Recycling system for manipulation of intracellular NADH availability

• US 7,709,261 B2
• Filed: 07/04/2007
• Issued: 05/04/2010
• Est. Priority Date: 11/02/2001
• Status: Active Grant

• Current Assignee: Rice University
• Original Assignee: Rice University
• Inventors: Berrios-Rivera, Susan J., Bennett, George N., San, Ka-Yiu
• Primary Examiner(s): Qian; Celine X
• Application Number:
  US11/773,408
• Publication Number:
  US 20080009034A1
• Time in Patent Office: 1,035 Days
• Field of Search: None
• US Class Current: 435/455
• CPC Class Codes: C12N 9/0036 acting on NADH or NADPH (1.6)
• CPC Class Codes: C12P 1/00 Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
• CPC Class Codes: C12P 7/625 Polyesters of hydroxy carboxylic acids

Abstract

The present invention describes a novel recombinant NADH recycling system that is used as a process for producing reduced compounds. In a specific embodiment, the reduced compounds include ethanol, succinate, lactate, a vitamin, a pharmaceutical and a biodegraded organic molecule. The NADH recycling system effects metabolic flux of reductive pathways in aerobic and anaerobic environments.

Claims

1. A method of producing an NADH dependent product comprising:

a) culturing an engineered cell comprising a recombinant NADH-recycling system comprising a native cofactor-independent formate dehydrogenase genetically engineered to reduce its activity as compared with a wild type cell, and an expression construct comprising an expressible gene encoding a heterologous NAD+-dependent formate dehydrogenase (FDH) that increases the enzymatiac activity of NAD+-dependent FDH activity in said cell as compared with a wild type cell, wherein said FDH converts NAD+ to NADH in the presence of formate;

b) inducing production of an NADH dependent product; and

c) isolating the NADH dependent product.

2. The method of claim 1, wherein said NADH dependent product is selected from the group consisting of an amino acid, organic acid, hydroxy acid, ester, alcohol, vitamin, antimicrobial, pharmaceutical, polypeptide, biopolymer, mineral, or biodegraded organic compound.

3. The method of claim 1, wherein said engineered cell is cultured with formate.

4. The method of claim 1, wherein said engineered cell is cultured with at least about 100 mM formate.

5. The method of claim 1, wherein said engineered cell is cultured under aerobic conditions.

6. The method of claim 1, wherein said engineered cell is cultured under anaerobic conditions.