Engineering Escherichia Coli Coculture System For De Novo Biosynthesis Of Betalains

Betalain is a kind of water-soluble and safe food pigment,with rich antioxidant and optical activity.It has potential application value in the field of health food and fluorescence imaging.This paper focuses on betalamic acid catalytic effect of the key cytochrome P450 enzyme BvCYP76AD1^LLand Bv CPR in Escherichia coli.Deviding betalains pathway into two cells,we design and construct a coculture system of engineered Escherichia coli for de novo synthesis of histidine-bextaxanthin.To study the effect of producing L-dopa,various N-terminal modifications are applied on the 22-amino acid-truncated BvCYP76AD1^LL and co-express with N-terminal truncated Bvt CPR in monocistron,bicistron and fusion protein strategy.The result shows hydrophilic N-modification of 2B1(MAKKTSS)significantly improved the production of L-Dopa,while the hydrophobic 17A modification only has slightly influence.The fusion protein linked with GST or GSTSSG shows no use for producing L-dopa.With 24h fermentation,co-expression of 2B1-Bvt CYP76AD1^L and Bvt CPR in monocistron strategy achieves a L-dopa titer of 28.56mg/L.Whereas,the bicistron performs best,a L-dopa titer of 40.41mg/L is achieved and shows an associative effects of N-terminal modification and CPR co-expression strategy.To construct a betalamic acid producing strain,a hydroxylase from Escherichia coli,hpa BC and a 4,5-DOPA dioxygenase extradiol from Mirabilis jalapa,Mj DODA are constructed into high or medium copy number vectors and screened various strains with high L-tyrosine production to obtain strain BTA6 with a best betalamic acid production from glucose.By the means of fementation supplement with 20 protein amino acids,10 amino acid-betaxanthins are detected.In the same way,two non-protein amino acids(PABA and L-dopa)and four amines(dopamine,tyramine,indoline and pyrrolidine)are tested and the corresponding betaxanthins are dectected.It is confirmed that BTA6 is capable of producing betalains with structural variety and different colors.To construct a strain producing L-histidine,with the help of genome engineering,site mutant(his G^E271K)to his G and replacement of his L with an artificial reading frame his L'release the feedback inhibition.Co-expressing his L'-his G^E271K to construct strain BHS3.With 24h fermentation,it can produce 422.60mg/L L-histidine.Coculture system of BHS3 and BTA6 enables de novo biosynthesis of histidine-betaxanthin.The coculture system tends to be stable after optimization.Ultimetly,the fermentation conducted in M9 containing yeast extract with an initial BTA6/BHS3 inoculation ratio of 12:1,OD₆₀₀of 3 without induction has de novo producing a histidine-betaxanthin titer of 287.69mg/L.The application of coculure strategy,betalains pathway is divided into two parts,which provides a promising new method for construct microbial coculuture factorty to effectively produce betalains.