Engineered Escherichia Coli Nissle 1917 With Urate Oxidase And An Oxygen-recycling System For Hyperuricaemia Treatment

Uirc acid(UA)is produced from purine metabolism and is not further degraded in humans.Normal serum UA level in humans is between 90 μM and 420 μM.Elevated UA in human bodies leads to hyperuricaemia,which can induce gout,chronic kidney diseases,cardiovascular diseases,and diabetes.With the risen consumption of highpurine diets,the incidences of hyperuricaemia are gradually increasing in many countries,and hyperuricaemia has become the second-largest metabolic disease after diabetes.Hyperuricaemia could be treated either by reducing UA production or promoting UA excretion through the kidney,which are two major factors leading to the development of hyperuricaemia.There are four clinical treatment options.First,diet control is used to reduce the intake of purines.Second,xanthine oxidase inhibitors such as febuxostat reduce UA production.Third,UA absorption in the renal tubules is inhibited by drugs such as benzbromarone.Fourth,UA is degraded in the blood by supplementing exogenous urate oxidase(UOX).These clinic therapies have limitations.Restricting diet can significantly reduce life quality of patients,and it must be adhered to for a long time to achieve the therapeutic effect.The use of medicines often has strong side effects.New strategies have been developed to treat hyperuricaemia.Several natural products have been tested to treat hyperuricaemia.One-third of the produced UA in humans is excreted through the intestinal tract and is further metabolized by the gut microbiota.Gut microbiota may be another way to treat hyperuricaemia.Several isolated Lactobacillus strains may degrade purines and UA in the gut to ameliorate hyperuricaemia in mice.Therefore,it is feasible to treat hyperuricemia by gut microbes.Most of studies on the treatment of hyperuricemia by gut microbes focus on the isolation with a few reports on utilization of engineered strains.The engineered strains have many advantages over the isolated wild strains,including the ability to direct therapeutics to particular niches,ultimately allowing for higher doses with lower systemic effects.In addition,engineered microbes can be used to explore the interactions between the microbiota,immune system and diet,opening up new therapeutic strategies.Relevant studies have proved that engineered probiotics can be used to treat metabolic diseases such as hyperacidemia and phenylketonuria.It is worth to explore whether hyperuricemia can be alleviated by engineered probiotics.UOX is a key enzyme for UA degradation.Several UOX enzymes obtained from microorganisms have been used as the clinical diagnostic reagent to detect UA and to therapeutically relieve excessive urate accumulation.The PucL and PucM from Bacillus subtilis are extensively studied,and they catalyze a 3-step UA degradation pathway.These enzymes are used to construct engineered bacteria for uric acid degradation.Because UOX requires oxygen for catalysis,its activity may be reduced in the oxygen-limiting gut.To overcome oxygen shortage in the gut,the bacterial hemoglobin(VHb)from Vitreoscilla sp.is used to improve bacterial oxygen.The E.coli catalase KatG efficiently removes H2O2 produced by UOX and replenish oxygen.Moreover,YgfU from E.coli is a proton-dependent transporter of UA,and it is used to improve the efficiency of UA degradation.E.coli Nissle 1917(EcN)is often used as an engineered host.EcN has been used to treat inflammatory bowel disease and irritable bowel syndrome for over a century.Because of its safety record and genetic malleability,it has been engineered to deliver therapeutic payloads to treat several disorders,such as bacterial infection and metabolic disorders.Recently,it has been developed as a vector to produce and deliver anticancer agents into tumors by intravenous injection.These applications prove that injecting a certain amount of EcN into the blood is relatively safe.We constructed an engineered EcN with PucL,PucM,KatG,VHb,and YgfU for UA degradation.The therapeutic effect of the engineered EcN strain was investigated in mice.The details are as follows:(1)The UriC domain of PucL(PucLT)catalyzes the first step in the UA degradation.We chose PucLT initially to construct engineered strain.Different plasmids and promoters were tested for the expression of PucLT.EcN::pMCS2-Ptrc-pucLT with the gene encoding PucLT under the control of the Ptrc promoter in pBBR1MCS-2 offered the highest activity in the cell extract.However,similar results were not obtained in whole cell assays.Then UOX was further optimized.We constructed EcN::pMCS2-Ptrc-pucLM that overexpressed the intact PucL and PucM,and we generated the two site-direct mutations in PucL to obtain EcN::pMCS2-Ptrc-pucLMM.UA degradation efficiencies by the cell extracts of these two strains significantly improved in comparison with EcN::pMCS2-Ptrc-pucLT.The Vmax of PucLMM increased about 3 times.However,the Km of PucLMM increased about 3 times too.The degradation rates of EcN::pMCS2Ptrc-pucLM and EcN::pMCS2-Ptrc-pucLMM whole cells were significantly faster than that of EcN::pMCS2-Ptrc-pucLT.The UA degradation ability by the whole cells of EcN::pMCS2-Ptrc-pucLMM was also weaker than EcN::pMCS2-Ptrc-pucLM when UA concentrations were low.Subsequently,the uric acid transporter YgfU was introduced.When YgfU was coexpressed with PucLMM,the UA degradation rate of EcN::pMCS2-Ptrc-pucLMM-ygfU whole cells was increased at high UA concentrations,but YgfU did not have any effect at low uric acid concentrations.The katG gene and vhb gene were further introduced into EcN::pMCS2-Ptr c-pucLMM-ygfU to construct EcN::pMCS2-Ptrc-pucLMM-vhb-ygfU-katG.EcN::p MCS2-Ptrc-pucLMM-vhb-ygfU-katG degraded UA at a slightly slower rate than EcN::pMCS2-Ptrc-pucLMM-ygfU did under normal oxygen conditions.The intes tinal environment was simulated by controlling dissolved oxygen.EcN::pMCS2Ptrc-pucLMM-vhb-ygfU-katG completed uric acid degradation when dissolved ox ygen was limited,but EcN::pMCS2-Ptrc-pucLMM-ygfU did not.