Optimization Of Fermentation Conditions For Production Of Arthrocolins By E.Coli

Xanthenes are promising class of compounds with a variety of applications in industrial,medical and food additives.Arthrocolins,a class of fluorescein compounds with an xanthene core,were identified in our laboratory by feeding Toluquinol to Escherichia coli,and are of great interest due to their good inhibitory activity against ovarian cancer cells A2780 with an IC₅₀ value of 0.5μM and their ability to restore the susceptibility of fluconazole-resistant Candida albicans to fluconazole-resistant Candida albicans with an IC₅₀ value of 1.41μM,and therefore has greater application value.However,due to its extremely low biosynthetic yield and even lower yield during fermentation in fermenters,resolution of its modular origin revealed that the phenyl and indole modules originated from Escherichia coli’s own mangiferin pathway.Therefore,in this study,based on the reports on improving the products of the mangiferous acid pathway in Escherichia coli,three aspects of the study were investigated,including optimising the fermentation conditions for Arthrocolins production in Escherichia coli,screening and construction of high yielding strains,and exploring the conditions affecting their yields,with the following main results1,Arthrocolins fermentation conditions optimization:YN broth（0.5%yeast extract,1%Na Cl）culture can improve the efficiency of Escherichia coli OP50 for the use of Toluquinol,thus improving the yield of Arthrocolins,its compared to the LB broth OP50production increased by 0.58;the most suitable p H is 6-8.Oxygen is essential for the synthesis of Arthrocolins.When bottle shaking fermentation,the incubation time should not exceed 72h,otherwise it will lead to a decrease in yield.The yield of Arthrocolins increased 1.82 with the addition of Toluquinol after 48h of incubation,followed by a decrease in yield.In fermenter fermentation,the yield of Arthrocolins increased under fermentation with the addition of precursors at OD₆₀₀=5 and batch replenishment.2.Screening and construction of single knockout strains:3901 mutant strains from the Keio collection,a library of single knockout strains of Escherichia col BW25113,were screened,among which 1310 single knockout strains with increased Arthrocolins production and 2587 with decreased production,indicating that most knockouts cause Arthrocolins production was reduced by most of the knockouts.KEGG enrichment analysis of the results showed that most of the knockout strains were enriched in four pathways,including ABC transport system,oxidative phosphorylation,arginine and proline metabolism andβ-lactam resistance,which may have affected their precursor transport and utilization.In contrast,most of the genes with reduced production in the knockout strains were enriched in seven pathways,including microbial metabolism,two-component system,sugar metabolism,glycolysis and sugar xenobiotic,flagellar assembly,starch and sucrose metabolism,and pentose phosphate pathway in different environments,suggesting that knockout of genes related to sugar metabolism all resulted in reduced production of Arthrocolins.Two single knockout strains,Δbcp andΔtrp D,were obtained by using the p Cas/p Target F dual plasmid knockout system to knock out the corresponding genes in Escherichia col OP50,and the Arthrocolins production of the mutant strains was increased 55.87 and 68.96,respectively,compared with the wild type OP50 strain under bottle shaking conditions.3.The conditions for Arthrocolins production were investigated:when the glucose content of the medium was 0.2%,the production of Arthrocolins was reduced by nearly30;when the glucose content reached 0.4%,Arthrocolins production was completely inhibited.Replacement of glucose with glycerol,maltose,lactose,D-sorbitol,sucrose,galactose,fructose and sucrose,respectively,revealed that all carbon sources,except sucrose,inhibited the production of Arthrocolins.The reasons for the inhibition of Arthrocolins production by carbon sources were investigated using glucose as an example.The redox potential of Escherichia col was found to increase upon the addition of glucose.The levels of the relevant synthetic precursors in the fermentation broth were examined and the levels of tyrosine,phenylalanine,tryptophan and Toluquinol were higher than those of the control without the addition of glucose for three days,while the levels of indole were lower than those of the control for three days and their production continued to be unrecoverable after supplementation with indole;Toluquinone was added as a precursor instead of Toluquinol to the medium supplemented with Toluquinone was added to the medium with glucose as a precursor,but no Arthrocolins were produced and no Toluquinone was detected in the fermentation broth,instead a large amount of Toluquinol was detected,suggesting that glucose did not inhibit Arthrocolins production by inhibiting its precursors,but probably by reducing the free radicals needed for the reaction,thus preventing Arthrocolins production.The correlation between the production of Arthrocolins and the redox potential of Escherichia col and redox substances was investigated and it was found that the production of Arthrocolins was not directly linked to changes in redox potential,but the addition of the free radical trapping agent GSH reduced the production of Arthrocolins,suggesting that that a decrease in free radicals could reduce the production of Arthrocolins.This result corresponds to the previous result.