Construction Of A Melatonin-producing Engineered Probiotic Nissle 1917

Sleep is essential for animals.Sleep deprivation leads to accumulation of reactive oxygen species in the intestine.Reactive oxygen species are one of the inducing factors of intestinal diseases such as enteritis and tumors,which seriously endanger human health.Melatonin is a strong antioxidant that can effectively remove reactive oxygen species.The intestine is an important place for the synthesis and storage of melatonin.Sleep deprivation leads to a decrease in the concentration of melatonin in the intestine,so it is impossible to prevent the accumulation of reactive oxygen species.Therefore,sleep-deprived populations need to supplement melatonin.Melatonin is also a hormone,which can lead to inattention,drowsiness and lower body temperature,and is not conducive to maintaining the working state of night shift people.Because the accumulation of reactive oxygen species is a slow process,the dosage of melatonin should be consistent with the production rate of reactive oxygen species,otherwise excessive melatonin will enter the blood,causing side effects.Engineered probiotics as functional molecular carriers have the advantages of targeting,continuity and controllability,and are expected to become a new means to maintain intestinal health in night shift populations without affecting their working status.The number of reported microorganisms with melatonin production capacity is limited and there are biological safety issues.The engineered probiotic chassis bacteria represented by Nissle 1917(EcN)has not yet been reported to produce melatonin.This study proposes to develop new genetically engineered bacteria using serotonin to synthesize melatonin through genetic engineering and protein engineering with EcN as the chassis bacteria.The main contents and research results of this study are as follows:(1)Construction and whole-cell biocatalysis of single expression Psmf and OsCOMT genetically engineered EcN.Animals usually use tryptophan as a substrate to synthesize melatonin through four cascade reactions.The second metabolic intermediate product serotonin in this process can be produced and secreted in large quantities in the intestine.In order to minimize the synthesis pathway of melatonin,we chose Psmf and OsCOMT to construct a metabolic pathway that utilizes serotonin to synthesize melatonin.The proteins encoded by Psmf(from Streptomyces griseofuscus)and OsCOMT(from Oryza sativa)are key enzymes in the last two steps of this cascade.Psmf and OsCOMT catalyze the reactions that convert serotonin into N-acetylserotonin and convert N-acetylserotonin into melatonin,respectively.In order to make the melatonin synthesis pathway the shortest,we chose Psmf and OsCOMT to construct a metabolic pathway for synthesizing melatonin using serotonin.Psmf(derived from Streptomyces griseofuscus Psmf gene cluster)and OsCOMT(derived from Oryza sativa,encoding O-methyltransferase)encode proteins that are key enzymes in the last two steps of the cascade reaction.In order to explore the exogenous expression of the two genes in EcN and the activity after expression,we constructed the p BAD-Psmf and p BAD-OsCOMT expression vectors,which were separately introduced into EcN for induced expression and whole-cell biocatalysis separately and compared with wild-type EcN.The results show that EcN could express proteins encoded by these two genes.With 1000 μg/m L serotonin as the substrate for whole-cell catalysis of engineered EcN expressing the Psmf gene alone,the concentration of N-acetylserotonin in the product reached 807.63 ± 25.46μg/m L as detected by HPLC.With 100 μg/m L N-acetylserotonin as the substrate for wholecell catalysis of engineered EcN expressing the OsCOMT gene alone,the concentration of melatonin in the product reached 3.80 ± 0.25 μg/m L as detected by HPLC.No relevant substances were detected in the whole-cell biocatalysis products of wild-type EcN,indicating that wild-type EcN does not have the ability to convert serotonin to Nacetylserotonin or N-acetylserotonin to melatonin and the proteins eroded by Psmf and OsCOMT are active when they were expressed in EcN.(2)Construction and whole cell biocatalysis study of co-expressing Psmf and OsCOMT genetically engineered EcN.In order to construct a metabolic pathway for synthesizing melatonin using serotonin as a substrate in EcN,we constructed a co-expression system of Psmf and OsCOMT to investigate the combined catalytic activity after co-expression of Psmf and OsCOMT.The co-expression system of Psmf and OsCOMT was obtained by constructing their single transcriptional unit co-expression vector.Since the start order of downstream genes of the single transcriptional unit co-expression vector is related to their expression,we constructed p BAD-Psmf-OsCOMT and p BAD-OsCOMT-Psmf,which were separately introduced into EcN.And whole-cell biocatalysis was carried out to explore the co-catalytic activity of Psmf and OsCOMT.EcN carrying these two co-expression vectors can express Psmf and OsCOMT,indicating that the co-expression vector was successfully constructed;However,the difference in their expression was not significant.Whole-cell biocatalysis can synthesize 645.6 ± 13.4 μg/m L N-acetylserotonin and 7.8 ± 0.15 μg/m L melatonin under the condition of 1000 μg/m L serotonin.Since the yield of melatonin in the whole-cell catalysts of EcN with p BAD-OsCOMT-Psmf was higher than that of EcN with p BAD-Psmf-OsCOMT,EcN with p BAD-OsCOMT-Psmf was used in subsequent experiments.(3)Optimization study of co-expression Psmf and OsCOMT genetically engineered EcN whole cell biocatalysis efficiency of melatonin production.OsCOMT is the ratelimiting enzyme in the cascade enzymatic production of melatonin.Compared with Psmf,its activity is far from meeting the needs of cascade enzymatic reactions.In order to further increase the yield of melatonin in whole-cell biocatalysis,we performed sequence alignment and structural analysis of the reported COMT,and used protein engineering methods to modify OsCOMT to increase its ability to convert N-acetylserotonin,thereby increasing the yield of co-expression Psmf and mutant OsCOMT.Genetically engineered EcN whole cell biocatalysis produces melatonin.Compared with genetically engineered EcN without optimization,the final concentration of melatonin increased by about 1 time to 14.7 ± 0.6μg/m L with 1000 μg/m L serotonin as the substrate for whole cell biocatalysis.In summary,this study constructed single expression and co-expression Psmf and OsCOMT genetically engineered EcN.The co-expression genetically engineered EcN can synthesize melatonin in vitro with serotonin as the substrate.By modifying OsCOMT,the whole cell biocatalysis efficiency of co-expressing Psmf and OsCOMT genetically engineered EcN that convert serotonin to melatonin was further improved.This study may provide a new means to explore the reduction of intestinal damage caused by sleep deprivation.