Co-expression of P450 BM3 and glucose dehydrogenase by recombinant Escherichia coli and its application in an NADPH-dependent indigo production system.
P450 BM3 mutant can catalyze indole to indoxyl, and indoxyl can dimerize to form indigo. But the reaction catalyzed by P450 BM3 requires NADPH, as coenzyme regeneration is very important in this system. As we know, when glucose dehydrogenase oxidizes glucose to glucolactone, NADH or NADPH can be formed, which can contribute to NADPH regeneration in the reaction catalyzed by P450 BM3. In this paper, a recombinant Escherichia coli BL21 (pET28a (+)-P450 BM3-gdh0310) was constructed to co-express both P450 BM3 gene and glucose dehydrogenase (GDH) gene. To improve the expression level of P450 BM3 and GDH in E. coli and to avoid the complex and low-efficiency refolding operation in the purification procedure, the expression conditions were optimized. Under the optimized conditions, the maximum P450 BM3 and GDH activities amounted to 8173.13 and 0.045 U/mg protein, respectively. Then bioconversion of indole to indigo was carried out by adding indole and glucose to the culture after improved expression level was obtained under optimized conditions, and 2.9 mM (760.6 mg/L) indigo was formed with an initial indole concentration of 5 mM.
Lu
Yan
Y
Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, People's Republic of China.
Mei
Lehe
L
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005948:Glucose Dehydrogenases / Q000096:biosynthesis* / Q000235:genetics; D007203:Indigo Carmine; D007211:Indoles / Q000378:metabolism*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000235:genetics; D009249:NADP / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase; D011994:Recombinant Proteins / Q000096:biosynthesis; D013696:Temperature; D013997:Time Factors
D001426:Bacterial Proteins; D007211:Indoles; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D007203:Indigo Carmine; D006899:Mixed Function Oxygenases; D005948:Glucose Dehydrogenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases