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

Inhaltsverzeichnis (Table of Contents)

• Abstract
• Introduction
• Materials and Methods
  • Bacterial Strains and Plasmids
  • Media and Growth Conditions
  • Enzymatic Assays
  • DNA Techniques
  • Protein Analysis
  • Whole-Cell Biotransformation
  • Analytical Methods
• Results
  • Deletion of the genes yrfE and yjaD
  • Overexpression of pncB and nadE
  • Mannitol Biotransformation
  • Influence of Mannitol Transport on the Stability of Whole-Cell Biotransformation
  • Cloning and Characterization of a Putative Mannitol Transporter from Leuconostoc pseudomesenteroides
• Discussion

Zielsetzung und Themenschwerpunkte (Objectives and Key Themes)

This study aimed to increase the intracellular NAD(H) concentration in Escherichia coli to enhance the productivity and long-term stability of reductive whole-cell biotransformations. The research focused on manipulating the NAD metabolism within the cells, specifically by targeting NAD degradation and biosynthesis pathways.

• Enhancement of NAD(H) pool in Escherichia coli for biotransformation
• Impact of NAD turnover and concentration on whole-cell biotransformation productivity
• Investigation of NADH-pyrophosphatase activity and its role in NAD degradation
• Optimization of NAD biosynthesis through gene overexpression
• Study of the influence of mannitol transport on the stability of whole-cell biotransformation

Zusammenfassung der Kapitel (Chapter Summaries)

The introductory chapter provides an overview of the research area, highlighting the significance of NAD(H) in biotransformations and the challenges associated with maintaining high intracellular NAD levels. The Materials and Methods section details the experimental procedures employed, including bacterial strains, plasmids, growth conditions, enzymatic assays, DNA techniques, protein analysis, whole-cell biotransformation, and analytical methods.

The Results chapter presents the findings of the study, including the effects of deleting the genes yrfE and yjaD on NADH-pyrophosphatase activity, the impact of overexpressing pncB and nadE on the intracellular NAD concentration, and the influence of mannitol transport on the stability of whole-cell biotransformation. The chapter also discusses the cloning and characterization of a putative mannitol transporter from Leuconostoc pseudomesenteroides.

The Discussion chapter provides a comprehensive interpretation of the results, highlighting the significance of the findings in the context of improving whole-cell biotransformation processes. It discusses the potential applications of the engineered strains and suggests future research directions.

Schlüsselwörter (Keywords)

The keywords of this study include: whole-cell biotransformation, NAD(H), Escherichia coli, NADH-pyrophosphatase, NAD biosynthesis, mannitol, mannitol dehydrogenase, formate dehydrogenase, glucose facilitator, mannitol transporter, long-term stability, productivity.