| Sulfur is an important element in living organisms.Hydrogen sulfide(H2S)is a toxic gas with the smell of rotten eggs and is a common product of yeast metabolism.In mammals,H2S inhibits the function of the respiratory chain and thus affects the normal function of the animal's respiratory and central nervous systems.H2S in microorganisms can inhibit the function of cytochrome c,affecting their normal metabolism.In recent years,many organisms have been found to produce H2S endogenously,suggesting that H2S has a certain physiological role in the body.H2S is often referred as the third member of gas signal molecules after CO and NO.H2S plays a very important role in the body,regulating a variety of processes,including vasodilation,anti-blood pressure,anti-inflammation,anti-apoptosis,and metabolism regulation;abnormal H2S metabolism in the body can also cause atypical embryo transfer(commonly known as ectopic pregnancy).H2S can help Escherichia coli and Staphylococcus aureus resisting external oxidative stress.In S.cerevisiae,H2S is involved in the regulation of ultradian respiratory oscillation as an inter-bacterial signaling molecule.H2S may affect the function of a protein by modifying the sulfhydryl group of the protein,thereby regulating the related activity.It is generally believed that the regulatory role in the body is via reactive sulfur species(RSS),rather than H2S.The sulfur atom in RSS is a sulfane sulfur,which is more active and easier to modify the target.In mammalian cells,cystathionine beta synthase(CBS),cystathionine gamma lyase(CSE)and 3-mercaptopyruvate transferase(3-MST)can produce H2S.In microorganisms,in addition to CBS,CSE and 3-MST,sulfite reductase is also an important way to produce H2S.The most common sulfur oxidation enzymes are sulfide:quinone oxidoreductase(SQR),persulfide dioxygenase(PDO)and sulfur transferase(ST).SQR oxidizes H2S to polysulfide,which reacts with glutathione(GSH)to form glutathione persulfide(GSSH),which is then oxidized by PDO to sulfite.GSSH and polysulfides can react with sulfite to form thiosulfate.ST can accelerate the reaction of GSSH with sulfite to form thiosulfate,and can also catalyze the reaction of polysulfide with GSH to form GSSH.Human Tstd1 and yeast Rdl1 can also catalyze the degradation of thiosulfate.Human red blood cells can oxidize H2S by iron ions to ensure that the concentration of H2S in the blood is maintained at a suitable concentration.Catalase(CAT)and superoxide dismutase(SOD),as well as myoglobin,can also oxidize H2S to reduce the concentration of H2S in the body.In microorganisms,H2S is mainly oxidized by the SQR/PDO/ST system.It has recently been discovered that flavocytochrome c-sulfide dehydrogenases(FCSD)systems can also oxidize H2S.During the H2S oxidation process,SQR can produce a large amount of sulfane sulfur.SOD,CAT and myoglobin also produce sulfane in the process of oxidizing H2S.CBS,CSE and 3-MST can catalyze the production of cysteine persulfide from cysteine,which is an important way to produce cellular reactive sulfur species.It has recently been discovered that cysteinyl-tRNA synthetase can catalyze the production of cysteine persulfide from cysteine.The yield of H2S in different strains of S.cerevisiae varies widely,ranging from 0 to 290 ?g/L,which is much higher than that of humans.The main forms of sulfur in yeast cells are cysteine and GSH,and the concentration of GSH in yeast is up to 10 mM.Yeast can directly absorb cysteine or assimilate sulfate to synthesize cysteine for normal cellular metabolism.Yeast absorbs cysteine through Yct1,and the expression of YCT1 is significantly up-regulated when the extracellular sulfur source is insufficient.Sulfate enters yeast cells through Sul1 and Sul2,and it is reduced to H2S that is used to synthesize cysteine.Sul1 and SuI2 are redundant in function,and the absence of either one does not affect the assimilation of sulfate.When the extracellular sulfur source is insufficient,the mRNA levels of both are sharply increased.Sul1 and Sul2 can also sense the concentration of extracellular sulfate.They are the first transceptor to be discovered and activate the intracellular PKA pathway.After the sulfate enters the cell,it is activated by ATP sulfurylase(Met3)to form adenosine 5'-phosphosulfate(APS),which is phosphorylated by APS kinase(Metl4)to 3'-phosphoadenylsulfate(PAPS).Each of the above two steps comsumes one molecule of ATP.PAPS reductase(Met16)consumes 1 molecule of NADPH,reduces PAPS to sulfite,and then reduces