Metabolic Engineering Of Escherichia Coli For Production Of Three Auxins

The large-scale use of chemical fertilizers has become more and more harmful to biodiversity and environmental pollution,which is not in line with the environmentally-friendly and sustainable development advocated today.For this reason,various agricultural microorganisms are being actively developed at domestic and overseas to reduce dependence on chemical fertilizers.The auxin-producing microorganisms can effectively promote plant growth and reduce chemical fertilizer use.Indole-3-acetic acid is the main natural auxin in plants.At present,researchers at home and abroad have evaluated the ability of many microorganisms to synthesize indoleacetic acid based on tryptophan as a substrate.Although many microorganisms have been found to be able to efficiently synthesize indole acetic acid using tryptophan,since free tryptophan is rare in nature,it is necessary to add tryptophan to organic fertilizer in the process of using these microorganisms to regulate plant growth,which is greatly increases the cost and makes it difficult to achieve economic feasibility.Taking into account that cellulose and its derived monosaccharide-glucose exist widely in nature,it can be more feasible for the microorganism to directly use glucose to efficiently synthesize auxin.In this paper,it have carried out the work of constructing a complete pathway for de novo synthesis of three auxins in Eescherichia coli,including the following:Earlier studies reported that E.coli aldehyde dehydrogenase Ald H can accept multiple aldehydes as substrates.This study first carried out the expression and purification of E.coli aldehyde dehydrogenase Ald H.In vitro enzyme activity experiments confirmed the enzyme can effectively catalyze the conversion of indole-3-acetaldehyde to indole-3-acetic acid（IAA）.It was confirmed that Ald H chose NAD⁺as a cofactor when indole-3-acetaldehyde was oxidized.The Km and Kcat values of the enzyme determined were 3.78 mmol/L and 8.96 s^-1,respectively.Based on the confirmation that Ald H can effectively catalyze the indole-3-acetaldehyde to indole-3-acetic acid,we further constructed a de novo synthetic pathway of IAA in E.coli RARE strains which can accumulate aromatic aldehydes.First,0.5 g/L L-tryptophan was added exogenously and Saccharomyces cerevisiae aminotransferase ARO8,S.cerevisiae decarboxylase KDC and E.coli aldehyde dehydrogenase Ald H were expressed.Feasibility,the results showed the recombinant strain produced IAA as high as 387±19.8 mg/L in 24 h,and its molar conversion was 0.90 mol/mol.Then by knocking out the L-tryptophan transcription repression gene trp R,over-expressing Trp ABCD,aro G^fbr,ser A^fbr,and anthranilate synthase trp E^fbr,the cells were able to accumulate L-tryptophan by glucose metabolism.Finally,further co-expression of ARO8,KDC and Ald H will link the upstream and downstream parts to establish a complete IAA biosynthetic pathway.Recombinant strains were cultured in shake flasks for 24 h and produced about 744±26.9 mg/L of IAA from 20 g/L glucose.In addition to indole-3-acetic acid,phenylacetic acid（PAA）and p-hydroxyphenylacetic acid（4-HPA）are also common auxins,which play a role in regulating plant growth in many plants.To this end,we further constructed de novo synthesis pathways of PAA and 4-HPA.By expressing aro G^fbr and Tyr A^fbr,the upstream pathway regulates the accumulation of L-tyrosine in glucose through shikimic acid metabolism;then,co-expression of AR08,KDC and Ald H in the downstream pathway converts L-tyrosine to 4-HPA.The final output of the shake flask is about 991±48.8 mg/L.Similarly,replacing Tyr A^fbr with phe A^fbr,a gene that synthesizes L-phenylalanine,and then co-expressing AR08,KDC,and Ald H can convert L-phenylalanine to PAA,and eventually produce up to 1217±45.2 mg/L of PAA.Studies on the de novo synthesis of three auxins,IAA,PAA,and 4-HPA,were conducted through glucose.The synthetic pathway is divided into two parts,upstream and downstream,using aromatic amino acids as intermediates.The formation of the final product can be changed by regulating upstream aromatic amino acid synthesis genes.And,the shake flask output of the three auxins is ideal.Therefore,the construction of a path for E.coli to synthesize auxin opens the way for the future development and utilization of agricultural microorganisms to synthesize auxin and promote plant growth.