The aim of this work was to limit the intracellular NAD turnover and to increase the intracellular NAD concentration in Escherichia coli in order to construct a strain characterized by higher productivity and improved long-term stability in the reductive whole-cell biotransformation. To this end, first the genes yrfE and yjaD were deleted, which code for the NADH-pyrophosphatases that probably play a part in intracellular NAD degradation. After deletion of these genes, the specific activity of the NADH-pyrophosphatase in the raw extract was reduced by 70%. By overexpressing the genes pncB and nadE from the NAD biosynthesis it was possible to double the intracellular NAD concentration. The joint overexpression of the two genes increased the NAD pool by a factor of seven.
A recombinant strain of E. coli for obtaining mannitol from fructose was the reference system for studying whether the mutations in the NAD metabolism influenced the long-term stability of whole-cell transformations. Here, fructose was reduced to mannitol by a mannitol dehydrogenase from Leuconostoc pseudomesenteroides. The cofactor NADH was regenerated by a formate dehydrogenase from Mycobacterium vaccae N10. The uptake of fructose was made possible by a glucose facilitator from Zymomonas mobilis. In the present study, it was foand that during the biotransformation the cells lost NAD(H) via a permeabilized cell membrane. The cause of cell permeabilization was foand to be an accumulation of mannitol in the cell, which led to bursting of the cell due to increased osmotic pressure. By cloning a putative mannitol transport gene from L. pseudomesenteroides, it was possible to increase long-term productivity of the recombinant E. coli, so that after three days of biotransformation the mannitol yield was increased by 20%. Biochemical characterization revealed that the putative mannitol transporter is a secondary carrier specific for fructose.
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
1. Reductive Biocatalysis with whole Cells
The interest in enzymatic substrate conversion in industrial processes rises rapidly nowadays (Faber, 2000; Koeller and Wong, 2001; Schmid, et al., 2001; Zhao, et al., 2002). Oxido reductases which catalyze the asymmetric reduction of carbonyl groups to alcohols and amines or the oxygenation of carbon-hydrogen-bondages are in the focus of chemical industries (Li et al., 2002; Stewart, 2001).
The employment of whole cells instead of isolated enzymes is advantageous since a laborious purification of enzymes is avoided and the product can be separated by the product without ease (Buchholz and Gröger, 2006). Often enzymes are more stable in their natural environment. Finally, cells possess a natural cofactor pool; employing isolated enzymes often requires the addition of cofactors increasing the process costs. Most oxido reductases require pyridine nucleotides as cofactors. By using isolated enzymes a stoichiometric addition of NAD(P)H would not be reasonable for economic considerations (van der Donk and Zhao, 2003). That is why a cofactor consuming reaction is always coupled to a cofactor regenerating reaction in industrial processes. Either this redox reaction can be catalyzed by one enzyme alone or the cofactor regenerating reaction has to be catalyzed by a second enzyme (Buchholz and Gröger, 2006).
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.
- Quote paper
- Florian Heuser (Author), 2007, Increasing the Productivity of Whole Cell Biotransformation by Enhancing the intracellular NAD(H) Concentration, Munich, GRIN Verlag, https://www.grin.com/document/137750
