Increasing the Productivity of Whole Cell Biotransformation by Enhancing the intracellular NAD(H) Concentration

Table of Contents

Introduction

1. Reductive Biocatalysis with whole Cells

2. Stability of nicotinamide dinucleotide cofactors

3. NAD+ Biosynthesis and Recycling

4. Aims of this Thesis

Material and Methods

1. Organisms and Plasmids

2. Chemicals and Reagents

3. Growth Media, Buffers and Additives

4. Gene Expression in E. coli

5. DNA Isolation

6. Quantitation of DNA Concentrations

7. DNA Restriction and Ligation

8. Agarose Gel Electrophoresis

9. Isolation of E. coli Membrane Proteins

10. SDS Polyacrylamide Gel Electrophoresis

11. Identification of Proteins with MALDI-TOF Mass Spectrometry

12. Polymerase Chain Reaction (PCR)

13. DNA Sequencing

14. Inactivation of NADH pyrophosphatase gene yrfE in E. coli BL21(DE3)

15. Inactivation of NADH Pyrophosphatase Gene yjaD in E. coli BL21(DE3) and Genomic Integration of pncB

16. Competent cells and DNA transfer

17. Solubilisation of Cells

18. Quantitation of Enzyme Activity and Protein Concentration

19. Quantitation of intracellular NAD(H) Concentrations

20. Quantitation of Fructose and Mannitol via HPLC

21. Quantitation of Ketones and Chiral Alcohols with Gas Chromatography

22. Determination of Intracellular Mannitol Concentration

23. Determination of intracellular pH of E. coli

24. Whole Cell Biotransformation

25. Quantitation of Fructose and Mannitol Transport

Results

1. Enhancement and Stabilization of the NAD(H) Pool in E.coli

1.1 NAD(H) Degradation in E. coli

1.2 Deletion of NADH Pyrophosphatase Genes yrfE and yjaD

1.3 Overexpression of genes within the NAD(H) recycling pathway

1.4 Genomic Integration of pncB

1.5 Overexpression of Anorganic Pyrophosphatase ppa

1.6 Overexpression of Genes nadB and nadA from the NAD(H) de novo Synthesis

2. Influence of an increased NAD(P)(H) Pool on the Productivity of Whole Cell Biotransformations

2.1 Mannitol Productivity of NADH Pyrophosphatase Mutant Strains

2.2 MannitolPproductivity with Plasmid bound Overexpression of pncB

2.3 Mannitol Productivity after Genomic Integration of pncB

2.4 Mannitol Productivity by Overexpression of Genes pncB and nadE

2.5 Case study 2: Influence of NAD(P)(H) Pool Size on the Reaction Rate of Methyl Acetoacetate to (R)-Methyl-3-Hydroxybutanoate

2.6 Case Study 3: Influence of the NAD(H) Pool Size on Productivity in a Biotransformation System of Formate Dehydrogenase and Alcohol Dehydrogenase

3. Reasons for the Decrease of intracellular NAD(H) Concentration in Fructose Mannitol Biotransformation

3.1 Detection of extracellular NAD(H) during Biotransformation

3.2 Influence of Formate on Stability of the NAD(H) Pool

3.3 Participation of Formate Dehydrogenase in Cell Permeabilization

3.4 Influence of CO2 Production and Expression of glf on Cell Integrity

3.5 Influence of Mannitol Formation on Cell Permeabilization

3.6 Cloning of a putative Mannitol Permease from Leuconostoc pseudomesenteroides

3.7 Determination of intracellular Mannitol Concentrations in Cells Expressing ORF2

3.8 Cloning and examination of a putative permease from Leuconostoc pseudomesenteroides

3.9 Biotransformation with expression of fupL

Discussion

1. NAD(H) Turnover in Resting Cells of Escherichia coli

2. Overexpression of Genes from the NAD(H) Synthesis Pathway

3. Influence of an enhanced NAD(P)(H) Pool on the Reductive Biotransformation

4. Characterization of FupL and its beneficial effect on biotransformation

Research Objectives and Topics

This work aims to mitigate intracellular NAD(H) depletion in Escherichia coli whole-cell biotransformations to enhance both enzymatic productivity and long-term process stability. The research investigates enzymatic causes for NAD(H) degradation, implements genetic modifications to stabilize the cofactor pool via deletion of pyrophosphatase genes and overexpression of biosynthetic pathway genes, and characterizes transport proteins to improve substrate availability.

• Engineering of E. coli strains to minimize intracellular NAD(H) turnover.
• Overexpression of genes within the NAD(H) biosynthetic and recycling pathways.
• Evaluation of increased NAD(H) cofactor pools on whole-cell biotransformation productivity.
• Analysis of cell membrane integrity and the role of sugar transporters in cofactor loss.
• Characterization of a putative mannitol/fructose permease for biotransformation optimization.

Excerpt from the Book

Summary of Chapters

Introduction: Provides an overview of reductive whole-cell biocatalysis, the importance of cofactor stability, and the specific challenges of NAD(H) metabolism in E. coli.

Material and Methods: Details the strains, plasmids, molecular cloning techniques, enzymatic assays, and biotransformation protocols used throughout the study.

Results: Presents findings on the stabilization of the NAD(H) pool through genetic engineering, the impact of these changes on biotransformation productivity, and the investigation of factors causing cell permeabilization.

Discussion: Interprets the experimental results, specifically focusing on the limited success of pyrophosphatase deletion compared to biosynthetic pathway overexpression and the discovery of FupL as a fructose transporter.

Keywords

Escherichia coli, whole-cell biotransformation, NAD(H) metabolism, cofactor regeneration, metabolic engineering, D-mannitol, formate dehydrogenase, NADH pyrophosphatase, pncB, gene overexpression, membrane permeabilization, fructose transport, FupL, bioprocess stability.

Frequently Asked Questions

What is the primary goal of this research?

The main objective is to stabilize the intracellular NAD(H) cofactor pool in recombinant Escherichia coli to prevent productivity loss in whole-cell biotransformation processes.

Which scientific methods were primarily utilized?

The work employs metabolic engineering, including gene deletion and overexpression, analytical HPLC and GC for metabolite quantification, SDS-PAGE for protein expression analysis, and kinetic assays for enzyme activity.

What role does the NAD(H) pool play in biotransformations?

The NAD(H) pool serves as a critical cofactor for oxidoreductases used in these biotransformations; its depletion directly correlates with a reduction in the long-term productivity of the cell strain.

What were the findings regarding NADH pyrophosphatases?

While yrfE and yjaD were suspected of causing NAD(H) degradation, their deletion did not significantly stabilize the NAD(H) pool or increase mannitol productivity, suggesting they are not the primary drivers of turnover.

What is the significance of the gene pncB?

Overexpression of pncB was found to be effective in increasing the intracellular NAD(H) concentration, acting as a key "switch" in regulating the cofactor pool size.

What characterizes the identified FupL protein?

FupL, initially identified as a putative mannitol permease from Leuconostoc pseudomesenteroides, was characterized as a secondary fructose transporter that enhances fructose uptake in E. coli.

How does formate concentration affect cell integrity?

High concentrations of formate correlate with a faster loss of intracellular NAD(H), suggesting that formate, likely acting as a weak acid, influences membrane stability in conjunction with other process parameters.

Why was the strain Tuner(DE3) used in certain experiments?

Tuner(DE3) is a lacY mutant, which allows for finer, concentration-dependent tuning of gene expression via the addition of IPTG, preventing potential toxicity from overexpressing heterologous membrane proteins.