RESUMO
Based on the principle of the pathway engineering, a novel pathway of producing glycerol was built in E. coli. The gpd1 gene encoding glycerol 3-phosphate dehydrogenase and the hor2 gene encoding glycerol 3-phosphatase were cloned from Saccharomyces cerevisiae, respectively. The two genes were inserted into expression vector pSE380 together. A recombinant plasmid pSE-gpd1-hor2 containing polycistron was constructed under the control of the strong trc promoter. Then it was transformed into E. coli BL21. The result showed the recombinant microorganism GxB-gh could convert glucose to glycerol directly. And the recombinant microorganism GxB-gh was incubated to produce glycerol from D-glucose in the fermentor. The maximal concentration of glycerol was 46.67g/L at 26h. Conversion rate of glucose was 42.87%. The study is about "green" producing glycerol by recombinant microorganism and is also useful for further working in recombining microorganism of producing 1,3-propanediol.
Assuntos
Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Glicerolfosfato Desidrogenase/genética , Monoéster Fosfórico Hidrolases/genética , Saccharomyces cerevisiae/genética , Clonagem Molecular , Escherichia coli/genética , Fermentação , Proteínas Fúngicas/biossíntese , Engenharia Genética , Glicerol/metabolismo , Glicerolfosfato Desidrogenase/biossíntese , Monoéster Fosfórico Hidrolases/biossíntese , Saccharomyces cerevisiae/enzimologiaRESUMO
A new open reading frame in Thermobifida fusca sequenced genome was identified to encode a new trehalose synthase, annotated as "glycosidase" in the GenBank database, by bioinformatics searching and experimental validation. The gene had a length of 1830 bp with about 65% GC content and encoded for a new trehalose synthase with 610 amino acids and deduced molecular weight of 66 kD. The high GC content seemed not to affect its good expression in E. coli BL21 in which the target protein could account for as high as 15% of the total cell proteins. The recombinant enzyme showed its optimal activities at 25 degrees and pH 6.5 when it converted substrate maltose into trehalose. However it would divert a high proportion of its substrate into glucose when the temperature was increased to 37 degrees, or when the enzyme concentration was high Its activity was not inhibited by 5 mM heavy metals such as Cu2+, Mn2+, and Zn2+ but affected by high concentration of glucose. Blasting against the database indicated that amino acid sequence of this protein had maximal 69% homology with the known trehalose synthases, and two highly conserved segments of the protein sequence were identified and their possible linkage with functions was discussed.