to H2S by sulfite reductase(Met5 Met 10).There is no serine acetyltransferase activity in S.cerevisiae and no acetylserine is present.Therefore,cysteine synthetase(Met15)uses H2S and acetylhomoserine to form homocysteine.It is then converted to cystathionine by cystathionine beta synthase(Cys4)and then catalyzed by cystathionine gamma lyase(Cys3)to form cysteine.Thiosulfate is widely present in the environment and is a sulfur oxidation product of various sulfur-oxidizing bacteria.The concentration of soil in the upper layer of vegetation can reach 150 ?M.S.cerevisiae can also assimilate thiosulfate to synthesize cysteine.When S.cerevisiae is fermented with thiosulfate as a sulfur source,it can produce more biological materials,which means thiosulfate is a good sulfur resource.At present,little research has been done on H2S and sulfane sulfur in yeast,and it is unclear how yeast can assimilate thiosulfate.In this paper,S.cerevisiae was used as the research object to study the H2S metabolism and H2S function in yeast,assimilating thiosulfate to synthesize cysteine and the tolerance of yeast to thiosulfate(1)Metabolism and function of H2S in yeastThe function of H2S and sulfane in S.cerevisiae was studied by measuring the concentration changes of H2S and sulfane sulfur in S.cerevisiae by lead-acetate test strip and the sulfane sulfur probe(SSP4).The amount of H2S accumulated in the two strains of S.cerevisiae BY4741 and BY4742 was different.BY4741 is a mutant with cysteine synthetase(Met15)being deleted and it cannot use H2S to synthesize homocycteine.The yield of H2S in the sulfate-rich SD medium of the BY4741 strain was much higher than in the rich YPD medium.Moreover,after the sulfite reductase was knocked out,the H2S production of the yeast decreased significantly,indicating that the sulfate assimilation pathway in S.cerevisiae is a main route for the synthesis of H2S.Exogenously expressed SQR from S.pombe and PDO from human in BY4741,and the recombinant yeast did not accumulate H2S,indicating that different sulfur oxidation systems can be combined to function in yeast.H2S was produced mainly when the yeast is about to enter the stationary phase of the growth.The SSP4 probe detected that there was abundant sulfane sulfur in the yeast cells.And the intracellular sulfane sulfur concentration was high in the log phase and gradually decreased when the cell entered the stationary phase.The accumulation of H2S is inversely related to the concentration of intracellular sulfane sulfur,possibly due to the aboundance of intracellular reducing power in the late stage of yeast,which reduces sulfane sulfur to H2S and then releases it to the extracellular.After knocking out sulfite reductase(Met5),the tolerance of yeast to copper ions and zinc ions was significantly increased,and the Met5 deletion strain accumulated less copper ions in the cells.After exogenous expression of SQR and PDO,the tolerance of yeast to heavy metals also changed significantly.It can be seen that H2S and sulfane sulfur are closely related to the tolerance of yeast to heavy metals,and the specific mechanism still needs to be studied.(2)Sulfur transferase is involved in yeast assimilation of thiosulfateSince thiosulfate and sulfate have similar structures,some genetic manipulations of yeast were carried out with reference to the metabolism of sulfate,and the pathway of yeast assimilation of thiosulfate was found finally.Compared with assimilating sulfate,when yeast BY4742 uses thiosulfate as the sulfur source,the growth rate is faster,and the ethanol yield is higher,indicating that thiosulfate is a good sulfur source.Yeast absorbs thiosulfate by Sul1,Sul2 and Soal,and then catalyzes by intracellular sulfur transferase(Rhodanese,Rhod):Rdl1,Rdl2,Tum1 and Ych1,and uses GSH as a coenzyme to form sulfite and H2S.Sulfite is converted to H2S by sulfite reductase.Cysteine synthetase(Met 15)catalyzes the reaction of H2S with acetylhomoserine to form homocysteine,which is then catalyzed by cystathionine beta synthase(Cys4)and cystathionine gamma lyase(Cys3)to form cysteine.,Unlike CysM of Escherichia coli,Met15 cannot directly catalyze the reaction of thiosulfate with acetyl homoserine,and only H2S,a product of thiosulfate metabolism,can be used as a substrate.Sequence analysis revealed that the homologous protein of CysM is mainly distributed in bacteria and is not present in fungi.The homologous protein of Met15 is mainly distributed in fungi and is closer to the evolutionary relationship of