Study On Carbon Metabolism In Extremely Acidophilic A Cidithiobacillus Ferrooxidans

Acidithiobacillus ferrooxidans is a chemolithoautrophic,Gram-negative,y-proteobacteriumthat thrives optimally at pH 2,butcan grow at pH 1 or lower,using energy from oxidation of iron-and sulfer-containing minerals for CO2 fixation and growth.It has important values in industrial application such as bioleaching and bio-desulfurization.Previous experiments showed that A.ferrooxidans can only use little amount of glucose added to the culture medium possibly for theincompleteness of its centrol carbon metabolism pathways.In this paper,some key enzyme encoding genes in the centrol carbon metabolism pathways of Acidithiobacillus ferrooxidanswerestudiedEarly experimental result confirmed the PFK activity ofAFE₁807 gene,but the activity was much less than that of PFKB or PFKA of E.coli K12.By replacing AFE₁807 with the pfkBfrom E.coli K12,the mutant RK12B showed better growth than the wild type,but it didn't ultilize more glucose than the wild type.So,by using the markerless mutagenesis system established in our lab,another mutant RK12A was successfully constructed byreplacing the AFE₁807 withpfkA,a higher activity PFK encoding gene from E.coli K12.A comparative study was performed betweenA.ferrooxidansRK12A,wild type,?1807 mutant and RK12B on growth characters,glucose ultilization and ferrous oxidation ability.The results showed that RK12A grew better than the other three strains and used more glucose both in the cultures with sulfur and ferrous iron as energy source,and got higher specific activity of PFK.No diferrence in ferrous oxidation ability was found between RK12A and the other three strains.Two genes,AFE₁667 and AFE₂053.were predicted to encode phosphoketolase in A.ferrooxidans.To verify their functions,the protein sequences were firstly blasted in the website NCBI.The results showed that both sequences owned the C-terminal conserved sequence of phosphoketolase.Then,the functional phosphoketolase was characterized by the expression of the two genesin E.coli Rosetta?DE3?and measurement of the specific activities..Next.AFE₁667 and AFE₂053 were cloned into the plasmid pJRD215.and transferred into A.ferrooxicidans by conjugation to construct two engineering strains of A.ferrooxidans?pJRD215-1667?and A.ferrooxidans?pJRD215-2053?.Finally,the two strains were compared with the control strain of A.ferrooxida ns?pJRD215?.Although the higher phosphoketolase activities ere detected with the two engineering strains,no better growth was detected than that of A.ferrooxidans?pjRD215?,and A.ferrooxidans?pJRD215-1667?showed even worse.However,when adding glucose in the cultures,all strains grew better than those without glucose.The transcriptional results showed that pgi?glk?zwf and pfkB were upregulated,suggesting that AFE₁667 and AFE 2053 could acceleratee the centrol carbon metabolism.And the functions of AFE₁667 and AFE₂053 were predicted to be F6PPK and F6PPK/X5PPK,respectively.The HMP pathway was intact in A.ferrooxidans with two key enzymes?PGD,ZWF?encoded by AFE₂024 and AFE 2025.The two genes were co-transcripted and cloned together into plasmid pJRD215 with their own promoter.An engineering strain A.ferrooxidans?pJRD215-2024-2025?was constructed by transferring the recombinant plasmid pJRD215-2024-2025 into A.ferrooxidans.A comparative study was performed between A.ferrocoxidans?pJRD215-2024-2025?and control strain A.ferrooxitdans?pJRD215?.'Ihe groxwth of A.ferrooxidans?pJRD215-2024-2025?showed worse than the control without glucose,but was better than the control when adding 1 g/L glucose.The transcriptional results showed that glk,zwfandwere upregulated.and pgi.xfpl were downregulated.It suggested that more carbon flux was disrtibuted into HMP by the overexpression of AFE₁667 and AFE-2053.A comparative study was performed with the adding of glucose,galactose,sucrose,maltose and fructose in the medium on the influence of growth of A.ferrooxidans.Sucrose showed most promotion on the growth of A.ferrooxidans among the tested sugars and different concentrations of sucrose were studied in details.The cell yield increased along with the enhancement of sucrose concentration when the concentration was under 4%,and 4%was the best both for cell yield and the consumption of sucrose.The transcriptional results revealed that 1 some genes involed in sugar degradation were upregulated,but pgi,glk and xfpl were downregulated,probably suggesting the inhibition of EMP by F-1,6-BP,a quick product from fructose.In addition,four recombinant plasmids pJRD215-Ptac-gusA were constructed,with four different start codons of ATG/TTG/GTG/CTG initializing the translation of gusA.The GusA specific activities were measured with A.ferrooxidans carrying different recombinant plasmid.The highest GusA specific activity was obtained with initial codons of ATG,next the TTG,GTG,and the least was CTG,which was the same in E.coli.It provides a possible way to control gene expression in A.ferrooxidans besides the promoters.