One step DNA assembly for combinatorial metabolic engineering.

The rapid and efficient assembly of multi-step metabolic pathways for generating microbial strains with desirable phenotypes is a critical procedure for metabolic engineering, and remains a significant challenge in synthetic biology. Although several DNA assembly methods have been developed and applied for metabolic pathway engineering, many of them are limited by their suitability for combinatorial pathway assembly. The introduction of transcriptional (promoters), translational (ribosome binding site (RBS)) and enzyme (mutant genes) variability to modulate pathway expression levels is essential for generating balanced metabolic pathways and maximizing the productivity of a strain. We report a novel, highly reliable and rapid single strand assembly (SSA) method for pathway engineering. The method was successfully optimized and applied to create constructs containing promoter, RBS and/or mutant enzyme libraries. To demonstrate its efficiency and reliability, the method was applied to fine-tune multi-gene pathways. Two promoter libraries were simultaneously introduced in front of two target genes, enabling orthogonal expression as demonstrated by principal component analysis. This shows that SSA will increase our ability to tune multi-gene pathways at all control levels for the biotechnological production of complex metabolites, achievable through the combinatorial modulation of transcription, translation and enzyme activity.

[1]  M. Smit,et al.  Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis. , 1990 .

[2]  W. Stemmer.  Rapid evolution of a protein in vitro by DNA shuffling , 1994, Nature.

[3]  T. D. Schneider,et al.  Quantitative analysis of ribosome binding sites in E.coli. , 1994, Nucleic acids research.

[4]  H. Salis,et al.  Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites , 2013, Nucleic acids research.

[5]  Harri Savilahti,et al.  Critical evaluation of random mutagenesis by error-prone polymerase chain reaction protocols, Escherichia coli mutator strain, and hydroxylamine treatment. , 2009, Analytical biochemistry.

[6]  D. G. Gibson,et al.  Enzymatic assembly of DNA molecules up to several hundred kilobases , 2009, Nature Methods.

[7]  D. Shcherbo,et al.  Bright far-red fluorescent protein for whole-body imaging , 2007, Nature Methods.

[8]  Drew Endy,et al.  Precise and reliable gene expression via standard transcription and translation initiation elements , 2013, Nature Methods.

[9]  Manfred T Reetz,et al.  Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes , 2007, Nature Protocols.

[10]  P. K. Ajikumar,et al.  The future of metabolic engineering and synthetic biology: towards a systematic practice. , 2012, Metabolic engineering.

[11]  P. R. Jensen,et al.  Synthetic promoter libraries--tuning of gene expression. , 2006, Trends in biotechnology.

[12]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[13]  A. Zeng,et al.  Protein design in systems metabolic engineering for industrial strain development , 2013, Biotechnology journal.

[14]  Ernst Weber,et al.  A Modular Cloning System for Standardized Assembly of Multigene Constructs , 2011, PloS one.

[15]  Dan S. Tawfik,et al.  Enzyme promiscuity: a mechanistic and evolutionary perspective. , 2010, Annual review of biochemistry.

[16]  Joachim Goedhart,et al.  Bright monomeric red fluorescent protein with an extended fluorescence lifetime , 2007, Nature Methods.

[17]  K. Hammer,et al.  The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters , 1998, Applied and Environmental Microbiology.

[18]  Jingdong Tian,et al.  Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries , 2011, Nature Protocols.

[19]  Doheon Lee,et al.  Bioinformatics Applications Note Gene Expression Rbsdesigner: Software for Designing Synthetic Ribosome Binding Sites That Yields a Desired Level of Protein Expression , 2022 .

[20]  J Craig Venter,et al.  Chemical synthesis of the mouse mitochondrial genome , 2010, Nature Methods.

[21]  Carola Engler,et al.  A One Pot, One Step, Precision Cloning Method with High Throughput Capability , 2008, PloS one.

[22]  Gregory Stephanopoulos,et al.  Synthetic biology and metabolic engineering. , 2012, ACS synthetic biology.

[23]  W. Edelmann,et al.  SLiCE: a novel bacterial cell extract-based DNA cloning method , 2012, Nucleic acids research.

[24]  J. Forment,et al.  GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology1[C][W][OA] , 2013, Plant Physiology.

[25]  Rainer Breitling,et al.  Computational tools for the synthetic design of biochemical pathways , 2012, Nature Reviews Microbiology.

[26]  Kevin W Eliceiri,et al.  NIH Image to ImageJ: 25 years of image analysis , 2012, Nature Methods.

[27]  Christopher M Pirie,et al.  Integrating the protein and metabolic engineering toolkits for next-generation chemical biosynthesis. , 2013, ACS chemical biology.

[28]  Jo Maertens,et al.  Construction and model-based analysis of a promoter library for E. coli: an indispensable tool for metabolic engineering , 2007, BMC biotechnology.

[29]  J. Keasling,et al.  High-level semi-synthetic production of the potent antimalarial artemisinin , 2013, Nature.

[30]  J. Liao,et al.  Protein engineering for metabolic engineering: Current and next‐generation tools , 2013, Biotechnology journal.

[31]  C. Collins,et al.  Modular optimization of multi-gene pathways for fatty acids production in E. coli , 2013, Nature Communications.

[32]  Sylvestre Marillonnet,et al.  Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system , 2012 .

[33]  Torsten Schwede,et al.  The SWISS-MODEL Repository and associated resources , 2008, Nucleic Acids Res..

[34]  M. De Mey,et al.  Promoter knock-in: a novel rational method for the fine tuning of genes , 2010, BMC biotechnology.

[35]  Peng Xu,et al.  ePathBrick: a synthetic biology platform for engineering metabolic pathways in E. coli. , 2012, ACS synthetic biology.

[36]  G. Stephanopoulos,et al.  Tuning genetic control through promoter engineering. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[37]  Yosephine Gumulya,et al.  Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates , 2008, Applied Microbiology and Biotechnology.

[38]  T. Terwilliger,et al.  Engineering and characterization of a superfolder green fluorescent protein , 2006, Nature Biotechnology.

[39]  They know why the caged bird sings... slower , 2009, Nature Methods.

[40]  Christopher A. Voigt,et al.  Automated Design of Synthetic Ribosome Binding Sites to Precisely Control Protein Expression , 2009, Nature Biotechnology.

[41]  Dan S. Tawfik,et al.  The moderately efficient enzyme: evolutionary and physicochemical trends shaping enzyme parameters. , 2011, Biochemistry.