Modification Of Sulfur Metabolic Pathway In Saccharomyces Cerevisiae And Its Application In The Treatment Of Heavy Metal Lead

Lead is a biotoxic heavy metal pollutant,and its massive emission seriously disturbs the ecological balance and endangers people’s health,which has become a global priority ecological and environmental problem and an important challenge for China’s green and sustainable development.So lead pollution has become a kind of heavy metal toxic pollution strictly controlled in China.Biology plays an important role in the treatment of lead pollution,among which the remediation technology of using sulfate-reducing bacteria to produce hydrogen sulfide to control lead pollution provides a new way for the treatment of heavy metal pollution,but the characteristics of anaerobic growth and slow growth of sulfate-reducing bacteria limit the efficiency of industrial application.The use of synthetic biology technology to construct new highly efficient hydrogen sulfide producing strains is the key to solve the bottleneck of "strains".Saccharomyces cerevisiae,as a model organism with simple culture and mature gene editing technology,has an alternative sulfate assimilation pathway that can reduce SO42-to H2S,which in turn can combine with various heavy metal ions to form sulfide precipitates for the purpose of heavy metal pollution control,and has important practical application potential.In this paper,the key genes and influence pathways of sulfate assimilation pathway in Saccharomyces cerevisiae were genetically modified to construct efficient hydrogen sulfide producing strains,and the physiological efficacy and application effects of the strain were studied.The main findings are as follows:1.Construction of the high hydrogen sulfide producing strains based on the modification of sulfur metabolic pathway and their growth phenotype and identification of hydrogen sulfide productionA knock-out strain CEN.PK2-1 C(MET17Δ)(short for MET17Δ)was constructed using MET17,a key gene in the sulfate assimilation pathway,as the target gene.The recombinant strain CEN.PK2-1C(MET17Δ,OEACS1)(short for MET17Δ&OEACS1)was obtained by overexpressing A CS1,a key gene for acetate catabolism,in the knock-out strain MET17Δ.It was found that the growth of the mutant strains did not differ significantly from WT under basal medium YPD conditions.In CSM medium culture,the mutant strain showed a more obvious growth lag in the middle and late stages of logarithmic growth when methionine(Met)was absent,indicating that methionine plays a key role in normal physiological metabolism of the mutant strains.And the differences in growth of the mutant strain were not significant in the case of cysteine(Cys)deficiency and complete of both amino acids.The H2S production study found that the mutant strains produced H2S significantly,and the gas production pattern was similar to the cell growth trend,showing a lag period-logarithmic period-stable period,with rapid gas production in the logarithmic period.And the H2S production was obvious in the medium with single deficiency of methionine,double deficiency of cysteine and methionine:MET17Δ was 491 ppm,594 ppm;MET17Δ&OEACS1 was 545 ppm,755 ppm,respectively.The results indicated that MET17,the key gene of sulfur metabolism,and Met,the downstream product of sulfur metabolic pathway,had significant effects on the release of H2S.Meanwhile,the overexpression of ACS1,a gene related to acetate catabolism,could help to effectively utilize acetic acid and promoted H2S release without affecting the normal growth of the strain.2.Exploration of optimal conditions for lead removal and identification of reaction products in high hydrogen sulfide producing engineering strainsLead acetate trihydrate was used as the substrate to simulate industrial lead wastewater environment,and the optimal conditions for the ability of MET17Δ to remove lead were investigated in terms of medium components,sulfur-containing amino acid concentration,cultivation time and substrate concentration.The most significant lead removal ability of MET17Δ was nearly 91.75%when the strain was cultured to the 8th h with 0.5 mM of lead acetate trihydrate as substrate in the CSM-M medium(50 mM Cys).Applying this condition to MET17Δ&OEACS1,its lead removal rate increased slightly,showing around 92.88%.The STEM analysis of the reaction product identified its component as PbS,which verified the principle of Pb-sedimentation reaction.And