(2)To determine whether the engineered EcN strain degraded uric acid in the mice gut,uric acid was injected into the gastrointestinal tract of mice by intragastric administration.At the beginning,uric acid accumulated in the stomach and duodenum of mice;after 30 minutes,uric acid mainly accumulated in the jejunum,while uric acid accumulated in the jejunum was degraded after intragastric administration of the engineered EcN strain.It is proved that the engineered strain could directly digest UA efficiently in gut.Four reported methods to establish hyperuricaemia in mice models were tested.The serum UA reached 2.2-fold higher than the control group only when the 7d-1PO method was used.Mice induced by the 7d-1PO method were induced by oral administration of adenine(75 mg/kg)for 7 consecutive days,and potassium oxonate at 250 mg/kg was intraperitoneally injected 1 hour after the last adenine administration.The induced EcN cells that were administered intragastrically did not ameliorate hyperuricaemia,nor did it affect the UA concentration in the gut.Further inspection revealed that the UA concentration in the 7d-1PO model was was 45.1 μM,still 10-fold lower than that in humans.Degradation ability of the engineered EcN strain under different UA concentrations were measured,at 48.6 μM UA,UA degradation ability of the engineered EcN strain was significantly lower than that at 1095.8 μM UA.And potassium oxonate,an inhibitor of UOX,further decreased the UA degradation rate by the engineered EcN cells.These two factors suggest that the traditional hyperuricemia mice model is not suitable for testing engineered EcN strains and a suitable hyperuricemia mice model needs to be developed.(3)To increase serum UA in mice to the levels comparable to that in humans,a UA solution was injected into the blood vessel at 70 mg/kg of the testing mice.Serum UA was promoted up to～1 mM,The concentration can be maintained for about 1 hour,and gradually decreased over time.This method could construct acute hyperuricemia mice,and it was referred to as the UA-injection method.This method was used to test the function of the engineered strain.The therapeutic effect of the engineered strain by intragastric administration by was measured.UA level of mice group with orally administration of total 1.0×101l CFU of EcN::pMCS2-Ptrc-pucLMM-vhb-ygfU-katG was sharply decreased.This indicated that the engineered EcN strain by intragastric administration could effectively alleviate hyperuricemia in UA-injection mice.Whether the injection of EcN::pucLMM-vhb-ygfU-katG cells into the blood could treat hyperuricaemia was tested.The induced cells of 1.0×107 CFU/mL,1.0×108 CFU/mL and 1.0×109 CFU/mL were added in a buffer,commercial mice serum,human serum and blood,respectively,and the background or additional added UA were rapidly degraded by the engineered strain.With the increase of bacterial dosage,the degradation rate also increased.When the EcN::pucLMM-vhb-ygfU-katG cells(1.0×108 CFU/mL)were added in the human blood of youth and older gruops,UA was rapidly degraded by the strain in both groups.UA was repeatedly added in the whole blood samples,and the UA degradation rates were not altered.The induced cells at 5.0×108 CFU and 1.0×109 CFU were released into the UA-injection hyperuricaemia mice at the same time.The serum UA level decreased faster in the treated group than the untreated group.More engineered EcN offered faster UA degradation.However,1.0×109 CFU cells were not suitable for hyperuricaemia treatment,as injection of high doses of the EcN cells increased mouse mortality.Mice with injection of 5.0 × 108 CFU cells were in good condition.5.0×108 CFU EcN cells were injected into the vein of the mice for 8 hours,the UA level decreased much faster in the co-injection group.When a UA solution was injected into the blood vessel at 5.7 mg/kg of the testing mice,the serum UA increased to 80 μM at the initial point and was quickly metabolized to normal level in 10 min.The engineered EcN strains by intravenous administration or intragastric administration also alleviate the increase of UA at the start point.Then we compared therapeutic effects of the EcN strains with or without vhb and katG gene for UA-injection hyperuricemia mice.The EcN strain with vhb and katG also helped the bacterium to degrade UA and remove H2O2 faster than the EcN strain without vhb and katG.These results further indicated that VHb and KatG helped the strain maintain oxygen concentration and reduce ROS.In summary,a recombinant EcN strain was constructed to efficiently degrade UA under both normal oxygen and hypoxic conditions.The bacterium degrades UA in the gut and blood of mice that was induced with hyperuricaemia by intravenously injecting UA.The finding supports the application of bacteria in the gut to degrade UA for hyperuricaemia treatment.Applying EcN directly in the blood is a new idea to treat metabolic disorders,but more optimizations and efforts are required to achieve the goal.