coli For Enhancing The Production Of Heme

5-Aminolevulinic acid,a five-carbon amino acid,is a vital intermediate involving in biosynthesis of tetrapyrrole compounds.Heme,an iron-containing porphyrin,is synthesized from ALA in the body,and involves in oxygen transfer.The aim of this dissertation is to identify the key genes and regulatory mechanisms of ALA and heme biosynthesis so as to enchance the production of heme.The main results were described as highlighted below:(1)Mutation of gene hemB.hemB(encoding ALA dehydrogenase)is the first gene down the ALA biosynthesis pathway.In order to increase the production of ALA,Ser near the HemB active site was altered to Tyr to inhibite the expression of hemB.Together with the strains preserved in our lab,we analyzed the influence of hemB mutation on ALA production.The result showed that ALA production of BL21(DE3)-pfli C-1 was increased by 24.5% compared with the control strain.However,ALA concentration had fallen off a cliff when the strains had intragenic mutations.Further experiments showed that the decreased activity of HemA was the main reason leading to the decreased yields of ALA.Therefore,a tentative inference on this result was that there was an interaction between HemA and HemB.Once the sequence of HemB was changed,the activity of HemA was lowered and the yield of ALA decreased finally.(2)The expression of sRNA MicC.To investigate the regulatory mechanism of ALA and heme biosynthesis,small non-coding RNA MicC was chose to inhibit the expression levels of gene sucA,hemC,hemG,hemH.The results indicated that the cell growth was better after using MicC to down-regulate the genes’ expression.When expression of sucA,hemG and hemH were inhibited,the production of ALA reached 1180 mg·L-1,1224 mg·L-1,1080 mg·L-1 respectively.Compared with the control strain,it was a 46.6%,52.0% and 34.2% raise respectively.Nonetheless,a sharp decrease in heme production appeared when inhibiting the expression of hemC,hemG and hemH.It showed that there was a negative feedback between ALA and heme.Meanwhile,it also proved that overexpression of hemC,hemG and hemH were beneficial to heme accumulation.(3)Identification of gene promoters in heme biosynthesis pathway.Gene gfp(encoding green fluorescent protein)was selected to analyze the expression time and intensity of gene promoters in heme biosynthesis pathway.Among this,all of the genes down the ALA biosynthesis pathway were expressed at later growth stage of cell,which revealed that heme was accumulated at later growth phase.What’s more,the intensity of hemF and hemG promoter was the strongest,hemE,hemH and gltX promoter took the second place and hemA,hemL,hemB,hemCD promoter came to the last position.Thus,it’s necessary to improve the expression level of hemA and hemL(committed genes for ALA production)so as to increase the yields of ALA and heme.(4)On the basis of previous experiments and the analysis of enzyme structure and function in heme biosynthesis pathway,the modular combination strategy was applied for enhancing the production of heme.Gene hemA and hemL as the first module was overexpressed by the high copy number plasmid.In parallel,the downstream heme biosynthesis pathway genes were divided into the second(hemB,hemC,hemD and hemE)and third(hemF,hemG,and hemH)modules,and were combinatorially expressed by the plasmids of different copy numbers.In comparison,the recombinant strain E.coli D6,achieved the highest heme titer(0.820 mg·L-1)in shake flask cultures and 1.399 mg·L-1 in 3 L fermenter.