Main description:

This volume outlines key steps associated with the design, building, and testing of synthetic metabolic pathways for optimal cell factory performance and robustness, and illustrates how data-driven learning from these steps can be used for rational cost-effective engineering of cell factories with improved performance. Chapters are divided into four sections focusing on the four steps of the iterative design-build-test-learn cycle related to modern cell factory engineering. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and practical, Synthetic Metabolic Pathways: Methods and Protocols aims to ensure successful results in the further study of this vital field.

Contents:

Part I: Parts and Models for Designing Synthetic Metabolic Pathways

1. Parts characterization for tunable protein expression

Michael S. Klausen and Morten O. A. Sommer

2. Enzyme Nicotinamide Cofactor Specificity Reversal Guided by Automated Structural Analysis and Library Design

Jackson K.B. Cahn, Sabine Brinkmann-Chen, and Frances H. Arnold

3. Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability

Ida Lauritsen, Virginia Martinez, Carlotta Ronda, Alex Toftgaard Nielsen, and Morten H. H. Norholm

4. An Automated Pipeline for Engineering Many-Enzyme Pathways: Computational Sequence Design, Pathway Expression-Flux Mapping, and Scalable Pathway Optimization

Sean M. Halper, Daniel P. Cetnar, and Howard M. Salis

5. Computational Approaches on Stoichiometric and Kinetic Modeling for Efficient Strain Design

Mohammad Mazharul Islam and Rajib Saha

6. Extended Metabolic Space Modeling

Pablo Carbonell, Baudoin Delepine, and Jean-Loup Faulon

7. Computational Methods to Assess the Production Potential of Bio-based Chemicals

Miguel A. Campodonico, Sumesh Sukumara, Adam M. Feist, and Markus J. Herrgard

Part II: Building and Refactoring Synthetic Metabolic Pathways

8. Multiplex Genome Editing in Escherichia coli

Sheila Ingemann Jensen and Alex Toftgaard Nielsen

9. Designing and Implementing Algorithmic DNA Assembly Pipelines for Multi-gene Systems

Szu-Yi Hsu and Michael J. Smanski

10. An adaptive Laboratory Evolution Method to Accelerate Autotrophic Metabolism

Tian Zhang and Pier-Luc Tremblay

11. CRISPR-Cas9 toolkit for Actinomycete Genome Editing

Yaojun Tong, Helene Lunde Robertsen, Kai Blin, Tilmann Weber, and Sang Yup Lee

12. Assembly and Multiplex Genome Integration of Metabolic Pathways in Yeast using CasEMBLR

Tadas Jakociunas, Emil D. Jensen, Michael K. Jensen, and Jay D. Keasling

13 A modified Gibson assembly method for cloning large DNA fragments with high GC contents

Lei Li, Weihong Jiang, and Yinhua Lu

14 Coupling Yeast Golden Gate and VEGAS for efficient assembly of the violacein pathway in Saccharomyces cerevisiae

James Chuang, Jef D. Boeke, and Leslie A. Mitchell

Part III: Screening and Selection of Synthetic Metabolic Pathways

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15 Multi-capillary Column Ion Mobility Spectrometry of Volatile Metabolites For Phenotyping Of Microorganisms

Christoph Halbfeld, Joerg Ingo Baumbach, Lars M. Blank, and Birgitta E. Ebert

16 Selection of Highly Expressed Gene Variants in Escherichia coli using Translationally-coupled Antibiotic Selection Markers

Maja Rennig, Daniel O. Daley, and Morten H. H. Norholm

17. Design, Engineering, and Characterization Of Prokaryotic Ligand-binding Transcriptional Activators as Biosensors in Yeast

Francesca Ambri, Tim Snoek, Mette L. Skjoedt, Michael K. Jensen, and Jay D. Keasling

18. A Capture-SELEX Strategy for Multiplexed Selection of RNA Aptamers against Small Molecules

Lasse H. Lauridsen, Holger B. Doessing, Katherine S. Long, and Alex T. Nielsen

19. High-throughput Microfluidics for the Screening Of Yeast Libraries

Mingtao Huang, Haakan N. Joensson, and Jens Nielsen

20. Growth-Coupled Carotenoids Production Using Adaptive Laboratory Evolution

Luis H. Reyes and Katy C. Kao

Part IV: Learnings from Design-Build-Test-based Projects

21. Two scale 13C Metabolic Flux Analysis for Metabolic Engineering

David Ando and Hector Garcia Martin