the TEM analysis observed a cluster-like distribution of PbS,and the size and shape were closely related to the concentration of the substrate,with a decrease in the concentration of the substrate resulting in an enlarged size(around 60-100 nm)and a regular shape(needle and rod).3.Proteomic analysis of the strains affecting the ability of lead removalProteomic analysis of MET17Δ and WT under CSM medium culture conditions with Met deficiency revealed that,in the case of MET17 mutation,on the one hand,the upstream protein expression of sulfur metabolism was increased to weaken the effect of downstream MET17 mutation,with various ion-binding and signal transduction-related proteins playing an active role in it,thus achieving a significant increase in MET17Δ H2S production;on the other hand,the downstream synthesis of sulfur-containing amino acids and their derivatives was blocked,and the expression of related proteins was significantly down-regulated,especially the low expression of thiamine and its compound synthesis-related proteins,which could not meet the demand of MET17Δ for amino acids and constituted a negative impact on its normal growth,resulting in the growth retardation phenomenon.Proteomic analysis of MET17Δ under unsupplemented and methionine-supplemented conditions revealed that,in the case of methionine compensation,on the one hand,the upstream pathway of sulfur metabolism was weakened and the conversion of SO42-to S2-was decreased,while the expression of thiamine synthesis proteins was increased,which steadily promoted thiamine biosynthesis,resisted sulfite interference and drove S2-metabolism to proceed normally,resulting in the reduction of MET17Δ hydrogen sulfide production;on the other hand,membrane transport and constituent proteins,as well as thiamine synthesis proteins related to nucleic acid synthesis and energy metabolism were significantly up-regulated to maintain membrane stability of the cell and restore the normal growth of MET17Δ.Meanwhile,the expression of ethanol metabolism-related proteins was elevated,which provided directions for solving MET17Δ acetic acid handling problems.4.Optimization of medium based on response surface methodology in the high hydrogen sulfide producing strain to enhance its lead removal abilityBased on the more economical YPD medium,the response surface methodology was applied to optimize each component of the medium.Single-factor experiments were conducted to identify glucose,Corn Steep Liquor(CSL),KH2PO4,MgSO4 and Cys as the best substances for each single factor.The effect of partial analysis and Box-Behnken experiment on the lead removal effect of MET17Δ was found:CSL>glucose>KH2PO4;the best medium component formulation:glucose 31.45 g/L,CSL 17.89 g/L,KH2PO4 0.97 g/L(MgSO4 1 g/L,Cys 50 mg/L),and the theoretical lead removal rate was 68.87%.The actual lead removal rate of 67.91%was obtained through experimental tests,which was close to the theoretical value and 1.94 times as that of the YPD medium before optimization,providing a scientific basis for the preparation of an economical industrial fermentation medium for yeast with high lead removal performance.When the optimized medium was applied to the actual treatment of lead-acid battery waste liquid,the lead removal rate of MET17Δ was 37.15%,which was higher than that of YPD medium before optimization by 13.99%,indicating that in the actual treatment of heavy metal wastewater represented by lead-acid battery waste liquid with complex composition,the lead removal capacity of the strain can be greatly improved by culture component optimization,which provided theoretical guidance for the application of the strain.In summary,this study obtained an engineered strain with high hydrogen sulfide production and optimized conditions by modifying key gene of sulfate assimilation pathway in Saccharomyces cerevisiae;combined with proteomic analysis to investigate the detailed mechanism of lead removal ability of the mutant strain in response to the mutation of key gene MET17 and the supplementation of sulfur metabolite Met;and optimized the medium formulation to improve the lead removal effect of the engineered strain and carried out a study on the treatment of actual lead-containing waste liquid.All the results provide scientific basis and theoretical guidance for the efficient bioremediation of heavy metal lead pollution based on the modification of sulfur metabolic pathway in Saccharomyces cerevisiae.