Construction Of Synthetic Indigoidine Engineering Strain And Whole-cell Conversion

Indigoidine,originated from microorganisms,is non-toxic and harmless.It is produced by the oxidation of two molecules of glutamine.It belongs to blue pigment and has a very important commercial value in printing and dyeing,medical treatment,cosmetics and other industries.With the increase of demand,the chemical synthesis of blue pigment is widely used.However,the chemical synthesis caused great damage to the environment,so the microbial synthesis of blue pigment,a green and environmental-friendly method,has attracted the attention of scholars at home and abroad.At present,the most reported synthesis of blue pigment by microorganisms is produced with indole as substrate.This reaction will produce indirubin as well as other by-products,and the substrate is expensive.Moreover,the stability of indigo is obviously worse than that of indigoidine.Therefore,indigoidine with lower cost,high efficiency and high yield has become the focus of current research that can replace indigo in industry.In this study,phylogenetic trees were constructed from 24 homologs of indigoidine synthetase from different species,and 5 indigoidine synthetase genes were selected and cloned from different branches.The plasmid contained 5 indigoidine synthetase along with4'-phospho-pantoylthioethylamine transferase(PPTase)from Streptomyce were expressed in Escherichia coli.The results showed that ind1 B from Streptomyces lavendulae had the best ability to synthesize indigoidine when fermented at 30? for 24 h,and the yield reached14.4mg/L.Then,PPTase homologs from different species were also screened.The results showed that sfp from Bacillus subtilis had the highest yield of indigoidine production,which reached 24.3mg/L during 24 h fermentation.After optimizing of fermentation conditions,the production of indigoidine reached 354.9mg/L at 20? for 48 h with the engineered strain BW/pYB-ind1B-sfp as the starting strain.When 20 mM glutamine was added at the beginning,the yield of indigoidine increased to 368.4mg/L after 48 h fermentation.In order to increase the yield of indigoidine,the whole-cell conversion was used to synthesize indigoidine.The engineered strain BW?gln A/pYB-ind1B-sfp was constructed by knocking out gln A gene in genome,and glutamine was used as substrate for whole-cell conversion.The induction temperature(20?,25?,30?),whole-cell conversion temperature(20?,25?,30?),pH value of buffer(6.0,7.0,8.0,9.0),the addition amount of L-glutamine(5,10,15,20,40,60,80,100 mM)and the conversion biomass were optimized.As a result,when the induction temperature was 30?,the whole-cell conversion temperature was 20?,the pH of buffer was 9.0,the L-glutamine addition amount was40 mM,and the conversion biomass was 30 OD,the indigoidine production was achieved at2.02g/L with the glutamine conversion ratio of 40.70% after 24 hours conversion.Glutamate has a cost advantage over glutamine.In this experiment,glutamate was substituted for glutamine to produce indigoidine.Firstly,the glutamine synthetase(gluton)from Bacillus subtilis was cloned to obtain the engineered strain BW/pYB-ind1B-sfpgluton.After 24 h fermentation at 20?,the yield of indigoidine was 192mg/L,and with exogenous addition of 5mM glutamate,the indigoidine production increased slightly to202mg/L after 48 h fermentation.Then,the whole-cell conversion was used to produce indigoidine of the engineered strain BW/pYB-ind1B-sfp-gluton.When 50 mM glutamate,10 mM magnesium chloride,10 mM glucose and 50 mM ammonium chloride were added exogentically in the pH 8.0 buffer system and catalyzed at 20? for 22 h.The yield of indigoidine was 5.48g/L,and the conversion ratio of glutamate was up to 88.5%.Compared with the engineered strain BW?gln A/pYB-ind1B-sfp,the yield of indigoidine and the conversion ratio of glutamate increased by 162.4% and 47.8%,respectively.