利用大肠杆菌细胞工厂生产吲哚-3-乙酸的研究

Study on the Production of Indole-3-acetic Acid Using E.coli Cell Factory

目的:利用重组大肠杆菌全细胞转化色氨酸生产IAA。方法:在大肠杆菌胞内构建两条全新的IAA合成途径,即吲哚-3-乙酰胺(indole-3-acetamide,IAM)途径和色胺(tryptamine,TRP)途径。结果:IAM途径涉及两个酶,分别是色氨酸-2-单加氧酶(IAAM)和酰胺酶(AMI1),构建好的重组大肠杆菌TPA-4以2g/L的色氨酸为底物,可以产生0.803g/L的IAA;敲除控制色氨酸合成副产物吲哚的tnaA基因后,菌株MPA-3的IAA产量达到1.43g/L,提高了78%。第二条TRP途径合成IAA涉及三个酶:左旋色氨酸脱羧酶(TDC),二胺氧化酶(AOC1)和吲哚-3-乙醛脱氢酶(IAD1)。包含这条途径的重组大肠杆菌TPTA-2以2g/L的色氨酸为底物能够合成13.0mg/L的IAA。在菌株MPTA-3中,最终产生了21.0mg/L的IAA,产量增加了61.5%。结论:首次通过IAM途径和TRP途径利用重组大肠杆菌全细胞催化生产IAA,其中IAM途径的IAA产量较高,有较高的工业化应用前景。

Objective:Indole-3-acetic acid(IAA)was produced from tryptophan in the metabolically engineered E.coli MG1655 using whole-cell catalysis.Methods:Two novel IAA biosynthetic pathways,the indole-3-acetamide(IAM)pathway and the tryptamine(TRP)pathway,were constructed in E.coli MG1655.Results:The IAM pathway involves two enzymes,tryptophan-2-monooxygenase(IAAM)and amidase(AMI1).2 g/L tryptophan as a substrate was used by the constructed recombinant E.coli strain TPA-4.TPA-4 can produce 0.803 g/L of IAA;however,in the strain MPA-3 that was knocked out the gene tnaA which divert flux from tryptophan synthesis,the yield of IAA reached 1.43 g/L,an increase of 78% compared with the control.The second TRP pathway biosynthesis of IAA involves three enzymes:L-tryptophan decarboxylase(TDC),diamine oxidase(AOC1)and indole-3-acetaldehyde dehydrogenase(IAD1).The recombinant E.coli TPTA-2 that included the TRP pathway can only synthesize 13.0 mg/L IAA with 2 g/L tryptophan as substrate.In the strain MPTA-3 with disruption of tnaA gene,21.0 mg/L of IAA was finally produced,and the yield increased by 61.5%.Conclusion:It is the first report to realize production of IAA using the metabolically engineered E.coli through the IAM pathway and TRP pathway via whole-cell catalysis.IAA production from the IAM pathway is relatively higher,and it probably has an industrial application prospect.