Improvement of P450(BM-3) whole-cell biocatalysis by integrating heterologous cofactor regeneration combining glucose facilitator and dehydrogenase in E. coli.
Escherichia coli BL21, expressing a quintuple mutant of P450(BM-3), oxyfunctionalizes alpha-pinene in an NADPH-dependent reaction to alpha-pinene oxide, verbenol, and myrtenol. We optimized the whole-cell biocatalyst by integrating a recombinant intracellular NADPH regeneration system through co-expression of a glucose facilitator from Zymomonas mobilis for uptake of unphosphorylated glucose and a NADP(+)-dependent glucose dehydrogenase from Bacillus megaterium that oxidizes glucose to gluconolactone. The engineered strain showed a nine times higher initial alpha-pinene oxide formation rate corresponding to a sixfold higher yield of 20 mg g(-1) cell dry weight after 1.5 h. The initial total product formation rate was 1,000 micromol h(-1) micromol(-1) P450 leading to a total of 32 mg oxidized products per gram cell of dry weight after 1.5 h. The physiological functioning of the heterologous cofactor regeneration system was illustrated by a sevenfold increased alpha-pinene oxide yield in the presence of glucose compared to glucose-free conditions.
Biochemical Engineering Group, DECHEMA e.V., Karl-Winnacker-Institut, Theodor-Heuss-Allee 25, 60486, Frankfurt, Germany.
Hendrik
H
Schewe
Bjoern-Arne
BA
Kaup
Jens
J
Schrader
D001410:Bacillus megaterium / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D000081008:Bicyclic Monoterpenes; D018533:Biomass; D001711:Biotransformation / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D042843:Glucose 1-Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D039821:Monoterpenes / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D013729:Terpenes / Q000378:metabolism*; D013997:Time Factors; D017355:Zymomonas / Q000201:enzymology / Q000235:genetics
D001426:Bacterial Proteins; D000081008:Bicyclic Monoterpenes; D039821:Monoterpenes; D013729:Terpenes; C086512:glucose facilitator protein, Zymomonas; C053997:2-pinene oxide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042843:Glucose 1-Dehydrogenase; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D005947:Glucose; C005451:alpha-pinene