Optimization Of Fermentation Process Of Ergothioneine By Methylobacterium Oryzae

Ergothioneine as a natural amino acid,which has the functions of antioxidant and scavenging free radicals and maintaining the balance of redox in vivo,is an important physiological active substance in vivo.Ergothioneine is widely used in food,medicine,cosmetics,health care and other fields because of its unique antioxidant properties.In particular,it has a broad prospective application in the treatment of neurological diseases,preservation of viscera and biomedical materials.Methylobacterium is a kind of widespread microbe which use methanol as their sole carbon and energy sources,can produce high value-added products from methanol,in the meanwhile relieve the pressure of?competing with food?by microbial cell factory.Previous studies have shown that ergothioneine can be synthesized by fermentation of Methylobacterium,however the current reported yield is very low.In this study,Methylobacterium oryzae was employed as an original strain.Using adaptive laboratory evolution to obtain methanol-resistant strains,raising a method of detecting ultraviolet absorption wavelength as a high-throughput screening strategy to efficiently screen the mutated strain with high yield by applying atmospheric and room temperature plasma(ARTP)mutagenesis.Moreover,the titer of EGT accumulated by M.oryzae was further enhanced by optimizing medium components and fermentation conditions.In addition,in order to further verify the M.oryzae?s ability of EGT production,we rebuilt the E.coli which contain Egt ABCDE from M.oryzae,the result shown that Egt ABCDE can well express in E.coli,then test the yield of the strain to confirm the gene synthesis ability,the main results are listed as following:1.An automagical and continuous adaptive laboratory evolution experiment(ALE)performed in a Microbial Microdroplet Chemostat(MMC),to generated a methanol-tolerant Methylobacterium oryzae strain.We determined the starting concentration for the ALE experiments was set at 30 g/L of methanol,and progressively increased to promote a continuous improvement in the tolerance fitness during the evolution process.The experiments ended at 45 g/L of methanol,for the growth of the strain was strongly inhibited even after in dozens of inoculations.Six droplets which have a good growth rate under 45 g/L of methanol were then plated to isolate individual clones.Finally,a methanol-tolerant strain(M.oryzae-33)was isolated with OD₆₀₀ were improved about 10 times than wild type and ergothioneine titer were 35.9%higher than the wild type under 40 g/L of methanol.2.An atmospheric room temperature plasma(ARTP)jet was employed to treat cell culture media and obtain ergothioneine high-producer.For the antioxidant properties of ergothioneine,50 mg/L of potassium permanganate was used as the screening pressure.A high-throughput screening method based the linear relationship of ergothioneine and OD₂₅₄ was established.The mutagenic strains was fermented by deep-well multiwell plate then detected OD₂₅₄ by microplate reader and selected dominant strain to shake flask fermentation.Finally selected a strain(M.oryzae-336)with 37.5%ergothioneine titer higher than the parent was obtained,and the genetic stability was confirmed by successive passages.3.According to the growth characteristics of M.oryzae and the synthesis characteristics of ergothioneine fermentation medium were investigated included carbon source,nitrogen source,metal ion,and microelement on the production of ergothioneine.Complex carbon sources,complex nitrogen source and iron ion was found to influence the synthesis of ergothioneine significantly.The optimal culture conditions for ergothioneine production were as follows:yeast extract 2.5 g/L,ammonium chloride 6 g/L,methanol 30 g/L,xylose 8 g/L,ferrous sulfate 4 mg/L,ferrous sulfate 4 mg/L and pyridoxal phosphate 6 mg/L respectively.As a result,ergothioneine titer were improved about 4.5 times than before the optimization and ergothioneine titer of M.oryzae-336 were 37.5%higher than the wild type.In a 5 L fermenter,the p H optimal control strategy was initial at 7.0,and maintained at 5.5 for the whole fermentation process.The intermittent feeding fermentation strategy was added to fermenter 10 g/L methanol when the fermenter methanol concentration below 10 g/L,in this way,ergothioneine titer were 35.9%higher than the batch fermentation.4.Ergothioneine synthetic genes Egt A-E was were cloned into the p ET21 vector and expressed in E.coli BL21.The expressed of Egt A-E was confirmed by gel electrophoresis map,LC-MS verified the presence of ergothioneine and the obtained recombinant strain can produce 20 mg/L of ergothioneine,which is higher than the original Methylobacterium oryzae.This result shows the superior catalysis capacity of Egt A-E for the synthesis of ergothioneine.