Bacterial P450-catalyzed polyketide hydroxylation on a microfluidic platform.

The incorporation of a multicomponent, cofactor-dependant P450 into a microfluidic biochip is demonstrated. The PikC hydroxylase Streptomyces venezuelae was incorporated into a PDMS-based microfluidic channel. The enzyme was immobilized to Ni-NTA agarose beads via in situ attachment following the addition of the beads to the microchannel. The enzyme loading was approximately 6 microg per mg of beads resulting in a microchannel loading of 10.7 mg/mL. This high enzyme loading enabled the rapid hydroxylation of the macrolide YC-17 to methymycin and neomethymycin in about equal amounts with a conversion of >90% at a flow rate of 70 nL/min. This high reactivity allowed rapid hydroxylation reactions to be performed with short residence times, which is critical for complex enzymes with limited inherent stability.

[1]  H. Yamada.  Natural products of commercial potential as medicines. , 1991, Current opinion in biotechnology.

[2]  S. Jacobson,et al.  Integrated microdevice for DNA restriction fragment analysis. , 1996, Analytical chemistry.

[3]  D. Cane,et al.  Erythromycin biosynthesis: exploiting the catalytic versatility of the modular polyketide synthase. , 1996, Bioorganic & medicinal chemistry.

[4]  S. Jacobson,et al.  Microchip device for performing enzyme assays. , 1997, Analytical chemistry.

[5]  J. Kealey,et al.  Characterization of the macrolide P-450 hydroxylase from Streptomyces venezuelae which converts narbomycin to picromycin. , 1998, Biochemistry.

[6]  B Wilkinson,et al.  Engineering broader specificity into an antibiotic-producing polyketide synthase. , 1998, Science.

[7]  D. Sherman,et al.  Hydroxylation of macrolactones YC-17 and narbomycin is mediated by the pikC-encoded cytochrome P450 in Streptomyces venezuelae. , 1998, Chemistry & biology.

[8]  C. Walsh,et al.  Harnessing the biosynthetic code: combinations, permutations, and mutations. , 1998, Science.

[9]  A Manz,et al.  Chemical amplification: continuous-flow PCR on a chip. , 1998, Science.

[10]  M. Neuman,et al.  Amperometric Microcell for Enzyme Activity Measurements , 1998 .

[11]  M. Cima,et al.  A controlled-release microchip , 1999, Nature.

[12]  G. Whitesides,et al.  Fabrication of microfluidic systems in poly(dimethylsiloxane) , 2000, Electrophoresis.

[13]  D. Clark,et al.  Stable sol-gel microstructured and microfluidic networks for protein patterning. , 2001, Biotechnology and bioengineering.

[14]  F. Regnier,et al.  Channel-specific coatings on microfabricated chips. , 2001, Journal of chromatography. A.

[15]  A. Srinivasan,et al.  Microfluidic peroxidase biochip for polyphenol synthesis. , 2003, Biotechnology and bioengineering.

[16]  Alexander Steinbüchel,et al.  Metabolic engineering and pathway construction for biotechnological production of relevant polyhydroxyalkanoates in microorganisms , 2003 .

[17]  A. Srinivasan,et al.  Multienzyme catalysis in microfluidic biochips. , 2003, Biotechnology and bioengineering.