Recycling system for manipulation of intracellular NADH availability

• US 7,256,016 B2
• Filed: 11/01/2002
• Issued: 08/14/2007
• Est. Priority Date: 11/02/2001
• Status: Active Grant

• Current Assignee: Rice University
• Original Assignee: Rice University
• Inventors: Bennett, George N., San, Ka-Yiu, Berrios-Rivera, Susana J.
• Primary Examiner(s): Woitach; Joseph
• Assistant Examiner(s): MITCHELL, LAURA MCGILLEM
• Application Number:
  US10/286,326
• Publication Number:
  US 20030175903A1
• Time in Patent Office: 1,747 Days
• Field of Search: None
• US Class Current: 435/69.1
• 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. An isolated engineered cell, comprising a recombinant NADH-recycling system having:

(a) a native cofactor-independent formate dehydrogenase genetically engineered to reduce its enzymatic activity as compared with the activity in a non-engineered cell, and

(b) an expression construct comprising an expressible gene encoding a heterologous NAD+-dependent formate dehydrogenase (FDH) that increases the enzymatic activity of NAD+-dependent FDH in the cell as compared with the activity of NAD+-dependent FDH in a non-engineered cell,

wherein said FDH converts NAD+ to NADH in the presence of formate.

2. The cell of claim 1, wherein said cofactor-independent formate dehydrogenase is engineered to eliminate its activity by deletion, inactivation, or mutation.

3. The cell of claim 2, wherein said heterologous FDH is selected from the group consisting of Candida boidinii FDH, Candida methylica FDH, Pseodomonas sp 101 FDH, Arabidopsis thaliana FDH, Staphylococcus aureus FDH, Saccharomyces bayanus FDH, Saccharomyces exiguus FDH, Saccharomyces servazzii FDH, Zygosaccharomyces rouxil FDH, Saccharomyces kluyveri FDH, Kluyveromyces thermotolerans FDH, Kluyveromyces lactis FDH, Kluyveromyces marxianus FDH, Pichia angusta FDH, Debaryomyces hansenii FDH, Pichia sorbitophila FDH, Candida tropicalis FDH, and Yarrowia lipolytica FDH.

4. The cell of claim 3, wherein said FDH comprises a sequence selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, and SEQ ID NO;

6.

5. An isolated engineered bacterial cell with a recombinant NADH-recycling system comprising:

(a) a native cofactor-independent formate dehydrogenase that has been inactivated by mutation or deletion, and

(b) an expression construct comprising an expressible gene encoding a heterologous NAD+-dependent formate dehydrogenase (FDH) that results in overexpression of the NAD+-dependent FDH as compared with the expression of NAD+-dependent FDH in a non-engineered cell, wherein said FDH converts NAD+ to NADH in the presence of formate.

6. The bacterial cell of claim 5, wherein said heterologous FDH is selected from the group consisting of Candida boidinii FDH, Candida methylica FDH, Pseodomonas sp 101 FDH, Arabidopsis thaliana FDH, Staphylococcus aureus FDH, Saccharomyces bayanus FDH, Saccharomyces exiguus FDH, Saccharomyces servazzii FDH, Zygosaccharomyces rouxii FDH, Saccharomyces kluyveri FDH, Kluyveromyces thermotolerans FDH, Kluyveromyces lactis FDH, Kluyveromyces marxianus FDH, Pichia angusta FDH, Debaryomyces hansenii FDH, Pichia sorbitophila FDH, Candida tropicalis FDH, and Yarrowia lipolytica FDH.

7. The bacterial cell of claim 6, wherein said FDH comprises a sequence selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, and SEQ ID NO;

6.

8. A bacterial cell comprising an expression vector encoding a heterologous NAD+-dependent formate dehydrogenase (FDH) and lacking cofactor-independent formate dehydrogenase activity, wherein said FDH converts NAD+ to NADH in the presence of formate.