| Indole-3-acetic acid(IAA),also known as auxin,is a plant endogenous hormone that is ubiquitously present in plants and microorganisms and is one of the most widely distributed indole derivatives in the environment.IAA involves almost all aspects of plant growth and development,cell division and elongation,and physiological metabolism,such as promoting the differentiation and formation of plant organs;promoting gamete,embryogenesis,seedling growth,and flower development;stress alleviation and response;maintenance of plant phototropism,geotropism,and polar transport,etc.Not only that,but ghrelin can also regulate by interacting with other hormones.Five tryptophan-dependent IAA biosynthesis pathways have been reported,these pathways use L-tryptophan(L-trp)as a substrate,and these pathways pass through indole-3-acetamide(IAM),indole-3-pyruvic acid(IPA),tryptamine(TAM),indole-3-acetaldoxime(IAOx)and indole-3-acetaldehyde(IAAl)synthesis of IAA.It has been reported that many strains can synthesize IAA through the IPA pathway,and the yield is high.Although the IAM pathway synthetic pathway is relatively more concise,it has been neglected due to the low yield of IAA.Tryptophan can be converted into IAA through a two-step reaction.First,tryptophan undergoes an oxidation reaction under the action of tryptophan-2-monooxygenase(TMO)to generate indole-3-acetamide(IAM),and then generate IAA under the action of amidase(Amidase).The studies on the heterologous expression of other synthetic pathways in Escherichia coli are rarely reported.In this stduy,the IAM pathway was recombinantly expressed in Escherichia coli by means of metabolic engineering,by knocking out some genes related to the pathway that competes with tryptophan accumulation and strengthening the genes related to the intracellular ATP production pathway,enhancing the cellular TCA cycle pathway,strategies to achieve de novo synthesis of IAA in Escherichia coli strains.In this study,we first enhanced the use of glucose to generate and accumulate L-Trp in engineered bacteria cells.Using E.coli MG1655 as the starting strain,the gene ppc encoding phosphoenolpyruvate carboxylase was knocked out by CRISPR/Cas9 geneediting technology;the tryptophanase encoded by tna A can catalyze the degradation of LTrp to generate pyruvate and indole and ammonia to deplete L-Trp and reduce its accumulation in cells,therefore knocking out the gene tna A encoding tryptophanase reduces the effect of substrate depletion on the yield of IAA;since L-serine deaminase II can catalyze L-Serine produces acetone,and the synthesis pathway of L-serine competes with L-Trp synthesis,so the sda B gene encoding L-serine deaminase II was knocked out;the transcription of L-Trp biosynthesis and transport genes in E.coli is mainly regulated by Tyr R and Regulation of Trp R,as a transcriptional repressor,Trp R affects the synthesis of L-Trp in E.coli,so knockout of the trp R gene further promotes the accumulation of L-Trp in cells.The results show that these modifications to the metabolic pathway of E.coli MG1655 can significantly improve the synthesis of L-Trp in recombinant engineered bacteria cells.The biosynthetic metabolism of cells is closely related to the consumption and balance of energy.The intracellular synthesis of L-Trp in E.coli is a process that requires a large amount of ATP.If the enhanced supply of intracellular ATP can be achieved,it is possible to increase the synthesis of L-Trp and further enhance the production of IAA.In order to increase the ATP concentration in E.coli cells,this study screened three different sources of phosphoenolpyruvate carboxykinase(PEPCK),namely the pepck gene from Ricinus communis,Actinomyces succinates,and Escherichia coli.Enhance the expression of pepck on the genome to improve the energy metabolism of cells and obtain a pepck strain with higher energy metabolism to achieve the purpose of enhancing IAA production in Escherichia coli.The three genes were inserted into the sda B locus by CRISPR/Cas9 technology,and three strains that overexpressed pepck on the genome were constructed.The contents of ATP in the three genotype strains were measured respectively,and finally,an MEP strain was obtained with the highest ATP content in cells.Knock out the gene where it is in the above-mentioned strains knocking out ppc,trp R,and tna A,and then use the strain with the highest intracellular ATP measured among these three strains as the chassis cell,and continue to put high-yield tryptophan and high-yield IAA plasmids After transferring into this strain of chassis cells for fermentation,and optimizing the culture conditions,the yield of IAA reached 134 mg/L.Finally,by strengthening the TCA cycle,knocking out the gene adh E encoding the fusion of acetaldehyde-Co A dehydrogenase and iron-dependent alcohol dehydrogenase aldehyde/alcohol dehydrogenase,inserting the gene icd encoding isocitrate dehydrogenase,knocking out the gene encoding lemon The acid dehydrogenase kinase gene ace K was inserted into the encoding citrate synthase gene glt A,and three strains were constructed,namely MGΔadh E::icd,MGΔace K::glt A,MGΔadh E::icdΔace K::glt A,and the plasmids with high production of tryptophan and high production of IAA were constructed.It was transformed into MGΔadh E::icd,MGΔace K::glt A,MGΔadh E::icdΔace K::glt A strains for fermentation,and it was found that the MGΔadh E::icdΔace K::glt A double-knockout strain had a stronger ability to produce IAA than the single-knockout strain.The maximum yield of IAA was 128mg/L,which was 50% higher than that of the control group.Finally,these optimal genetic modifications were combined to obtain a strain of MNP-M-IAA.By optimizing the fermentation conditions,substrate concentration,p H,culture temperature,etc.,the IAA yield was further increased to 284 mg/L,which was the same as the control group MGIAA.3.19 times the M-IAA.In this paper,by using metabolic engineering methods and synthetic biology techniques,Escherichia coli expresses the exogenous IAM pathway to produce IAA.The constructed MNP-M-IAA strain has good industrial application prospects.It has laid a theoretical and practical foundation,and more importantly,provided new ideas and directions for the efficient use of microbial cell factories to produce amino acid derivatives. |
