In vitro metabolic engineering on microscale devices

• US 7,427,497 B2
• Filed: 11/01/2002
• Issued: 09/23/2008
• Est. Priority Date: 11/01/2001
• Status: Active Grant

• Current Assignee: Rensselaer Polytechnic Institute, Regents of the University of California (University of California), University of Minnesota
• Original Assignee: DARPA, Rensselaer Polytechnic Institute, Regents of the University of California (University of California)
• Inventors: Dordick, Jonathan S., Clark, Douglas S., Sherman, David H., Srinivasan, Aravind, Kim, Jungbae
• Primary Examiner(s): Redding; David A
• Application Number:
  US10/287,440
• Publication Number:
  US 20040062687A1
• Time in Patent Office: 2,153 Days
• Field of Search: None
• US Class Current: 435/175
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 19/0093 Microreactors, e.g. miniaturised or microfabricated reactors laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00364 Pipettes
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00533 essentially rectangular
• CPC Class Codes: B01J 2219/00536 in the shape of disks
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00691 using robots
• CPC Class Codes: B01J 2219/0072 Organic compounds
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B01J 2219/00743 Cells
• CPC Class Codes: B01J 2219/00835 Comprising catalytically active material
• CPC Class Codes: B01J 2219/00837 comprising coatings other than catalytically active coatings
• CPC Class Codes: B01J 2219/0086 Dimensions of the flow channels
• CPC Class Codes: B01L 2300/069 Absorbents; Gels to retain a fluid
• CPC Class Codes: B01L 2300/0819 : Microarrays; Biochips
• CPC Class Codes: B01L 2300/0829 : Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 3/5027 : by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/5085 : for multiple samples, e.g. microtitration plates

Abstract

Disclosed herein is a microfluidics device that can be used to prepare natural products and their analogs. The device comprises the enzymes of a biosynthetic pathway immobilized thereon and a means for sequentially directing a starting material and each ensuing reaction product to the enzymes of the biosynthetic pathway in the order corresponding to the steps of the biosynthetic pathway. The device can thus be used to prepare the natural product using the natural starting material of the biosynthetic pathway or analogs of the natural product using an unnatural starting material. Alternatively, artificial pathways can be created by immobilizing an appropriate selection of enzymes on the device in an order whereby each subsequent enzyme can catalyze a reaction with the product of the prior enzyme. Novel chemical entities can be prepared from these artificial pathways.

Claims

1. An apparatus for conducting a plurality of microscale reactions, comprising

a. a solid support comprising a first and a second reaction site, wherein each site comprises a distinct immobilized biocatalyst;

b. a micro-directing element to direct an intermediate composition from the first site to the second site,

wherein the biocatalyst at the first reaction site and the biocatalyst at the second reaction site are each a functional catalytic unit from sequential steps in a natural biosynthetic pathway, the apparatus further comprising a micro-directing element to direct a composition from a source to at least one said reaction site, and

wherein

1) the apparatus comprises n additional reaction sites;

2) n is an integer from 1 to 50;

3) each reaction site comprises a distinct immobilized biocatalyst; and

4) the apparatus additionally comprises one or more micro-directing elements to sequentially direct a composition from the second reaction site to each of the n additional reaction sites.

2. The apparatus of claim 1, wherein each said biocatalyst is a catalytic antibody, an enzyme, an enzyme domain, a multi-enzyme complex, a catalytic peptide or a catalytic RNA molecule.

3. The apparatus of claim 1, wherein the apparatus additionally comprises at least one reaction site which comprises a catalyst of non-biological origin.

4. The apparatus of claim 1, wherein:

a. the biocatalysts at the first reaction site, the second reaction site and each of the n additional reaction sites are each a functional catalytic unit from the steps of a natural biosynthetic pathway; and

b. the apparatus additionally comprises one or more micro-directing elements to direct a composition sequentially to each of the reaction sites in the order of the sequence of the catalytic units in the biosynthetic pathway.

5. The apparatus of claim 4, wherein said biosynthetic pathway is the picromycin polyketide synthase pathway, the erythromycin polyketide synthase pathway, the nonribosomal peptide synthetase pathway, or the β

-lactam nonribosomal peptide synthetase pathway.

6. The apparatus of claim 1, wherein each biocatalyst is immobilized by a material comprising a polyacrylamide, a polyacrylate, a sugar-substituted polyacrylate, a polyvinyl alcohol, a polyvinylene, or a polyvinyl silicate.

7. The apparatus of claim 6, wherein each said material comprises a polyacrylate substituted with sucrose, lactose, mannose, an alkylmannoside, glucose, an alkylglucoside, galactose, an alkylgalactoside, fructose, an alkylfructoside, or trehalose.

8. The apparatus of claim 7, wherein said micro-directing elements comprise a channel or a conduit between each reaction site in the sequence.

9. The apparatus of claim 8, additionally comprising a device to propel a fluid between the reaction sites using electroosmotic force.

10. The apparatus of claim 9, further comprising a device to control the temperature of at least a portion of the apparatus.

11. The apparatus of claim 10, further comprising a micro-directing element to direct at least one said intermediate composition from one said reaction site to a detector.

12. The apparatus of claim 11, further comprising a detector, wherein said detector comprises an aspiration probe, a laser desorption probe, an ion beam desorption probe, a gas desorption probe, a liquid desorption probe, a contact probe, an optical spectrometer, a microscope, an imager, a mass spectrometer, a chromatography apparatus, an electrochemical detector, a particle detector, a radiation detector or a magnetic resonance spectrometer.

13. A microfluidics chip, comprising

a. a solid support comprising a first and second reaction site, wherein

i. the first and second reaction sites each comprise a distinct biocatalyst that is a functional catalytic unit of a natural biosynthetic pathway;

ii. each said biocatalyst is immobilized by a polyacrylate substituted with sucrose, lactose, mannose, an alkylmannoside, glucose, an alkylglucoside, galactose, an alkylgalactoside, fructose, an alkylfructoside, or trehalose;

b. microfluidics elements to direct a substrate composition from a source to the first reactive composition, and microfluidics elements to direct an intermediate composition from the first reaction site to the second reaction site, wherein the biocatalysts at the first and second sites are from sequential steps in the natural biosynthetic pathway.