E.coli CysK.This result suggests that the fungus may utilize thiosulfate by a metabolic pathway similar to yeast.For the microorganism that does not contain the CysM type cysteine synthetase,thiosulfate can be utilized with a cysteine synthase synthesis system similar to yeast.When the extracellular sulfur source of yeast is insufficient,the expression levels of SUL1 and SUL2 are significantly up-regulated;after the addition of thiosulfate,the mRNA level is drastically reduced and the transport capacity is rapidly decreased.After knocking out SUL1 and SUL2,the addition of thiosulfate still activates the PKA pathway of yeast,indicating that there are other sensory proteins that sense extracellular thiosulfate in yeast.Rhod can catalyze the reaction of thiosulfate and GSH,presumably through the ping-pong mechanism,accompanied by the formation of persulfides.In addition to GSH,Rdl2 can select cysteine,DTT and coenzyme A as sulfur acceptors,eventually producing H2S.Rdl1 plays a major role in yeast assimilation of thiosulfate.The ability to use thiosulfate after knockout is reduced.After knocking out other Rhod,the phenotype is more obvious.The ability to utilize thiosulfate:Wt>?rdl1>?rdl1?rdl2>?rdl1 ?rdl2 ?tum1 ?ych1.The ability of yeast to utilize thiosulfate can be restored when Rhod from other strains are expressed in knockout of four Rhod strains.The assimilation pathway of thiosulfate is highly coincident with the assimilation pathway of sulfate,except that additional Rhod participation is required.Since Rhod is widely distributed,the sulfate-assimilating organism can assimilate thiosulfate,all that is required is a Rhod.Thiosulfate contains a sulfane sulfur atom,which requires only one NADPH to produce H2S,and the sulfate to form H2S requires 2 ATPs and 4 NADPHs.Microorganisms may therefore prefer to use thiosulfate as a source of sulfur in the environment.Thiosulfate is widely present in the environment because heterotrophic bacteria can oxidize H2S to thiosulfate or thiosulfate to sulfate.Since thiosulfate consumes less energy than sulfate,thiosulfate may be a better choice in industrial fermentation.(3)The tolerance of yeast to thiosulfateCompared with sulfites,thiosulfate contains a sulfane sulfur atom,and sulfite could be referred to when looking for the target site of thiosulfate toxicity.Moreover,Rhod can degrade thiosulfate,and it is speculated that Rhod in yeast is related to yeast tolerance to thiosulfate.S.cerevisiae BY4742 could effectively utilize thiosulfate,but excess thiosulfate was toxic to yeast and was significantly more toxic than sulfite.The lower the pH of the medium,the lower the tolerance of the yeast to thiosulfate.Thiosulfate caused yeast cell lysis at low pH,killing yeast cells,but only inhibited yeast cell growth at high pH.In the SD medium,the final pH of the yeast culture solution could reach 2.1,and a large amount of thiosulfate was still present,and thiosulfate did not substantially degrade in the buffer of pH 3.4,indicating that it was thiosulfate acted directly on yeast cells at low pH.After deleting RDL1,the yeast was very sensitive to thiosulfate,and the double deletion of RDL1 and RDL2 made the mutant more sensitive;however,knocking out RDL2 did not affect the tolerance of yeast to thiosulfate,indicating that the toxicity of thiosulfate to yeast was mainly relieved by Rdl1.Rdll and Rdl2 contributed to the major sulfur transferase activity of yeast.It can be seen that yeast is resistant to thiosulfate by Rhod.Moreover,the processes of Rdll and Rdl2 in catalyzing thiosulfate were accompanied by the production of sulfane sulfur.Exogenous expression of Rhod from other species in the Ardl1 strain did not restore yeast tolerance to thiosulfate.Rdl1 was localized to mitochondria,and the addition of thiosulfate inhibited the oxygen consumption of yeast,so it was speculated that the target site of thiosulfate was mitochondria.Rdl1 and Rdl2 can catalyze both the reaction of thiosulfate with GSH to form GSSH and sulfite,and accelerate the reaction between GSSH and sulfite to form thiosulfate.Moreover,PspE and GlpE from E.coli can also catalyze this reversible reaction,indicating that Rhod can affect the degradation and synthesis of thiosulfate.Thiosulfate is widely distributed in the environment,which is an important sulfur metabolite and can also be used as a sulfur source.Therefore,Rhod may play a very important role in the sulfur cycle of the natural environment. |
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